PHYSIOLOGICAL REVIEWS
Vol. 81, No. 4, October 2001
Printed in U.S.A.
Ion Channels and Their Functional Role
in Vascular Endothelium
BERND NILIUS AND GUY DROOGMANS
Department of Physiology, KU Leuven, Campus Gasthuisberg, Leuven, Belgium
1416
1416
1418
1418
1419
1422
1426
1427
1427
1427
1427
1431
1431
1436
1436
1437
1438
1439
1439
1440
1440
1441
1443
1446
1446
1447
1447
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
I. Introduction
II. Variability of Ion Channel Expression in Endothelial Cells
III. Calcium Entry Channels
A. Nonselective cation channels
B. Store-operated or capacitative Ca2⫹ entry (SOC or CCE)
C. TRPCs: diverse Ca2⫹ entry channels in EC?
D. Ca2⫹ entry via a Na⫹/Ca2⫹ exchanger, NCX?
E. Conclusion
IV. Ion Channels Controlling Membrane Potential
A. NSC
B. K⫹ channels
C. Amiloride-sensitive Na⫹ channels
D. Cl⫺ channels
E. Conclusion
V. Voltage-Dependent Ion Channels in Endothelium?
VI. Mechanosensitive Channels in Endothelium
A. Mechanically induced Ca2⫹ entry
B. Conclusion
VII. Gap Junctional Channels
VIII. Functional Role of Ion Channels in Endothelium
A. Ion channels and electrogenesis in EC
B. Ca2⫹ signaling in EC
C. Secretion of vasoactive compounds: a potential role for ion channels
D. Ion channels and control of vessel permeation, leukocyte migration, and cell-cell contacts
E. Ion channels: a role in EC growth and angiogenesis?
F. Role of ion channels in dysfunction of endothelium?
G. Concluding remarks
Nilius, Bernd, and Guy Droogmans. Ion Channels and Their Functional Role in Vascular Endothelium. Physiol Rev 81:
1415–1459, 2001.—Endothelial cells (EC) form a unique signal-transducing surface in the vascular system. The abundance
of ion channels in the plasma membrane of these nonexcitable cells has raised questions about their functional role. This
review presents evidence for the involvement of ion channels in endothelial cell functions controlled by intracellular Ca2⫹
signals, such as the production and release of many vasoactive factors, e.g., nitric oxide and PGI2. In addition, ion
channels may be involved in the regulation of the traffic of macromolecules by endocytosis, transcytosis, the biosyntheticsecretory pathway, and exocytosis, e.g., tissue factor pathway inhibitor, von Willebrand factor, and tissue plasminogen
activator. Ion channels are also involved in controlling intercellular permeability, EC proliferation, and angiogenesis.
These functions are supported or triggered via ion channels, which either provide Ca2⫹-entry pathways or stabilize the
driving force for Ca2⫹ influx through these pathways. These Ca2⫹-entry pathways comprise agonist-activated nonselective Ca2⫹-permeable cation channels, cyclic nucleotide-activated nonselective cation channels, and store-operated Ca2⫹
channels or capacitative Ca2⫹ entry. At least some of these channels appear to be expressed by genes of the trp family.
The driving force for Ca2⫹ entry is mainly controlled by large-conductance Ca2⫹-dependent BKCa channels (slo), inwardly
rectifying K⫹ channels (Kir2.1), and at least two types of Cl ⫺ channels, i.e., the Ca2⫹-activated Cl⫺ channel and the
housekeeping, volume-regulated anion channel (VRAC). In addition to their essential function in Ca2⫹ signaling, VRAC
channels are multifunctional, operate as a transport pathway for amino acids and organic osmolytes, and are possibly
involved in endothelial cell proliferation and angiogenesis. Finally, we have also highlighted the role of ion channels as
mechanosensors in EC. Plasmalemmal ion channels may signal rapid changes in hemodynamic forces, such as shear
stress and biaxial tensile stress, but also changes in cell shape and cell volume to the cytoskeleton and the intracellular
machinery for metabolite traffic and gene expression.
www.prv.org
0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society
1415
1416
BERND NILIUS AND GUY DROOGMANS
I. INTRODUCTION
Physiol Rev • VOL
J Ca ⫽ 2 䡠 F 䡠 N 䡠 p 䡠 ␥Ca 䡠 共VM ⫺ ECa兲
(1)
where JCa is the Ca2⫹ influx, F is the Faraday constant,
and N is the number of channels. The open probability p
of the channel and its conductance ␥Ca for Ca2⫹ represent
inherent features of Ca2⫹ entry channels, which are discussed in the first part of this review. In addition, Ca2⫹
entry is regulated by the driving force for Ca2⫹, i.e., the
difference between membrane potential (VM) and equilibrium potential for Ca2⫹ (ECa). Thus Ca2⫹ entry will be
tuned by channels that modulate electrogenesis in EC,
which will be discussed in the second part of the review.
Figure 1 illustrates the dual function of ion channels on
Ca2⫹ entry in EC. Agonist stimulation induces in the
absence of extracellular Ca2⫹ only a fast release of intracellular Ca2⫹, which is accompanied by a transient hyperpolarization. In the presence of extracellular Ca2⫹ however, it also induces a plateaulike phase of [Ca2⫹]i due to
Ca2⫹ entry which leads membrane hyperpolarization and
an increased driving force for Ca2⫹ influx. Figure 1 gives
also an overview of the most important channels in EC
that are likely responsible for Ca2⫹ entry and modulation
of membrane potential. The third part of this review
discusses endothelial functions in which ion channels are
likely involved.
II. VARIABILITY OF ION CHANNEL
EXPRESSION IN ENDOTHELIAL CELLS
EC express a bewildering variety of ion channels.
Unfortunately, it is not clear which channels may be of
functional impact in the vascular tree in vivo because
some of these channels have only been detected in cultured endothelial cell lines or in primary cultured cells,
others in cells obtained by diverse cell dispersion methods, and only very few from in situ measurements in
blood vessels. It is therefore not surprising that the extensive amount of data on channel physiology in EC is
sometimes controversial and often questionable with regard to their physiological significance. In the future, it
might therefore be necessary to identify the candidate
channel in vivo. An elegant method to identify channels in
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
Endothelial cells (EC) form a multifunctional signaltransducing surface that performs different tasks dependent on its localization in the vessel tree. Arterial EC
provide a pathway for delivery of oxygen from blood to
tissue. They modulate the tone of vascular smooth muscle
cells (VSM), which in turn controls blood pressure and
blood flow by adjusting the caliber of arteries and arterioles. In the microvascular bed, EC regulate the permeation of various metabolites, macromolecules and gases,
as well as autocrine and paracrine factors and are involved in the regulation of cell nutrition. In all vessel
types, EC are involved in blood coagulation, control of the
transport between blood and tissue, movement of cells
adhering to EC, wound healing, and angiogenesis. Several
of these functions depend on constitutive properties of
EC, e.g., presenting an anticoagulative surface for blood
flow by secreting heparin sulfates and expressing ectonucleotidases and thrombomodulin (32, 33, 273, 295).
Other functions require an active response of EC to various signals of mechanical, chemical, or neuronal nature.
These signals originate from the blood or tissue side of EC
or from a coupling with other cells, such as neighboring
EC and adhering cells such as white blood cells and
thrombocytes, but also with cells at the abluminal side,
e.g., smooth muscle cells, mast cells, and fibroblasts. This
signal transduction is impaired during vessel disease (arteriosclerosis) and injury, inflammation, or hemodynamic
disturbances (hypertension).
The role of ion channels in the transduction of these
signals into cellular responses is still a matter of debate
and has received substantial attention only in the last few
years. Ion channels are mainly involved in “short-term
responses” (in the second and minute range) and affect
the synthesis or release of pro- and anticoagulants,
growth factors, and the release of vasomotor regulators.
Some of these compounds are produced “on demand”
[nitric oxide (NO), PGI2, and platelet activating factor
(PAF)], and others are released by exocytosis from storage granules [tissue plasminogen activator (tPA), tissue
factor pathway inhibitor (TFPI), von Willebrand factor
(vWF)] in response to various transmitters, such as acetylcholine, histamine, kinins (bradykinin), angiotensin,
ATP, ADP, the coagulation factor thrombin, growth factors, but also in response to mechanical stimuli, such as
shear stress and biaxial tensile stress. Fast responses
related to the regulation of the permeability of the bloodtissue interface, controlled by changes in the contractile
state of EC and their ability to modulate cell-cell contacts
are also controlled by ion channels. The “slow responses”
consist in the expression of several surface molecules,
adhesion molecules, gene expression, cytoskeletal changes,
remodeling, endothelial cell growth, and angiogenesis.
This review addresses the diversity of ion channels
and their functional role in EC. Our current knowledge is
limited to effects of ion channels on fast endothelial responses, these channels being mainly essential for the
regulation of Ca2⫹ signaling. The major part of the review
is therefore devoted to ion channels that function as
pathways for Ca2⫹ entry. These channels are mainly responsible for a long-lasting increase in the free intracellular Ca2⫹ concentration ([Ca2⫹]i) during various types of
stimuli. This Ca2⫹ entry is given by
ION CHANNELS IN VASCULAR ENDOTHELIUM
1417
endothelium by cell-specific single cell RT-PCR has been
published recently (337).
Another problem typical for EC is that ion channels
in EC adapt their appearance to the external conditions.
Apparently, ion channels or generally gene expression in
EC are highly malleable and vary with cell isolation, culture, and growth conditions (136). As a striking example,
human capillary EC grown in normal medium show TEAsensitive outwardly rectifying K⫹ currents and inwardly
rectifying K⫹ channels (IRK), but when grown in a “conditioned medium” (low cell density, medium changed only
during passages at days 5– 6 compared with controls in
Physiol Rev • VOL
which the medium was changed every second day) expressed different channels, such as slo BKCa or 4-aminopyridine-sensitive A channels (173). The quantitative features of volume-regulated anion currents in primary
explants of EC are different, i.e., activation during cell
swelling is delayed and inactivation at positive potentials
is accelerated as compared with cultured EC (381). The
expression of IRK channels in freshly isolated human
umbilical vein EC (HUVEC) changes with the number of
passages (290). The loss of different serpentine receptors
in EC during culturing is well documented (389). Likely,
EC might be especially predisposed for this variability.
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
2⫹
FIG. 1. Dual function of ion channels for Ca
entry in endothelial cells. Simultaneous recordings of intracellular
Ca2⫹ concentration ([Ca2⫹]i) and membrane potential (VM) in a freshly isolated mouse aorta endothelial cell in the
2⫹
presence and absence of extracellular Ca . Note the fast Ca2⫹ peak followed by a long-lasting plateau, which disappears
after washing out the agonist (10 ␮M ACh, 37°C). This plateau is absent when extracellular Ca2⫹ is removed. Thus the
functionally important plateau phase is mediated by Ca2⫹ entry. The increase in [Ca2⫹]i is accompanied by a hyperpolarization, which is maintained during the plateau phase, but transient in the absence of extracellular Ca2⫹ ([Ca2⫹]ext).
This hyperpolarization stabilizes the driving force for Ca2⫹ entry, VM ⫺ ECa (see text). Candidates for the Ca2⫹ entry
pathway are listed at the right together with channels that mainly control the driving force for Ca2⫹ entry (see text for
further explanation). RACC, receptor-activated cation channel; CNG, cyclic nucleotide-gated channel; HCN, hyperpolarization and cyclic nucleotide-gated channel; CCE, capacitative Ca2⫹ entry; SOC, store-operated channel; NCX,
Na⫹/Ca2⫹ exchanger; NSC, nonselective cation channel; ClCa, Ca2⫹-activated Cl⫺ channel; VRAC, volume-regulated
anion channel; CFTR, cystic fibrosis transmembrane conductance regulator.
1418
BERND NILIUS AND GUY DROOGMANS
EC also show regional heterogeneity, including differences in [Ca2⫹]i signaling, in immunological and metabolic properties, and in the pattern of released mediators
for endothelium-dependent vasorelaxation (202, 245, 273,
386, 393).
The uncertainty as to which types of ion channels are
present in a vessel type under physiological conditions is
a complicating factor for a comprehension of ion channel
function in EC.
III. CALCIUM ENTRY CHANNELS
A. Nonselective Cation Channels
Nonselective cation channels (NSC) poorly discriminating between cations have been detected in several ion
channel families. There was so far no compelling evidence for the existence of NSC members of the purinergic
ligand-gated receptor-channel complexes (the P2X family)
in EC, but very recently P2X4 receptors have been identified in several macrovascular primary cultured EC (448).
This receptor, which is involved in ATP and shear stress
induced Ca2⫹ influx (447), seems to be part of a far more
TABLE
1. STRP channels in EC
Type
Tissue
Reference No.
TRP1
BAEC
RPAEC
HPAEC
HUVEC
BPAEC
EA.hy926
MAEC
HMAEC
MAEC
BAEC
MAEC
HUVEC
EA.hy926
BAEC
MAEC
HUVEC
EA.hy926
BPAEC
BAEC
BAEC
MAEC
52, 107
257
195, 257
123
177
98, 177
195
TRP2
TRP3
TRP4
TRP5
TRP6
98
107
98
123, 177
177
52, 107
98
123, 177
177
177
52, 107
107
98
BAEC and MAEC, bovine and mouse aorta endothelial cells (EC),
respectively; RPAEC, HPAEC, and BPAEC, rat, human, and bovine
pulmonary EC, respectively; HUVEC, human umbilical vein EC;
EA.hy926, HUVEC-derived EC; HMAEC, human mesenteric artery EC.
Physiol Rev • VOL
1. Receptor-activated cation channels
Various functionally different receptor-activated cation channels (RACCs), gated by binding of agonists to
their membrane receptor, have been described in EC (123,
172, 177, 291, 295). Most of these channels are activated
via signaling cascades involving activation of phospholipase C (PLC), but it is not clear which second messengers are involved. Interestingly, TRP3 and -6, which are
members of the family of six transmembrane helix channels (STRPC, see below) that are present in endothelium
(98), are nonselective RACCs (132). Probably, TRP4 and
-5 are also nonselective RACCs (357), although it has been
shown that TRP4 might be at least part of a more Ca2⫹selective, store-operated channel (99). Endothelial RACCs,
activated by an increase in [Ca2⫹]i, are also permeable for
Ca2⫹ and provide a Ca2⫹-entry route that exerts a positive
feedback on their own activation. Their molecular structure is not known, although members of the TRP family
with Ca2⫹-calmodulin binding sites in their primary structure (see sect. IIIC) are possible candidates.
Ca2⫹-permeable NSC activated by vasoactive agonists, which have been already described in freshly isolated cells from umbilical vein (270, 271, 276, 289, 291),
have recently been characterized in more detail in cultured cell lines. The channel has a conductance of ⬃25 pS
and is permeable for Na⫹, Cs⫹, and Ca2⫹ with a permeation ratio PCa:PNa that varies between 0.03 and 2 (172,
177, 267, 272, 296). The current slowly activates during
agonist stimulation and has been observed only in the
presence of physiological [Ca2⫹]i. Buffering of [Ca2⫹]i
with 10 mM BAPTA completely prevents current activation. On the other hand, loading the cells with Ca2⫹ via the
patch pipette does not activate the current. Interestingly,
application of a store-depleting inhibitor of sarco/endoplasmic reticulum Ca2⫹-ATPase (SERCA) pumps, such as
tert-butyl-benzohydroquinone (tBHQ) or thapsigargin
(TG), can also activate this NSC. The influx of Ca2⫹
through this NSC is coupled to agonist stimulation and
depends on inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production. Block of PLC with the pyrrole-dione derivative
U73122 rapidly inhibits Ca2⫹ influx, whereas the pyrrolidine-dione derivative U73343, which does not inhibit PLC,
is ineffective. Also 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), Ni2⫹, ecanozole, and SKF-96365 inhibit
the agonist-induced Ca2⫹ entry (177). This channel shares
some properties with the hTRP3 channels expressed in
EC that do not express this NSC (178). Baron et al. (13)
described another nonselective Ca2⫹-permeable cation
channel, activated by intracellular Ca2⫹ and with a con-
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
Their role in Ca2⫹ signaling is obviously a fundamental
aspect of ion channels in EC. We therefore first describe the
various influx routes of Ca2⫹ in EC. Electrophysiological
properties of the presently known endothelial Ca2⫹ entry
pathways are summarized in Table 1.
complex Ca2⫹ entry system, because the ATP-induced
Ca2⫹ entry still persists in HUVECs treated with antisense
P2X4. NSCs belonging to the mammalian degenerins
(MDEG) family are apparently not expressed in EC.
ION CHANNELS IN VASCULAR ENDOTHELIUM
ductance of 44 pS for monovalent cations. This channel,
with a permeability ratio PCa:PNa of 0.7, is half-maximally
activated at 0.7 ␮M [Ca2⫹]i. A nonselective, agonist-induced current, which is also activated by [Ca2⫹]i and
suppressed by inhibitors of the cyclooxygenase pathway,
has been described in aortic EC (137).
2. Amiloride-sensitive NSC
arteries and forms a nonselective cation channel mediating entry of extracellular Ca2⫹ that might be involved in
the regulation of arterial blood pressure (452).
Agonist stimulation of rat pulmonary artery EC in
primary culture activates CNG channels, which resemble
CNG2 subunits of rat olfactory neurons. This activation is
subsequent to Ca2⫹ entry, probably via store-operated or
RACC channels, and is prevented in the presence of specific inhibitors of soluble guanylate cyclase. It has been
proposed that the increase of [Ca2⫹]i upon agonist stimulation activates endothelial NO synthase (eNOS), which
in turn enhances NO levels, stimulates soluble guanylate
cyclase, and increases cGMP levels, which then leads to
activation of CNG2 (see Fig. 2 and Ref. 441). This endothelial CNG is neither selective for K⫹, Na⫹, Cs⫹, nor Rb⫹.
Its activation contributes to membrane depolarization
and exerts a negative feedback on Ca2⫹ entry. Modulation
of CNG by cAMP via G protein-coupled receptors (GPCR)
has so far not been described in EC. It has to be mentioned that cGMP may also induce a negative feedback on
SOC/RACC via a PKG pathway (see sect. IIIB).
In summary, EC lack typical ligand-gated channels,
such as the P2X receptor. They express several Ca2⫹permeable, receptor-operated NSC with diverse activation mechanisms, superoxide-gated NSC, and cyclic nucleotide-gated channels. Background NSCs are probably
constitutively open. Some RACCs might be members of
the STRPC family.
3. Redox NSC
Oxidant stress, which induces superoxide anions,
activates a 28-pS NSC in a calf pulmonary artery cell line.
This channel is equally permeable for Na⫹ and K⫹ and
also for Ca2⫹. Oxidized glutathione, a cytosolic product of
oxidant metabolism, activates these channels, whereas
reduced glutathione reverses activation (198). This channel opens in two gating modes that do not depend on
intracellular Ca2⫹ stores or on [Ca2⫹]i. Activation of these
channels and the concomitant membrane depolarization
may limit Ca2⫹ influx (199). TRP3 has been proposed as a
molecular candidate for this channel (10).
4. Cyclic nucleotide-gated channels
NSC gated by cyclic nucleotides (CNG) have been
recently identified in cultured rat EC. NH2 and COOH
termini of these channel proteins are cytoplasmic with the
binding site for cyclic nucleotides located in the COOH
terminus. These channels are directly gated by binding of
cAMP or cGMP at the cyclic nucleotide-binding domain
(CNBD) and also open at hyperpolarized potentials. The
channels have a conductance of ⬃20 pS for monovalent
cations and are also Ca2⫹ permeable.
A CNG1 member of the CNG family has been recently
cloned in vascular endothelium. It is expressed in aorta,
medium-sized mesenteric arteries, and small mesenteric
Physiol Rev • VOL
B. Store-Operated or Capacitative Ca2ⴙ Entry
(SOC or CCE)
It is well known that compounds, such as tBHQ,
cyclopiazonic acid (CPA), and TG, which deplete intracellular Ca2⫹ stores without affecting Ins(1,4,5)P3 production, activate a Ca2⫹ influx pathway in EC (73, for a
review see Refs. 295, 358, 359, 398, 399). This influx is
apparently controlled by the filling degree of intracellular
Ca2⫹ stores and is presumed to represent the major pathway for Ca2⫹ influx during agonist stimulation (318).
Charged or filled stores prevent Ca2⫹ entry, whereas discharged or empty stores promote influx of extracellular
Ca2⫹. We will use the term SOC for electrophysiological
data and the term CCE for data derived from changes in
intracellular Ca2⫹ upon readmission of extracellular Ca2⫹
to store-depleted cells. This distinction is not only semantic since the latter signals also depend on other mechanisms, such as Ca2⫹ sequestration and buffering which
makes their interpretation often controversial. CCE signals are not much affected by mitochondrial Ca2⫹ buffering, but inhibition of the plasma membrane Ca2⫹ pump
(PMCA) inhibits their secondary decay phase, indicating a
modulating role of PMCA (194). Other reports also indicate a modulating role of a Na⫹/Ca2⫹ exchanger on CCE
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
An amiloride-sensitive, poorly selective cation channel (23 pS for Na⫹ and K⫹) has been observed in brain
microvessels (416). This channel seems to be important
for regulation of cation fluxes across the blood-brain barrier. Constitutively open, nonselective Ca2⫹-permeable
cation channels have been detected in both luminal and
abluminal membranes of endocardial EC. They consist
mainly of two types of nonselective outwardly rectifying
cation channels with a single-channel conductance of 36
and 11 pS, respectively. The permeability sequence for
monovalent cations is K⫹ ⬎ Rb⫹ ⬎ Cs⫹ ⬎ Na⫹ ⬎ Li⫹
which corresponds to the Eisenmann sequence IV and
indicates a weak-field strength binding site for these cations (83). Gd3⫹ and La3⫹ are efficient blockers, whereas
Ca2⫹ and Ba2⫹ but neither Mg2⫹ nor Mn2⫹ are permeable.
Vasoactive agonists induce a nonselective cation current
in endocardial EC with different ion selectivity and rectification properties (234, 235).
1419
1420
BERND NILIUS AND GUY DROOGMANS
signals: inhibition of the exchanger increases CCE and
delays the decay of store depletion-induced Ca2⫹ signals
after reapplication of extracellular Ca2⫹ (75, 118, 315).
Changes in membrane potential in non-voltage-clamped
cells will also affect the driving force for Ca2⫹ and hence
the CCE signals.
Store-operated Ca2⫹ channels (SOC) are obviously
distinct from classical voltage-operated Ca2⫹ channels
and Ca2⫹-permeable RACCs. In general, SOCs are much
more selective for Ca2⫹ than the above-described Ca2⫹
entry channels. Only two highly Ca2⫹-selective agonistactivated channels have been described in EC. The one is
activated by ATP and bradykinin and has been characterized at both the whole cell and single-channel level in
bovine aorta EC (BAEC) (227). It is very selective for
Ca2⫹ and Mn2⫹ and has a conductance of 2.5 pS for Mn2⫹.
Inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], but not
Ins(1,4,5)P3 increases its open probability, which is ⬃50%
lower at 1 ␮M [Ca2⫹]i than at 1 mM [Ca2⫹]i. The other
highly Ca2⫹-selective channel is gated by Ins(1,4,5)P3 and
has been observed in cell-attached, inside-out, and outside-out patches in BAEC. Ins(1,4,5)P3 increases its open
probability at a constant [Ca2⫹]i, whereas Ins(3,4,5,6)P4 is
ineffective. This channel is reversibly blocked by heparin
added to the intracellular side of inside-out patches and
might be directly gated by Ins(1,4,5)P3 binding (400).
The best-described SOC is the so-called “Ca2⫹-release-activated Ca2⫹-channel” (CRAC), which has been
studied extensively in mast cells and T lymphocytes (145,
146, 318). CRAC is a highly Ca2⫹-selective channel with a
single-channel conductance of ⬃30 fS for Ca2⫹ and of
⬃40 pS for monovalent cations under divalent-free conditions (188). The first “CRAC-like” current in endothePhysiol Rev • VOL
lium was published already in 1992. Bradykinin activates
a whole cell current in BAEC (⬃10 pA/cell at a potential
of ⫺60 mV) with characteristics similar to CRAC (251),
which is blocked by La3⫹ and inactivates rapidly if Ca2⫹ is
the main charge carrier but not if it is carried by Na⫹.
Inwardly rectifying CRAC-like currents have also been
recorded in other endothelial cells after depleting intracellular Ca2⫹ stores by dialyzing them with either a high
concentration of BAPTA to buffer [Ca2⫹]i at very low
concentrations or with Ins(1,4,5)P3, by extracellular application of ionomycin, or administration of the SERCA
blockers TG or tBHQ. (88, 109, 110, 306). Store depletion
protocols also activate Ca2⫹ channels in BAEC with a
conductance between 5 and 11 pS and which are not
highly selective for Ca2⫹, i.e., PCa:PNa ⫽ 10 (394, 398, 399).
The density of this current is approximately ⫺0.5 pA/pF at
0 mV, which is ⬃10 –20 times smaller than that reported
for Jurkat and peritoneal mast cells (318). Its permeability
sequence is Ca2⫹ ⬎ Na⫹ ⬎ Cs⫹ and Ca2⫹ ⬎ Ba2⫹ (88).
Elevation of extracellular Ca2⫹ increases the current amplitude, whereas removal of extracellular divalent cations
induces a large Na⫹ current that is blocked by micromolar concentrations of La3⫹.
In general, it seems that SOC currents in EC exhibit
much weaker inward rectification, are less Ca2⫹ selective,
and have a higher single-channel conductance than the
typical CRAC currents. A typical example for activation of
a SOC current in EA.hy926 cells, a cell line derived from
human umbilical vein, is shown in Figure 3. Breaking into
the cell with a patch pipette containing a high concentration of BAPTA, 30 ␮M Ins(1,4,5)P3, and 20 mM tBHQ
activates within 30 – 60 s an inward current, which reverses at a potential more positive than 50 mV and is
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
FIG. 2. Negative feedback of CNG-induced membrane depolarization on Ca2⫹ entry. Ca2⫹ entry via RACC and/or SOC elevates [Ca2⫹]i and stimulates endothelial
nitric oxide synthase (eNOS). The subsequent activation of soluble guanylate cyclase
(sGC) increases cGMP. cGMP and cAMP
[via agonist activation of G protein-coupled
receptors (GPCRs), Gs, and adenylate cyclase (AC)] activate CNG and/or HCN channels, which induces membrane depolarization. This depolarization exerts a negative
feedback on Ca2⫹ entry via RACC/SOC. In
addition, a feedback inhibition of Ca2⫹ entry
channels via activation of PKG has been described (203).
ION CHANNELS IN VASCULAR ENDOTHELIUM
1421
completely blocked by 1 ␮M La3⫹. This protocol bypasses
the agonist-mediated activation pathway via PLC activation described for RACC, but the exact activation pathway of this channel remains to be elucidated.
Pharmacological data on SOC currents in EC are rather
scarce. They are almost completely blocked by 1 ␮M La3⫹
and inhibited by econazol, NPPB, Ni2⫹, and carboxyamidotriazole (CAI), but a selective pharmacological
blocker is not available. It has recently been shown that
metabolites of the sphingomyelin pathway inhibit CRAC in
Physiol Rev • VOL
RBL cells (244). However, sphingosylphosphorylcholine,
which increases membrane capacitance in RBL cells, activates SOCs and NO production in EC (257). Likely, membrane lipids and metabolites of membrane breakdown are
directly involved in regulation of CCE (see also sect. IIIC).
The thiazolidinedione troglitazone and the imidazole-derivative SKF 96365 both block CCE in EC (186, 210, 366). It has
been shown recently that binding of capsaicin to vanilloid
receptor (VR) receptors, which are members of the OTRPC
subfamily, inhibits CCE (57).
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
FIG. 3. Store depletion activates a cation current in
mouse aorta endothelial cells (MAEC). A: activation of
2⫹
Ca entry can occur via depletion of intracellular Ca2⫹
stores. The mechanism of activation is unknown and
may involve a diffusible messenger, direct coupling between SOC and the inositol 1,4,5-trisphosphate receptor
(IP3R), or store-dependent exocytosis and membrane
incorporation. B: breaking into an isolated MAEC under
the conditions shown in the inset of C activates a cation
current. The current is blocked by 1 ␮M La3⫹ and shows
a typical delayed unblock. C: the current reverses
around ⫹60 mV, indicating a Ca2⫹-selective current.
(From S. H. Suh and B. Nilius, unpublished data.)
1422
BERND NILIUS AND GUY DROOGMANS
Physiol Rev • VOL
in amplitude if carried by Na⫹ in the absence of extracellular
Ca2⫹ (single-channel conductance of ⬃45 pS; Lepple-Wienhues, personal communication), a value similar to that observed in T lymphocytes (36 – 40 pS, Ref. 188).
C. TRPCs: Diverse Ca2ⴙ Entry Channels in EC?
The tremendous efforts in the last 3 years to identify
mammalian genes encoding for Ca2⫹ entry channels have
resulted in the discovery of the trp gene family (“transient
receptor potential”) in photoreceptors of Drosophila. The
genes of this still expanding family might be related to
several types of Ca2⫹-entry channels but in a much
broader sense than encoding for store-operated Ca2⫹ entry channels only (22, 29, 144, 457– 459). Trp genes encode
a superfamily of proteins with six transmembrane helices,
which can be divided into three subfamilies, i.e., S(hort)TRPCs, L(ong)TRPCs, and O(sm-9-like)TRPCs (Fig. 4,
Ref. 132). The latter group, named after its homology to
the osm-9 members in Caenorhabditis elegans which respond to changes in osmolarity, contains interesting
members, such as the epithelial Ca2⫹ channel (ECaC)
involved in Ca2⫹ reabsorption in the distal tubules of the
kidney, NSC such as the vanilloid receptor VR1, VRL1,
and the insulin-like growth factor regulated channel GRC
(132, 140, 183, 408). The LTRPC group contains proteins
of more than 1,600 amino acids, the function of which is
presently not understood.
The STRPC group of proteins between 700 and 1,000
amino acids comprises the mammalian isoforms of
TRP1–7, which are composed of six transmembrane helices with a pore-forming loop between the fifth and sixth
helix (Fig. 4). The NH2 terminus of all isoforms contains
three highly conserved ankyrin domains. The NH2-terminal part of the COOH terminus bears a proline-rich motif
(29, 132, 329, 333, 457). The TM4 region lacks the positive
charges typical for voltage-gated ion channels (16, 29,
457). STRPCs contain several calmodulin-binding regions,
which may mediate the Ca2⫹-dependent modulation of
store-related Ca2⫹ entry. However, the functional effect of
calmodulin is difficult to evaluate because it influences
many EC functions including the uptake of Ca2⫹ into
intracellular stores (437). Some STRPCs (e.g., TRP1) have
NH2-terminal coiled-coil structures and COOH-terminal
dystrophin motifs (for details see Ref. 333). STRPCs are
mainly activated by agonist stimulation of diverse receptors coupled to different PLC isoforms but some probably
by store depletion.
It is not clear how these channel proteins are organized, but the extreme functional heterogeneity observed
in expression experiments may point to a multiheteromeric structure. Proper heteromerization may depend on
unidentified auxiliary proteins similar to the Drosophila
InaD (from the mutant “inactivation no afterpotential”)
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
Mechanisms of CCE modulation are also still not
much elaborated. Stimulation of EC with vasoactive compounds, and application of TG results in tyrosine phosphorylation of 42- and 44-kDa isoforms of mitogen-activated protein kinase (MAP kinase). Inhibition of this
phosphorylation attenuates CCE. Tyrosine kinases may
therefore be associated with the regulation of CCE (94).
Calmodulin seems to inhibit CCE (394), whereas activation of protein kinase C (PKC) inhibits CRAC (318). Activation of protein kinase G (PKG) has been shown (204)
to inhibit both SOC (patch-clamp data) and CCE (Ca2⫹
entry).
The signal transduction mechanism linking changes
in intraluminal Ca2⫹ to the opening of plasma membrane
Ca2⫹ channels is still controversial. Various second messengers have been suggested, such as a low-molecularweight “Ca2⫹ influx factor” (CIF, 500 Da), an unidentified
tyrosine kinase or phosphatase, the arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET), cGMP,
1,2-diacylglycerol (DAG) or metabolized arachidonic acid
derivatives to explain the cross talk between Ca2⫹ stores
and membrane channels. Activation of cytochrome P-450
epoxygenase 2C, the endothelial isoform of the endoplasmic reticulum cytochrome P-450 monooxygenase (CytP450 MO), by ␤-naphthoflavone induces the biosynthesis of
5,6-EET and potentiates CCE in BAEC, porcine aorta and
human umbilical vein (119), whereas several P-450 inhibitors diminish CCE and induction of CytP-450 MO. In
addition, depletion of endothelial Ins(1,4,5)P3-sensitive
stores directly activates CytP-450 MO (139). It has therefore been proposed that CytP-450 MO-derived 5,6-EETs
may constitute a signal for CCE activation.
It has also been suggested that Ins(1,3,4,5)P4 or
Ins(1,4,5)P3 or a decrease of [Ca2⫹]i in a restricted subplasmalemmal space may directly gate the entry channels
and that a direct coupling between CRAC and the
Ins(1,4,5)P3 receptor may control channel gating (8, 14,
58, 141, 144, 193, 318, 342). A direct physical contact
between Ins(1,4,5)P3 receptors in the endoplasmic reticulum and COOH-terminal contact sites of the putative
plasma membrane TRP channels may be functionally involved (192, 193). Two short stretches of 54 (position
742–795) and 21 amino acids (position 777–797) that bind
strongly to specific sequences in the Ins(1,4,5)P3 receptor
have been identified in hTRP3. The binding domain in the
InsP3 receptor comprises 289 amino acids downstream of
the Ins(1,4,5)P3 binding site (position 638 –926) (37). This
protein-protein complex may fulfill a similar function as
the dTRP, PKC, receptor complex with the PDZ-domain
protein InaD in Drosophila (258, 339). More recently, it
has been shown that stimulation of vascular endothelial
growth factor (VEGF) receptor-2 by VEGF-E, which induces store depletion probably via phosphorylation of
PLC-␥, activates CRAC-like currents , which are blocked
by micromolar concentrations of La3⫹ and are enhanced
ION CHANNELS IN VASCULAR ENDOTHELIUM
1423
protein. This protein contains five PDZ domains (comprising DTSL motifs) and is physically linked to the rhodopsin
PLC (NorpA), a PKC (InaC), and the Gq␣-protein photoactivated by rhodopsin. The entire structure forms a signaling complex in which dTRP and dTRPL are integrated
(258). PDZ domains probably mediate the clustering of
membrane and membrane-associated proteins with TRP
channels. A similar protein hINAl (human InaD like),
which is twice as long as the Drosophila InaD (1,525 vs.
675 amino acids), has been cloned in human (330) and has
been detected in human EC (V. Flockerzi and B. Nilius,
unpublished data). In this context, formation of structural
complexes of Ca2⫹ entry channels could also be integrin
dependent, since an integrin-associated protein (IAP-50)
physically bound to integrin ␤3 has been proposed to
function as a Ca2⫹ entry channel (363). This signaling
pathway seems to be linked to the generation of inositol
phosphate and the production of Ins(1,4,5)P3 (328). INAF
is another 241-amino acid protein that is probably involved in Ca2⫹ signaling via STRPC proteins, since mutaPhysiol Rev • VOL
tions in the gene encoding for this protein directly affect
TRP channel function (215).
The activation steps following PLC stimulation are
unclear for all STRPC isoforms. One group of STRPCs
(still frequently described as TRPs) seems to be activated
via production of DAG and by binding of polyunsaturated
fatty acids (PUFAs; Refs. 58, 132, 141, 142). Stimulation of
EC via activation of the pertussis toxin-insensitive Gq
protein-coupled PLC-␤ causes Ins(1,4,5)P3 production
that initiates store depletion and probably STRPC-mediated Ca2⫹ entry. Also PLC-␥, which is activated by agonist-stimulated receptor tyrosine kinases and growth factor receptor tyrosine kinases, may activate STRPCs (98).
Figure 5 summarizes the activation pathways for STRPCs
that may be relevant for EC. It has to be noted that only
a few members of the putative STRPC family have been
analyzed at the functional level.
Phylogenetic analysis of the mammalian STRPCs
(TRPs) reveals four subfamilies (see Fig. 6A; Refs. 98, 132,
142, 333).
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
FIG. 4. The superfamily of TRP channels. Putative
domain structure of the TRP proteins consisting of 6
transmembrane helices. The NH2 terminus contains
several ankyrin repeats that may be important for
interaction with the cytoskeleton; the COOH terminus
contains a proline-rich motif. The putative pore region
is between helix 5 and 6. This basic structure is common to all members of the TRP channel superfamily
(131). This family can be divided on the basis of
homology into three subfamilies: short (STRPC), osm
(OTRPC), and long (LTRPC). These subfamilies have
structural differences, as indicated. For further explanation, see text and References 131, 141.
1424
BERND NILIUS AND GUY DROOGMANS
1) The first subfamily is a subclass that comprises
TRP4 and TRP5. Heterogeneously expressed mTRP4 and
mTRP5 are NSCs with a single-channel conductance of
42– 66 pS that can be stimulated by guanosine 5⬘-O-(3thiotriphosphate) (GTP␥S). The activation pathway may
integrate signaling pathways from G protein-coupled receptors and receptor tyrosine kinases independently of
store depletion. Their biophysical properties are inconsistent with those of CRAC channels. In other experiments,
however, TRP4 and -5 are clearly store dependent and
have a much higher Ca2⫹ selectivity (331, 332, 434).
2) TRP1, which is closely related to TRP4 and -5 in
terms of evolutionary distance, probably forms SOCs
(223, 224, 431) but is probably less Ca2⫹ selective than
TRP4 and -5 (329, 387, 434).
3) The short TRP3, TRP6, and TRP7 with ⬃800 amino
acids form store-independent NSCs probably activated
via the DAG pathway (141) and are also sensitive to
[Ca2⫹]i (178, 307, 461). TRP3 and -6 have a conductance
between 25 and 66 pS and are not very Ca2⫹ selective
(141, 178). However, also a store-dependent activation
mechanism has been considered for hTRP3 (124, 193).
FIG. 6. Overview of the TRPC superfamily
and expression pattern of STRPCs in mouse aorta
endothelial cells (MAEC). A: phylogenetic tree of
the mammalian STRPCs and the two members of
Drosophila (TRP and TRP-like, TRPL). Functionally, four different types are discussed (for the
controversial discussion see text). B: expression
pattern in MAEC revealed by RT-PCR. The ⫹ and
⫺ refer to presence or absence of reversed transcriptase. GAPDH, glyceraldehyde 3-phosphate
dehydrogenase. [From Freichel et al. (98). Copyright 1999 Karger, Basel.]
Physiol Rev • VOL
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
FIG. 5. Activation mechanisms of the
short TRP channels. Short TRP channels are
mainly activated via agonist receptors (G protein-coupled receptor, GPCR, and receptor
tyrosine kinases, RTK) (131). These receptors activate various phospholipase C. Some
STRPCs are directly activated by 1,2-diacylglycerol (DAG) or via still unknown interactions with G proteins, Ca2⫹, or even InsP3.
TRP3, -6, and -7 are probably receptor-activated cation channels or RACCs. Another
mechanism of activation is probably related
to emptying of intracellular Ca2⫹ stores (see
Fig. 3A).
1425
ION CHANNELS IN VASCULAR ENDOTHELIUM
4) The subfamily consisting of TRP2 is still uncertain
and forms probably truncated channel proteins.
Several TRPs are expressed in endothelium, as illustrated by their expression pattern in mouse aorta in Figure 6B. Their expression varies, however, considerably
between different types of EC, as shown in Table 2.
BPAEC express TRP1 and -4; BAEC two splice variants of
TRP1, -3, -4, and -5; HUVEC cells TRP1, -3, and -4; mouse
TABLE
aorta EC TRP1, -2, -3, -4, and -6; and rat pulmonary EC
TRP1 but neither TRP3 nor -6 (52, 124, 178, 259).
SOC currents have been identified in BPAEC cells
(88), but agonist stimulation does not activate Ca2⫹-permeable NSCs in these TRP3-deficient cells. Expression of
hTRP3 gives rise to functional expression of 25-pS NSCs
and enhances the agonist-induced Ca2⫹ increase in these
cells (Fig. 7, Ref. 178), suggesting that hTRP3 channels
2. Ca2⫹ entry pathways in EC identified by current measurements
Current
CCE
Conductance
0.02 pA/pF at ⫺40 mV,
at 1 mM Ca2⫹
0.1–0.2 pA/pF at 0 mV
SOC
NSCCa
Mol Cand
TRP3
HUVEC
123
Ca2⫹
Ca2⫹ ⬎ Ba2⫹ ⬎⬎
Na⫹
BHQ?
Ins(1,4,5)P3, TG,
VEGF-E
TRP1
257
210
Ca2⫹ ⬎ Na⫹
TG, Ins(1,4,5)P3,
BAPTA
TG, Ins(1,4,5)P3, 12
BAPTA
Agonists, TG, BHQ
?
Pulmonary EC
PTP-activated,
VEGFR-2,
HUVEC,
PAEC
HUVEC, BPAEC
Block by ␮M
La3⫹, BPAEC
Inhibition by
calmodulin,
BAEC
88
HUVEC
Gd3⫹ block,
BAEC
HUVEC
449
218
Ca ⬎ Ba
Na⫹
2⫹
2⫹
⬎⬎
CPA, [Ca2⫹]i???
Agonist, [Ca2⫹]i
?
⬃25 pS
Na⫹ ⬃ K⫹ ⬎ Ca2⫹
Agonist, [Ca2⫹]i
TRP3??
8 pS (100 Ca2⫹)
Ca2⫹ ⬎ Ba2⫹ ⬎ Na⫹
[Ca2⫹]i, Ins(1,4,5)P3
24 pS
24 pS
Na⫹ ⫽ K⫹ ⬎ Ca2⫹
Na⫹ ⬎ Cs⫹ ⬎ K⫹,
Ca:Na ⫽ 1.6
Ins(1,4,5)P3-R in
plasmalemma
2 pS (100 Mn2⫹)
Ca2⫹ ⫽ Ba2⫹, Mn2⫹
⬎ Na⫹
Na⫹ ⫽ K⫹ ⬎Ca2⫹
Na⫹ ⫽ K⫹ ⬎ Ca2⫹
[Ca2⫹]i, serotonin
Agonists, [Ca2⫹]i,
Ins(1,4,5)P3,
[Ca2⫹]i necessary,
Ca2⫹ activated?
[Ca2⫹]i, Ins(3,4,5,6)P4
24 pS, 16–19 pS in
Ca2⫹
21 pS, 6 pS in Ca2⫹
18 pS
BAEC
110, 303
249, 390, 394,
395
39, 268, 274,
287, 289,
443
396
Hepatic EC
EA, HUVEC,
BPAEC,
MAEC
38
176, 177, 377
InsP4-R
BAEC
225
?
CAEC, MAEC
BPAEC, EA,
13, 377
197, 198, 376
Na⫹ ⬎ K⫹ ⬎ Ca2⫹
Agonists via [Ca2⫹]i
Oxidant stressBuOOH, CN⫺,
[Ca2⫹]i?
Constitutively open
?
Endocardium
233, 234
K⫹ ⬎ Na⫹ ⬎ Ca2
Na⫹ ⬎ Ca2⫹
NO donors, cGMP
Stretch, cell swelling
CGN1, CGN2
BBBEC
BBBEC
154, 164, 446
332
Ca ⬎ Na ⫽ K
Na⫹ ⫽ K⫹ ⬎ Ca2⫹
Pressure
Pressure
RAEC
Upregulated in
SHR rats,
RTAEC
Endocardial EC
239, 240
147, 148
PAEC
HUVEC, HCEC,
HAEC
?
208
170, 264, 363
TRP3
Na⫹ ⫽ K⫹ ⬎ Ca2⫹,
Stretch
Ba2⫹
2⫹
2⫹
⫹
40 pS, 19 pS in Ca
Ca ⬍ Na
Stretch
Ca2⫹ ⬎ Na⫹ ⫽ Cs⫹,
Shear
NSC
Exchanger can work in reverse mode to import Ca2⫹
32 pS, 13 pS in Ca2⫹
NCX
Reference No.
Ins(1,4,5)P3, TG
Na⫹ ⬎ Ca2⫹
Na⫹ ⫽ K⫹ ⬎ Ca2⫹
44 pS
32 pS
Remarks
NSC, Ca2⫹ ⬎ Na⫹
5 pS in Ca2⫹, 11 pS
Ca2⫹ free, 0.3–2 pA/
pF in Ca2⫹ at ⫺60
mV (est.)
28 pS, 6 pS in Ca2⫹
28 pS, 13 pS in Ca2⫹
1 pA/pF at ⫺45 mV, 11
pS, 32 pS
CNG
Mech-NS
Activation
146
75, 381
SOC, store-operated channels; CCE, capacitative Ca2⫹ entry channel (STRPCs ?); NSCCa, nonselective, Ca2⫹-permeable channels; HCN,
hyperpolarization activated cation channels, nucleotide sensitive; CNG, cyclic nucleotide-gated cation channels; NCX, Na⫹/Ca2⫹ exchanger; HUVEC,
human umbilical vein EC; PAEC, porcine aorta EC; BPAEC, cultured (calf) bovine pulmonary artery EC; BAEC, bovine aorta EC; EA, EA.hy926
HUVEC derived EC (81); MAEC, mouse aorta EC; CAEC, pig coronary EC; BBBEC, blood-brain barrier EC; RAEC, excised rat aorta EC; RTAEC,
rat aorta EC; HCEC, human coronary EC; HAEC, human aorta EC; SHR, spontaneously hypertensive; TG, thapsigargin; Ins(1,4,5)P3, inositol
1,4,5-trisphosphate; [Ca2⫹]i, free intracellular Ca2⫹ concentration; NO, nitric oxide; InsP4-R, inositol tetrakisphosphate receptor.
Physiol Rev • VOL
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
⬃3 pA/pF at ⫺50 mV
(est.)
5 pA/pF at ⫺80 mV
⬃fS, 45 pS in Ca2⫹ free
Selectivity
1426
BERND NILIUS AND GUY DROOGMANS
may serve as a Ca2⫹-influx pathway during agonist stimulation. These channels share several properties with an
endogenous nonselective Ca2⫹ entry channel observed in
TRP3-expressing EA.hy926 cells (177). Expression of an
NH2-terminal fragment of hTRP3, which exerts a dominant negative effect on TRP channel function presumably
due to suppression of channel assembly (124), abolishes
activation of a store-dependent, agonist-activated nonselective cation current in HUVEC cells (124). These results
suggest a role of STRPC-related proteins as store-operated and/or agonist-activated cation channels identical to
TRP3. Other studies show that ␤-estradiol significantly
downregulates TRP4, and transretinoic acid dramatically
upregulates TRP5, whereas these hormones have little
effect on CCE (52). Expression of TRP1 and TRP4 in
BPAEC induces a cation current that is activated by tBHQ
and Ins(1,4,5)P3, suggesting that both STRPCs may form
functional but not highly Ca2⫹-selective channels (409).
Interestingly, activation of TRP1 in rat pulmonary EC
induces changes in EC shape and remodeling of the peripheral (cortical) filamentous actin (F-actin) cytoskeleton (259).
Another question refers to the nature of Ca2⫹ stores
involved in CCE activation. In addition to Ins(1,4,5)P3mediated Ca2⫹ release, ryanodine-sensitive Ca2⫹ release
(RsCR) seems also to be important for Ca2⫹ signaling in
EC. Several studies have presented evidence for endothelial ryanodine receptors (1, 62, 213, 460). In human aortic
EC, ryanodine-sensitive Ca2⫹ stores seem to be involved
in the regulation of agonist-sensitive, Ins(1,4,5)P3-depleted Ca2⫹ pools. Caffeine and ryanodine induce only a
small Ca2⫹ entry if the Ins(1,4,5)P3-sensitive stores are
filled. However, after store depletion, caffeine but not
ryanodine induces Ca2⫹ entry (62). One likely interpretation of these effects is that caffeine regulates CCE and/or
Physiol Rev • VOL
that caffeine also controls the extent of store depletion by
agonist stimulation. Also other experiments show that
RsCR may modulate but not initiate CCE. By using deconvolution microscopy to monitor simultaneously cytosolic, perinuclear Ca2⫹ by fura 2 and membrane near,
subplasmalemmal Ca2⫹ by the cell membrane impermeable dye FFP-18 in the same EC (315), it has been shown
that Ca2⫹ release initiated by ryanodine increases subplasmalemmal Ca2⫹ but not perinuclear Ca2⫹. In addition,
conditions that initiate RsCR also increase CCE. Although
these data support the existence of local Ca2⫹ gradients,
they are not indicative for CCE activation by the discharge of ryanodine-sensitive Ca2⫹ stores. Other findings
do not support any effect of the discharge of ryanodinesensitive stores on Ca2⫹ entry or Mn2⫹ quenching (354).
However, subplasmalemmal Ca2⫹ elevation could dramatically change the driving force for Ca2⫹ entry by activating
BKCa channels, which in turn would affect CCE (see sect.
IVA2).
D. Ca2ⴙ Entry Via a Naⴙ/Ca2ⴙ Exchanger, NCX?
The modulation of endothelial Ca2⫹ signaling via a
Na /Ca2⫹ exchanger (NCX) is an incompletely resolved
issue. Several groups have shown that a reduction of the
Na⫹ gradient increases [Ca2⫹]i via an NCX-mediated Ca2⫹
entry (352, 369, 378, 385). Evidence that the exchanger
may also shape Ca2⫹ transients activated by vasoactive
agonists comes from experiments in which [Ca2⫹]i transients are modulated by changing Na⫹ gradients, loading
EC with Na⫹ by using the ionophore monensin, application of NCX blockers, such as 3⬘,4⬘-dichlorobenzamil (in
comparison with the NCX ineffective amiloride) or La3⫹
(369). Recently, a NCX protein was detected in caveolinrich EC membrane fractions (385).
⫹
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
FIG. 7. Functional expression of hTRP3 in bovine
pulmonary artery endothelial cells (BPAEC), which do
not express TRP3. A and D: comparison of the effects of
ATP (1 ␮M) on membrane potential of wild-type and
htrp3 transfected BPAEC. Note the small hyperpolarization in the wild-type cells due to activation of a Ca2⫹activated Cl⫺ channel. In hTRP3 expressing cells, a clear
depolarization toward the reversal potential of a nonselective cation channel can be observed. Membrane potential was measured in current-clamp mode. B and E:
changes in [Ca2⫹]i in nontransfected and htrp3-transfected cells. Note the much stronger increase in [Ca2⫹]i
during agonist stimulation in the htrp3 transfected cell. C
and F: current (I)-voltage (V) curves obtained from voltage ramps (⫺100 to ⫹100 mV) in both cells types before
(a) and during application of ATP (b). Note the appearance of an IRK channel at negative potentials and a Cl⫺
channel at positive potentials (see also Fig. 11). In transfected cells, a clear activation of a cation current can be
observed which correlates with the increased Ca2⫹ plateau. [From Kamouchi et al. (178), with permission from
J Physiol.]
ION CHANNELS IN VASCULAR ENDOTHELIUM
E. Conclusion
IV. ION CHANNELS CONTROLLING MEMBRANE
POTENTIAL
Recently, a family of NSC has been cloned which
includes channels activated by hyperpolarization and by
cyclic nucleotides (HCN; Refs. 24, 25–27, 228). Four new
members of this family with a length between 780 and
⬎900 amino acids have been cloned recently. These channels consist of six transmembrane domains (TM) and are
modulated by binding of cyclic nucleotides to a CNBD.
TM4 is positively charged and contains at least 10 arginine
and lysine residues. These channels show a high overall
similarity with the cone CNG. They activate slowly at
hyperpolarized potentials and binding of cyclic nucleotides. They are permeable for monovalent but not for
divalent cations, Na⫹ being more permeable than K⫹
(PNa:PK ⬃0.15– 0.3). HCN channels play a role as mediators of NO- and cGMP-dependent cellular processes (25).
Therefore, they might be of potential importance in EC.
Indeed, in EC from the blood-brain barrier, a hyperpolarization-activated current carried by Na⫹ and K⫹ has been
described (165) that is modulated by NO, cGMP, and
cAMP. Its modulation by a cyclic guanosine nucleotide
may also explain previous results about a role for NO in
regulating blood-brain barrier function.
B. Kⴙ Channels
2⫹
A crucial role of ion channels in endothelial Ca
signaling is the fine-tuning of the electrochemical gradient
for Ca2⫹ (176, 226, 271, 272). The influence of membrane
potential, which as discussed in section VIIIA is determined by the expression pattern of various ion channels,
on Ca2⫹ entry in EC has been clearly demonstrated (170,
176, 226, 359). K⫹ channels, including Ca2⫹-activated K⫹
channels, inwardly rectifying K⫹ channels, and probably
also voltage-dependent K⫹ channels, are the major class
of ion channels in setting the membrane potential. In
addition, it has been shown recently that Ca2⫹ entry in
freshly isolated EC from rabbit aorta requires the presence of extracellular Cl⫺ to maintain a polarized membrane (433). Thus Cl⫺ channels, e.g., volume-regulated
anion channels (VRAC), Ca2⫹-activated Cl⫺ channels
(CaCC) and, interestingly, also cystic fibrosis transmembrane conductance regulator (CFTR) channels, recently
detected in EC (388), might be important modulators of
the inwardly driving force for Ca2⫹. In addition, NSC may
tune the membrane potential of resting and activated EC
(178, 419).
A. NSC
Some NSC are obviously impermeable for Ca2⫹
(419). They may induce a negative feedback on Ca2⫹ entry
by inducing depolarization of EC (see also Fig. 2). Little
information is available at the molecular level. Probably,
some TRP members might be candidates for background
Ca2⫹-impermeable NSC in resting endothelium.
Physiol Rev • VOL
1. Inwardly rectifying K⫹ channels (IRK or Kir)
IRK is one of the most important channels for the
control of the resting potential in nonstimulated cells. It
conducts inward currents at potentials more negative
than the K⫹ equilibrium potential but permits much
smaller currents at potentials positive to that potential.
However, this channel is quite heterogeneously expressed
in different EC types. Some macrovascular EC express
the inward rectifier channel (BPAEC, BAEC, coronary
EC), others do not (microvascular EC, EA.hy926 cells),
whereas in other cells only a fraction of the cells shows
IRK activity (HUVEC, Ref. 290). In general, the channel
appears to be more abundant in cultured than in primary
EC. In intact endocardial endothelium, it is mainly confined to the luminal side of the cells (235), but it appears
to be randomly distributed in monolayers of cultured
BAEC (60). If present, this conductance together with the
basal activity of the volume-regulated anion channel and
a background NSC determine the resting membrane potential of EC (46, 96, 419). As discussed in section VIIIA,
the impact of these channels on the resting potential is
often counteracted by that of Cl⫺ and background NSC
channels.
In addition to its well-understood role in controlling
electrogenesis, IRK is a K⫹ sensor, as shown in Figure 8.
A small increase in extracellular K⫹, e.g., resulting from
the activation of BKCa channels, shifts the reversal potential for K⫹ toward a more positive value and thereby
increases the conductance of IRK. This enhanced IRK
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
Ca2⫹ entry in EC occurs via different pathways, but
the molecular nature of these different entry pathways
and their mechanism of activation are still elusive. Likely,
endothelial Ca2⫹ entry channels belong to the family of
STRPC which comprise both receptor-activated, NSC and
more Ca2⫹-selective store-operated channels. Activation
of these channels occurs via both GPCR and receptor
tyrosine kinases. In addition, ligand-gated Ca2⫹-permeable channels, such as CNG channels and P2X receptors,
and probably endothelial isoforms of the NCX provide
alternative pathways for Ca2⫹ entry in EC. These mechanisms, which are controlled by various signaling cascades, are important for maintaining long-lasting responses of EC “on demand,” such as Ca2⫹-dependent
synthesis and release of NO, secretion of vasoactive compounds out of vesicular compartments, and probably also
during stimulation of EC by growth factors, such as
VEGF.
1427
1428
BERND NILIUS AND GUY DROOGMANS
conductance is sufficient to overcome the depolarizing
action of the nonselective cation and Cl⫺ conductance
(see also Fig. 11) and shifts the membrane potential toward more negative potentials.
The conductance of single endothelial Kir channels
ranges from 23 to 30 pS in symmetrical K⫹ solutions (84,
156, 180, 290, 295, 325, 372, 373). Typical features of this
channel are the increase in single-channel conductance
with the square root of the extracellular K⫹ concentration
(289, 373, 462) and a permeation profile of PK ⬎ PRb ⬎ PCs
(325, 373).
Extracellular Ba2⫹, TEA, TBA, and Cs⫹ all block Kir
(214, 322, 343, 419, 427). Extracellular Mg2⫹ causes a
time-dependent block at negative potentials, which can be
antagonized by extracellular K⫹. At positive potentials,
also intracellular Mg2⫹ induces a time- and voltage-dependent block (84).
The shear stress evoked hyperpolarization of EC has
been attributed to an enhanced outward whole cell K⫹
current, which can be blocked by Cs⫹. Shear stress also
enhances the open probability of IRK channels in luminal
cell-attached patches (312) and in inside-out patches
(163).
IRK seems to be metabolically regulated. Intracellular ATP but not its nonhydrolyzable analogs adenosine
5⬘-O-(3-thiotriphosphate) (ATP␥S) and adenylyl imidodiphosphate (AMP-PNP) prevent its rundown in whole
cell mode. Reduction of intracellular ATP and induction
of hypoxic conditions dramatically downregulate IRK
(180). The phosphatase inhibitor okadaic acid also prePhysiol Rev • VOL
vents rundown, but protamine, an activator of phosphatase 2A (PP2A), enhances the rate of rundown. Phosphorylation of the channel molecule therefore seems to be
essential for maintaining its activity, its rundown probably being due to dephosphorylation by PP2A (180).
An interesting observation is the inhibition of IRK in
capillary and macrovascular endothelial cells by the vasoactive agonists angiotensin II, vasopressin, vasoactive
intestinal polypeptide (VIP), endothelin (ET)-1, and histamine (150, 290, 322, 456). A similar inhibition has been
observed if GTP␥S is applied via the patch pipette (150,
180). This inhibition is probably due to activation of a
pertussis toxin (PTX)-insensitive G protein, which may
mediate these inhibitory actions by modulating PPA2.
IRK has been identified as Kir2.1, a member of the Kir
family (95, 179). Two highly conserved transmembrane
regions, consisting of 427 amino acids and a highly conserved TIGYG-H5 motif in the pore region, characterize
this channel. pH insensitivity, which is also characteristic
for the endothelial IRK, is conferred on the channel by a
methionine at site 84 (M84). Mg2⫹ and spermine, spermidine, and putrescine block appears to be linked to D172.
S425 in the COOH-terminal phosphorylation motif RRESEI, which is important for modulation of Kir2.1, might be
a candidate for the observed modulation by phosphatases
(180).
In addition to well-defined IRK channels, a number of
poorly characterized K⫹ channels, apparently not belonging to any of the cloned subfamilies of K⫹ channels, have
been observed in EC. An example is the 170-pS inward
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
⫹
FIG. 8. IRK (Kir2.1) channels are K
sensors. A:
topological scheme of the EC Kir 2.1 channel, showing
the GYG motif in the pore and the RRESEI phosphorylation site in the COOH terminus that is important for
channel modulation. B: typical I-V curves in BPAEC (see
also Figs. 7 and 11). The net current I-V relationships are
composed of several ionic current as discussed above.
Note the flat I-V relationship at low extracellular K⫹
concentrations, which are consistent with an unstable
membrane potential. C: increasing extracellular K⫹ to 12
mM, although shifting the reversal potential of IRK
(Kir2.1) toward more positive values, stabilizes the membrane potential at more negative values because of the
increase in conductance of IRK. After washing out of the
elevated K⫹, the bistable behavior of the membrane
potential is reflected in the transient fluctuations between two polarization levels. D: effect of changing extracellular K⫹ from 6 to 12 mM in nine individual EC is
also shown. Note the homogeneous response indicating
a K⫹ sensor function of IRK. (From B. Nilius and J.
Prenen, unpublished data.)
ION CHANNELS IN VASCULAR ENDOTHELIUM
rectifier in aorta EC activated by isoprenaline, adenosine,
forskolin, and membrane-permeable analogs of cAMP and
inhibited by PKA inhibitors (117).
2. Ca2⫹-activated K⫹ channels
Physiol Rev • VOL
erts an autocrine effect on BKCa in inside-out patches of
primary cultures of endothelial cells from pig coronary
artery (12). A similar autocrine effect of NO release is
however unlikely, since the NO donor S-nitrosocysteine
does neither directly activate nor modulate BKCa channels
activated by increased [Ca2⫹]i in cultured endothelial
cells (129). Interestingly, K⫹ efflux via endothelial BKCa
might activate K⫹-sensitive smooth muscle IRK channels
and induce hyperpolarization. Thus K⫹ itself may act as
EDHF (82).
BKCa has been identified by RT-PCR analysis as hslo
in EA.hy926 cells (179). Mammalian slo channels, consisting of ⬃1,200 amino acids, share a high degree of homology with voltage-dependent K⫹ channels and consist of
six transmembrane segments with a positively charged S4
segment. Five additional hydrophobic segments form a
unique secondary structure in the COOH terminus of the
mammalian slo channel. The origin of the voltage dependence is not completely clear, but hslo channels probably
contain a Ca2⫹-independent intrinsic voltage sensor (411).
An extra segment (segment 0) in the NH2 terminal is
probably a coupling site for the ␤-subunit. Four ␤-subunits have been cloned so far: ␤1 seems to be mainly
expressed in smooth muscle cells; ␤2 in endocrine cells;
␤3 probably in epithelium, liver, pancreas, and intestinal
tract; and ␤4 in the central nervous system (15). The
unique COOH terminus possibly contains four additional
helices, H7–H10, which represent almost 70% of the protein (80, 392). A 28-amino acid stretch between segment 9
and 10 contains highly conserved negatively charged
amino acid residues that possibly form a highly selective
Ca2⫹ binding site, the “Ca2⫹ bowl” (Fig. 9A, and Ref. 362).
Expression of ␣-hslo in EC that lack BKCa channels enhances the agonist-induced increase in [Ca2⫹]i and shifts
the membrane potential toward more negative values, a
functional example in support of the role of the driving
force for Ca2⫹ influx in EC. Figure 9, B and C, shows an
example of such an expression experiment in BPAEC,
which does not express functional slo channels. The plateau phase of the Ca2⫹ transient in BPAEC cells transfected with hslo is much more pronounced than in nontransfected cells. This finding correlates with the
hyperpolarizing action of vasoactive agonists in the transfected cells and indicates that activation of hslo-BKCa
exerts a positive feedback on the Ca2⫹ signals by counteracting the negative (depolarizing) effect of Ca2⫹-activated Cl⫺ channels (179).
The ␤-subunit of hslo could not be detected with
RT-PCR methods in EC, which does not exclude the
presence of other ␤-subunits. The BKCa opener DHS-I,
which only works in the presence of the ␤-subunit, is
ineffective in EC (248, 383), a functional indication for the
absence of the ␤1-subunit. Expression of the ␤-subunit in
EA.hy926 cells induces a leftward shift of the activation
curve and has a sensitizing effect on BKCa (316, 317).
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
The initial response to most EC stimuli is an increase
in [Ca2⫹]i that is often due to influx of Ca2⫹ and therefore
depends on the electrochemical Ca2⫹ gradient, primarily
set by the membrane potential. The depolarizing action of
Ca2⫹ influx on the membrane potential is in various EC
compensated by activation of Ca2⫹-dependent K⫹-channels (KCa), their increased open probability at elevated
[Ca2⫹]i causing membrane hyperpolarization. Different
types of Ca2⫹-activated K⫹ channels have been described,
e.g., BKCa or maxi-K channels, IKCa or intermediate-conductance channels, and SKCa or small-conductance channels. However, the expression of these channels between
different types of EC is also very heterogeneous (295).
High-conductance Ca2⫹-activated K⫹ channels, BKCa,
are expressed in most freshly isolated EC and EC in
primary culture. Their conductance ranges between 165
and 240 pS (129, 147, 176, 179, 220, 289, 349, 381). The fast
elevation in [Ca2⫹]i during stimulation with vasoactive
agonists induces a fast increase in the probability of the
channel being open. The open probability is enhanced at
more positive potentials, suggesting that the apparent
Ca2⫹ affinity of the channel is voltage dependent and that
the open probability depends on both [Ca2⫹]i and voltage.
An increase in [Ca2⫹]i shifts the voltage dependence of
activation toward more negative potentials (13, 63, 129,
176, 317).
The endothelial BKCa is blocked by charybdotoxin
(IC50 ⬃50 nM), iberiotoxin, TEA (IC50 ⬃1 mM, 100% block
at 10 mM), D-tubocurarine, and quinine. Extracellular
Mg2⫹ block is voltage dependent (13, 349, 350). The benzimidazolone compounds NS004 and NS1619 (in the ␮M
range) increase the open probability of BKCa in EC and
shift its activation curve toward more negative potentials
(121, 316). Nanomolar amounts of intracellular dehydrosoyasaponin (DHS-I) induce discrete episodes of high
channel open probability interrupted by periods of apparently normal activity. DHS-I decreases the Ca2⫹ concentration for half-maximal activation of the channel (113,
247). BKCa activators may be functionally important, since
it has been shown in many assays that inhibition of BKCa
interferes with NO release (42). It has not yet been shown
that cGMP-dependent kinases can activate BKCa in EC
like in smooth muscle (166, 445).
Ca2⫹-activated K⫹ channels in vascular smooth muscle cells are targets for various physiological factors released from the endothelium, including NO (31, 346, 384)
and endothelium-derived hyperpolarizing factor (EDHF;
probably a cytochrome P-450-derived arachidonic acid
metabolite; Refs. 92, 334). This latter compound also ex-
1429
1430
BERND NILIUS AND GUY DROOGMANS
Ryanodine-sensitive Ca2⫹ release during agonist
stimulation is the main activator of BKCa in an umbilical
vein-derived cell line, suggesting that these channels may
form a subplasmalemmal complex with Ca2⫹ release
units located in their close vicinity (100).
Intermediate-conductance, Ca2⫹-activated K⫹-channels, IKCa, are inwardly rectifying and have a conductance
between 30 and 80 pS in symmetrical K⫹ and 15 pS at
physiological extracellular K⫹. IKCa are activated by
Ins(1,4,5)P3-sensitive Ca2⫹ release induced by agonists,
such as bradykinin, acetylcholine, and ATP (355). ET-1
and ET-3 activate IKCa channels in brain microvascular
endothelium via endothelin-A receptors (404). A Ca2⫹dependent 30-pS channel in cultured BAEC is regulated
by G proteins (401). GTP together with Mg2⫹, as well as
GTP␥S stimulates IKCa, an effect that is reversed by
guanosine 5⬘-O-(2-thiodiphosphate) (GDP␤S). An agonistactivated IKCa with a single-channel conductance of 34 pS
Physiol Rev • VOL
and a dissociation constant (KD) of 0. 63 ␮M for Ca2⫹ has
been described in intact endocardial cells (234).
The pharmacology of the endothelial IKCa is not very
detailed. Charybdotoxin, quinine, and TBA are efficient
blockers of IKCa channels (220, 355, 356, 404). Noxiustoxin, a toxin purified from Centuroides scorpion toxin
(61, 396), and interestingly NS1619 (44), an activator of
BKCa channels, are potent inhibitors of IKCa channels. The
hydrophilic oxidative reagents 5,5⬘-dithio-bis(2-nitrobenzoic acid) and thimerosal reduce IKCa channel activity in
BAEC recorded in inside-out experiments but do not affect its unitary conductance. This activity is partly restored by the SH-group reducing agents dithiothreitol or
reduced glutathione. The lipid-soluble oxidative agent
4,4⬘-dithiodipyridine is less potent, suggesting that critical
SH groups localized at the inner face of the cell membrane
may be involved in channel gating (45).
The biophysical and pharmacological profiles of
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
FIG. 9. Functional expression of the
␣-subunit of hslo in BPAEC, which does
not express BKCa. A: topology of the hslo
structure. The ␤-subunit is probably not
expressed in EC. B: changes in membrane potential and [Ca2⫹]i in a control
BPAEC during stimulation with 1 ␮M
ATP (Ca2⫹-activated Cl⫺ channels inhibited with 100 ␮M niflumic acid) are
shown. [From Kamouchi et al. (178).
Copyright Harcourt Brace & Co. Ltd.
1997. Reprinted with permission of
Churchill Livingstone.] C: same as B, but
in a BPAEC transfected with the ␣-subunit of hslo. D: note the difference in
resting potential, the more pronounced
hyperpolarization, and enhanced plateau
Ca2⫹ level during agonist stimulation in
the transfected cell. [Modified from Kamouchi et al. (178).]
ION CHANNELS IN VASCULAR ENDOTHELIUM
3. ATP-sensitive K⫹ channels, KATP
The reduced intracellular ATP content during ischemic/hypoxic conditions can be mimicked by intracellular
dialysis with ATP-free solutions or application of extracellular glucose-free/NaCN solutions. Under these conditions, increased whole cell and single-channel currents
have been observed in EC from rat aorta and brain microvessels. Currents are reversibly blocked by glibenclamide or ATP in inside-out patches and activated by the
KATP channel opener pinacidil (164). Lowering intracellular ATP or applying the K⫹ channel activator levcromakalim evokes unitary currents with a conductance of
25 pS in rabbit aortic EC. They are inhibited by glibenclamide or in inside-out patches by increasing the ATP
concentration (184, 185). Low concentrations of the K⫹
channel openers diazoxide and rilmakalim cause a proPhysiol Rev • VOL
nounced glibenclamide-sensitive hyperpolarization in coronary capillary EC and induce a rapid Ca2⫹ transient
followed by a sustained elevation of [Ca2⫹]i. The high
sensitivity to diazoxide has been interpreted as a novel
type of KATP channel composed of a novel combination of
Kir and sulfonylurea receptor (SUR) subunits (208). At
present, Kir6.1 and Kir6.2 were detected in freshly isolated capillaries from guinea pig heart. It is not known
whether Kir6.1 and/or Kir6.2 ␣-subunits form the KATP
channels together with the ␤-subunit SUR2B in EC (249).
Langheinrich et al. (207) have also reported a glibenclamide-sensitive hyperpolarization induced by K⫹ channel openers and glucose deprivation in capillaries isolated
from guinea pig hearts. Interestingly, evidence for a role
of endothelial KATP channels in the flow- and shear stressmediated vasodilatation has been presented (152, 203).
The endothelium-dependent vasodilatation of coronary
microvessels in response to an abluminal increase in osmolarity is significantly attenuated by glibenclamide, but
not by low concentrations of BaCl2 that inhibit the inward
rectifier or by iberiotoxin, an inhibitor of KCa channels
(159).
However, the contribution of KATP channels to EC
function has been challenged in several reports (for a
review see Ref. 295). Effects of KATP blockers or activators might instead be transmitted via myo-endothelial
communication and originate from the vascular smooth
muscle cells (264).
C. Amiloride-Sensitive Naⴙ Channels
Amiloride-sensitive Na⫹ channels are essential for
controlling Na⫹ and fluid transport in epithelia, but have
also been described in endothelium (17). These channels
form a superfamily consisting of two TM helices, a short
intracellular NH2 and COOH terminus, and a long extracellular loop comprising two cysteine-rich domains. They
have been described in microvascular endothelium,
where they might be connected with water transport
through aquaporin-4, and in cornea endothelium (9, 254).
However, the expression pattern and also the functional
impact of these channels in endothelium are not clear
at all.
D. Clⴚ Channels
1. The volume-regulated anion channel, VRAC
Like most mammalian cells, EC also express anion
channels, which are mainly permeable for Cl⫺ under
physiological conditions and activated by classical “cellswelling” protocols. They are referred to as volume-regulated anion channels (VRAC; Refs. 270, 278, 279, 308, 377).
Cell shrinkage reduces the basal current in EC below its
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
these channels in EC are consistent with those of the
recently cloned hIK (158, 168) and mIK (403) channel,
which has an amino acid sequence related to, but distinct
from, the six-TM helix, small-conductance SKCa channel
subfamily (⬃50% conserved). Currents are inwardly rectifying, KD for Ca2⫹ activation is 0.3 ␮M, and their singlechannel conductance is 39 pS. The channels are reversibly
blocked by charybdotoxin (inhibitory constant ⫽ 2.5 nM)
and clotrimazole, minimally affected by apamin, iberiotoxin, or ketoconazole (158). Importantly, this channel
lacks putative Ca2⫹-binding sites, but the proximal COOH
terminus contains a Ca2⫹-dependent calmodulin (CaM)binding site that is probably associated with four CaM
molecules (87, 190).
Small-conductance Ca2⫹-activated K⫹ channels,
SKCa, with a conductance of ⬃10 pS in asymmetrical
conditions have also been observed in EC (122, 265, 353).
Unlike BKCa, these channels are not voltage sensitive.
They are blocked by TBA, apamin, clotrimazole, and
D-tubocurarine (122). Recently, two types of SKCa channels were identified in excised patches of rat aortic EC
with a conductance of 18 and 9 pS in symmetrical 150 mM
K⫹, and 6.7 pS and 2.8 pS at physiological extracellular K⫹
concentrations (241). The smaller one is completely
blocked by 10 nM apamin and 100 ␮M D-tubocurarine, and
the larger one is insensitive to apamin but inhibited by
charybdotoxin at concentrations ⬎50 nM. The topology of
SKCa is similar to that of voltage-gated K⫹ channels, exhibiting six TM helices and an intracellular COOH and
NH2 terminus. Primary sequences, however, are very different. Likely, four subunits form the channel. No EF
hand, C2A, or Ca2⫹ bowl motif indicative for Ca2⫹ binding
has been found. Gating of SKCa involves a Ca2⫹-independent interaction of calmodulin with the COOH-terminal
domain of the ␣-subunit and is mediated by binding of
Ca2⫹ to the first and second EF hand motifs in the NH2terminal domain of CaM (187, 442).
1431
1432
BERND NILIUS AND GUY DROOGMANS
Physiol Rev • VOL
possible candidate is the GTP-hydrolyzing RhoA-GTPase,
since VRAC is downregulated in cells pretreated with the
p21RhoA inactivating Clostridium limosum exoenzyme
C3, and enhanced by cytotoxic necrosis factor (CNF1), a
bacterial toxin that constitutively activates Rho GTPases.
Inhibition of the RhoA-associated protein kinase, a possible downstream target of p21 RhoA, with the specific
blocker Y-27632 also downregulates VRAC (277, 298). A
tyrosine phosphorylation step downstream of the Rhosignaling cascade may be critical for the swelling-induced
activation of the channel, since inhibitors of protein tyrosine kinases inhibit VRAC and the protein tyrosine
phosphatase inhibitors Na3VO4 and dephosphatin potentiate it. It has been shown recently that the tyrosine
kinase p56lck mediates activation of the swelling-induced
Cl⫺ currents in lymphocytes (212). Figure 10 shows a
typical example for activation of VRAC and a tentative
gating scheme of this channel.
Part of the activation cascade of VRAC seems to be
coupled to specialized cytoskeletal structures. The annexin II-p11 complex, which is part of the cortical cytoskeleton and involved in the formation of caveolae, is
important for activation of endothelial VRAC (280). The
current density of VRAC correlates with the content of
CAV-1 in various cell types. CAV-1 is the major protein in
caveolae, and linked to many other signaling proteins (see
sect. VIIIB3). In cells devoid of CAV-1, such as the cancer
cells FRT, MCF-7, T47D, and CaCo2, VRAC has a very low
current density (391), suggesting that targeting of VRAC
to caveolae may provide a microdomain for activation.
The pharmacology of VRAC is quite extensively studied, and the search for more selective high-affinity blockers is still underway. There is a huge variety of compounds that inhibit VRAC with moderate affinity (EC50
between 1 and 1,000 ␮M) and specificity. Among these
compounds are not only several classical Cl⫺ channel
blockers, such as DIDS, SITS, N-phenylanthranilic acid
(NPA), 9-aminocamptothecic acid (9-AC), NPPB, and niflumic acid, but also several “surprising” substances, such
as the P-glycoprotein inhibitors tamoxifen, 1,9-dideoxyforskolin, verapamil (281), quinine and quinidine (422),
antiallergic drugs from the chromone family (134),
Ins(3,4,5,6)P4 and Ins(1,4,5,6)P4 (285), the antihypertensive Ca2⫹-antagonist mibefradil (283), and the widely
used antidepressant drug fluoxetine, supposed to be a
selective serotonin (5-hydroxytryptamine) reuptake inhibitor (231). Most of these voltage-independent blockers
are relatively hydrophobic aromatic compounds, which
can easily permeate the cell membrane. It has been shown
that quinine, quinidine, and fluoxetine, which can be both
positively charged and neutral, exert their blocking effect
at nanomolar concentrations of the neutral form, supporting the idea that these blockers interact with VRAC within
the plasma membrane. Many of these blockers are propylamines with a -CF3 group, a group of compounds that
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
value in control conditions, indicating that VRAC is partially activated in resting cells and therefore important for
the electrogenesis of the resting potential in nonstimulated cells (419). VRAC is also activated by shear stress
(11, 266).
The swelling-activated Cl⫺ current (ICl,swell) shows
outward rectification and is time independent, except at
potentials positive to ⫹60 mV where it slowly inactivates.
An increase in volume but also a reduction of intracellular
ionic strength or dialysis of EC with GTP␥S in isovolumic
conditions are the main triggers for channel activation
(281, 286, 423, 424). In hypotonic conditions, the current
density of ICl,swell at ⫹100 mV ranges between 100 and 150
pA/pF in cultured endothelial cells (278). However, the
density is much smaller (40 pA/pS) in freshly isolated EC
and EC in primary culture (281, 293, 381). The singlechannel conductance is ⬃40 –50 pS at positive potentials
and 10 –20 pS at negative potentials (278, 279, 281, 289,
292, 308, 377), indicating that rectification is a property of
the open channel. VRAC currents are mainly carried by
Cl⫺ but also amino acids and organic osmolytes permeate
through the channel. The anion permeability sequence for
VRAC is SCN⫺ ⬎ I⫺ ⬎ NO3⫺ ⬎ Br⫺ ⬎ Cl⫺ ⬎ HCO⫺
3 ⬎
F⫺ ⬎ gluconate ⬎ glycine ⬎ taurine ⬎ aspartate, glutamate. The permeability sequence for amino acids is consistent with that for the rates at which these amino acids
are lost from cells during hyposmotic swelling. The pore
diameter estimated from the fit of the relative permeabilities of these anions to their Stoke’s diameter is ⬃11 Å. A
similar value has been obtained from the analysis of the
voltage-dependent open channel block by molecules, like
calixarenes and suramin, which block VRAC channels but
also permeate at large driving forces (76, 297). VRAC is
further characterized by inactivation at positive potentials
and a voltage-dependent recovery from inactivation. Its
kinetic properties are modulated by extracellular divalent
cations, extracellular pH, the permeating anion itself, and
various channel blockers. Inactivation is accelerated by
acidic extracellular pH and by increasing the extracellular
Mg2⫹ concentration (278, 308, 377, 421).
The mechanism of VRAC gating is a matter of extensive research. In most experimental approaches VRAC is
activated by hyposmotic cell swelling, but it can also be
activated in isovolumic conditions by reducing intracellular ionic strength at constant extracellular osmolality and
intracellular Cl⫺ concentration, a possible interpretation
being an influence of ionic strength on protein tyrosine
kinases (424). Such a decrease in ionic strength is to be
expected from the influx of water during cell swelling
(286, 423). Because these conditions are rather nonphysiological, the isovolumic activation, e.g., by shear stress,
may be more relevant for EC.
Intracellular dialysis with GTP␥S also activates
VRAC without any increase in cell volume, an effect that
is probably mediated via small G proteins (423, 424). A
ION CHANNELS IN VASCULAR ENDOTHELIUM
1433
may comprise promising candidates for highly selective
VRAC blockers (347). Additionally, phenol derivatives,
such as gossypol, a polyphenolic pigment found in cotton
plants, are generally rather potent blockers of VRAC (for
a review, see Ref. 297).
In addition to these voltage-independent blockers,
several compounds that contain sulfonyl groups have
been shown to block VRAC almost exclusively at positive
potentials. This group contains the classical Cl⫺ channel
blockers DIDS and SITS, as well as the family of calixarenes, para-substituted phenols conjugated by methylPhysiol Rev • VOL
enes to form macrocyclic basket (calix)-like molecules.
Calix[4]arenes induce a voltage-dependent inhibition of
ICl,swell, probably by occluding the pore region at moderate positive potentials, but this block is relieved at more
positive potentials where the negatively charged
calix[4]arene permeates the channel. Lowering external
pH potentiates the effect of calix[4]arenes, suggesting a
role for positively charged residues, presumably histidine,
within the channel pore. A similar voltage-dependent
block can be induced by negatively charged compounds,
including suramin, reactive blue (basilen blue), calix[6,8]-
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
FIG. 10. Activation of VRAC by cell
swelling. A: time course of VRAC activation and deactivation at ⫺80 mV in a
BPAEC. The bar indicates the application of a solution with 25% lower osmolality than the control solution. This hypotonic challenge induces cell swelling
(419). B: I-V curves obtained at the time
points 1 and 2 indicated in A. C: current
traces under isotonic, hypotonic, or hypertonic conditions evoked by a stepvoltage protocol. For all traces, a holding
potential of 0 mV was used. Steps are 2 s
long and range from ⫺80 to ⫹100 mV
(increment of 20 mV). D: tentative gating
scheme of VRAC, which is discussed in
detail in the text and in References 275,
284, 286, 295, 296, 419.
1434
BERND NILIUS AND GUY DROOGMANS
Physiol Rev • VOL
ClC-3 is responsible for the swelling-activated Cl⫺ channel in EC.
ClC-2 is obviously volume sensitive, but its biophysical and pharmacological properties are completely different from those of VRAC. Also another candidate, the
72-amino acid intrinsic membrane protein phospholemman with a single membrane-spanning domain, is obviously not VRAC (detailed discussion in Ref. 278).
The enigmatic molecular nature of VRAC does however not prevent us from speculating about its possible
functions in EC.
Because VRAC is a “housekeeping” Cl⫺ channel, its
block induces changes in the membrane potential of EC.
If these Cl⫺ channels are blocked, K⫹ channels may become dominant and strongly hyperpolarize the membrane
with potentially beneficial effects on agonist or shear
stress-induced Ca2⫹ influx. Block of Cl⫺ channels may
therefore be a strongly underestimated tool to modulate
the driving force for Ca2⫹ entry in EC and hence Ca2⫹
signaling (283, 297).
VRAC can also be activated by mechanical forces
(shear stress, biaxial tensile stress) that induce changes in
cell shape and possibly folding and unfolding of the
plasma membrane (11, 266, 277, 278, 295, 297). This function might be especially important for EC that sense
mechanical forces, a process in which a RhoA-mediated
pathway may be involved (see sect. VI). Activation of
RhoA inhibits myosin light-chain phosphatases (MLCP)
via a Rho/Rho-associated kinase (ROK), a pathway that
has been shown to modulate VRAC in cultured BPAEC.
One of the downstream effects is an increased myosin
light-chain phosphorylation, raising the possibility of a
functional link between myosin light-chain phosphorylation and VRAC activation. Application of myosin lightchain kinase (MLCK) inhibitors, including AV25, a specific
MLCK inhibitory peptide, inhibits preactivated VRAC. Cell
dialysis with a specific inhibitory peptide of MLCP,
NIPP1-(191O210), potentiates the activation of VRAC.
These results indicate that myosin phosphorylation is a
prerequisite for efficient activation of VRAC in BPAEC
cells and might be involved in the mechano-sensing pathway of VRAC by connecting the gating machinery to the
cytoskeleton (288).
VRAC is supposed to regulate EC proliferation.
Chemically completely different compounds that inhibit
VRAC also inhibit in the same concentration range seruminduced EC proliferation and [3H]thymidine incorporation
(278, 279, 283, 425). As discussed in section VIIIE, VRAC
blockers also inhibit angiogenesis. Interestingly, VRAC is
downregulated if cells switch from proliferation to differentiation (237, 426), which may also explain the different
current densities of VRAC in primary cultured and freshly
isolated cells (381). Thus VRAC could assist in regulating
EC growth and the transition from proliferation to differentiation.
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
arenes, and other compounds carrying sulfonic acidic
sites. The voltage-dependent block of VRAC by ATP is
probably also due to a similar mechanism. The blocking
effect of these compounds is enhanced by lowering extracellular pH and at moderate positive membrane potentials. The relief of the blocking effect at positive potentials
is only observed for compounds small enough to permeate the pore. Calix[6]arene and calix[8]arene are apparently not able to permeate the VRAC pore, from which a
pore diameter between 1 and 1.6 nm has been estimated,
a value in the same range as the values obtained above
(76, 77).
The molecular nature of the VRAC channel is still
unknown, and the putative candidates so far have been
disputed. P-glycoprotein, the product of the multidrug
resistance 1 gene which functions as a drug transporter
and possibly as an anion channel, has been proposed as a
putative candidate for VRAC, but now even its role as a
regulator of VRAC is questioned.
A second candidate for VRAC is pICln, whereby n
refers to a putative extracellular nucleotide-blocking site.
Because the primary amino acid sequence of pICln lacks
transmembrane helices, it has been proposed that the
pICln “channel” is a homodimer consisting of four
␤-strands which form an eight-stranded, antiparallel
␤-barrel transmembrane pore similar to that of porins.
The most compelling evidence for pICln being a plasma
membrane channel is the presence of a GXGXG motif,
being an essential component of the extracellular nucleotide binding site believed to be essential for the nucleotide sensitivity of the VRAC current. It has been reported
that mutations of this site (G54A; G56A; G58A) prevent
current inhibition by extracellular nucleotides, but these
data could not be reproduced, which undermines the idea
that pICln is VRAC (418). Recently, it has been shown that
pICln is a cytosolic rather than a membrane protein that
probably binds spliceosomes and regulates protein import into the nucleus (338). pICln also interacts with a
homolog of the p21(Cdc42/Rac)-activated protein kinases
(PAKs). The involvement of PAKs in cytoskeletal rearrangement suggests that pICln may be linked to a system
regulating cell morphology (201).
ClC-3, a membrane protein of the ClC family, is currently considered as a candidate for VRAC. These channels are opened by cell swelling or by inhibition of endogenous PKC, but closed by PKC activation, phosphatase
inhibition, or elevation of intracellular Ca2⫹. Site-specific
mutational studies indicate that a serine residue (serine51) within a consensus PKC phosphorylation site in the
intracellular NH2 terminus of the ClC-3 channel protein
represents an important volume sensor of the channel.
These results provide direct molecular and pharmacological evidence for channel phosphorylation/dephosphorylation (78, 79, 450). However, endothelial VRAC channels
lack these properties, and it is therefore unlikely that
ION CHANNELS IN VASCULAR ENDOTHELIUM
In addition, VRAC may also contribute to the control
of intracellular pH homeostasis (282, 351). Interestingly,
activation of VRAC induces a decrease in intracellular pH
(pHi) (282). A decrease in pHi stimulates prostanoid synthesis in EC from cerebral microvessel, which is possibly
mediated directly via a protein phosphorylation-dependent mechanism involving protein tyrosine kinases and
phosphatases as well as PKC. cPLA2 is the key enzyme
affected by a decreased pHi (319). Therefore, this mechanism could also link channel activity (VRAC) to the
production of vasoactive compounds.
2. Ca2⫹-dependent Cl⫺ channels, CaCC or CLCA
Physiol Rev • VOL
tein Lu-ECAM, the bovine bCLCA1, murine mCLCA1, and
human hCLCA1, -2, and -3 proteins that are believed to
represent putative Cl⫺ channels. Currents, showing some
similarity with CaCC, have been observed in HEK cells
expressing these proteins. These Ca2⫹-sensitive Cl⫺ currents, activated by extremely high, nonphysiological concentrations of [Ca2⫹]i, are outwardly rectifying and inhibited by DIDS, dithiothreitol, and niflumic acid. Cellattached patch recordings of transfected cells reveal
single channels with a slope conductance of 13.4 pS.
These findings suggest that members of the CLCA family
represent a Ca2⫹-activated Cl⫺ conductance. Proteins of
this family are characterized by a precursor of ⬃130 kDa
consisting of between 900 and 940 amino acid residues.
This precursor is cleaved to form heterodimers of ⬃90
and 35 kDa. The most likely topology is five TM with an
extracellular glycosylated NH2 terminus, containing a
number of conserved cysteine residues and an intracellular COOH terminus. The above-mentioned cleavage site is
located in the intracellular loop between TM3 and TM4.
The proteins contain several consensus sites for PKC
phosphorylation (105, 126 –128). RT-PCR shows a high
expression of mCLCA1 in mouse aorta EC. However, so
far we failed to record CaCC-like currents in cells expressing mCLCA1 and bCLCA1 (287; B. Nilius and J.
Papassotiriou, unpublished data).
A Ca2⫹-activated Cl⫺ channel with properties clearly
different from the above-described CaCC can be activated
by a Ca2⫹/CaM-dependent protein kinase II. Interestingly,
this channel is inhibited by Ins(3,4,5,6)P4, an endogenous
inositol tetrakisphosphate that also inhibits Ca2⫹-stimulated
Cl⫺ secretion. The effect seems to be specific for that particular isoform, since Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, Ins(1,3,
4,6)P4, and inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,
5,6)P5] [the latter being the immediate precursor of Ins(3,4,
5,6)P4] are all ineffective (444).
3. High-conductance Cl⫺ channels
Occasionally, high-conductance Cl⫺ channels (BCl),
which are obviously different from all other described Cl⫺
channels, have been observed in EC. These channels are
virtually silent in intact cells or in cell-attached patches,
but become active in excised inside-out and outside-out
patches. Their single-channel conductance ranges from
113 to 400 pS, and they exhibit several subconductance
states, which probably refers to a multi-barrel structure
(125, 310, 340, 395, 397). BCl is activated upon ␤-adrenergic stimulation in EC, which phosphorylates the channel
and shifts its activation range toward more negative potentials (397). It is also activated by the antiestrogen
tamoxifen and inhibited by 17␤-estradiol. This effect has
been discussed in relation to the protective effect of
estrogens on several cardiovascular diseases (218). A prolonged increase in intracellular Ca2⫹, as well as inhibition
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
The increase in [Ca2⫹]i during stimulation of EC exerts depending on the cell type two distinct effects on
membrane potential. In some EC, it is accompanied by a
prominent hyperpolarization due to activation of Ca2⫹dependent K⫹ channels, but in others, such as BPAEC, it
evokes only small changes in membrane potential due to
activation of Ca2⫹-dependent Cl⫺ channels (CaCC; Refs.
123, 138, 179, 284, 287). These channels inactivate rapidly
at negative potentials and activate slowly at positive potentials. Outward tail currents are slowly decaying, while
inward tail currents decay much faster (284, 287). They
show strong outward rectification. The permeability ratio
for anions is I⫺:Cl⫺:gluconate ⫽ 1.7:1:0.4 (283). The single-channel conductance is ⬃7 pS at 300 mM extracellular
Cl⫺ but only ⬃3 pS at physiological intra- and extracellular Cl⫺ concentrations (283, 287). Typical current densities of these Ca2⫹-activated Cl⫺ currents range from
10 –30 pA/pF at ⫹100 mV and are thus much smaller than
those of the volume-activated Cl⫺ currents. The channel
open probability is high at positive potentials, but very
small at negative potentials. [Ca2⫹]i for half-maximal activation is voltage dependent and ranges from 400 to 600
nM at ⫺100 mV to 50 – 80 nM at ⫹80 mV, pointing to
high-affinity binding sites for Ca2⫹. Steady-state and kinetic behavior of this current can be described by a model
which assumes activation of the channel by two identical,
independent, sequential Ca2⫹ binding steps preceding a
final Ca2⫹-independent transition from the closed to the
open state of the channel. The putative binding site for
Ca2⫹ is ⬃10 –15% within the membrane electric field from
the cytoplasmic side (7, 287).
Current activation requires intracellular ATP (438).
NPA, DIDS, Zn2⫹, and calmodulin antagonists (123, 284,
287) block CaCC currents. Inhibition by DIDS and niflumic acid is voltage dependent and more potent at positive
potentials. The block by NPA, NPPB, and tamoxifen is
voltage independent. Niflumic acid and tamoxifen are the
most potent blockers.
The molecular nature of this channel is not yet resolved. Recently, a number of related membrane proteins
have been cloned including the endothelial adhesion pro-
1435
1436
BERND NILIUS AND GUY DROOGMANS
of PKC, seems to increase the incidence of the channel in
cell-attached patches. Zn2⫹ from either side of the membrane blocks the channel (125). The physiological relevance of these channels is elusive.
4. CFTR in endothelium?
E. Conclusion
The membrane potential of EC is modulated by a
variety of ion channels. K⫹ channels, which are constitutively open (Kir type channels) or which are activated
during EC stimulation (BKCa, IKCa, SKCa, KATP), induce
hyperpolarization. Interestingly, EC express also a variety
of Cl⫺ channels (VRAC, CLCA, CFTR) whose inhibition
can also induce EC hyperpolarization. Activation of these
channels during EC stimulation may clamp the EC membrane potential at negative potentials close to the equilibrium potential for Cl⫺ (ECl). Importantly, K⫹ and Cl⫺
channels interacting with cation channels induce sometimes dramatic stepwise or oscillatory changes in the EC
membrane potential of up to 80 mV. Channels regulating
EC electrogenesis or inducing changes in the membrane
potential due to hormonal, neuronal, and mechanical
stimuli importantly influence several mechanisms. 1)
Physiol Rev • VOL
V. VOLTAGE-DEPENDENT ION CHANNELS
IN ENDOTHELIUM?
EC are nonexcitable, and it is therefore difficult to
reconcile the presence of voltage-gated ion channels with
the slow and often small changes in membrane potential
in these cells. Several reports have nevertheless clearly
shown the incidence of voltage-gated ion channels in both
cultured and freshly isolated EC. Voltage-activated Na⫹
channels with a low tetrodotoxin and high saxitoxin sensitivity have been observed in EC from human umbilical
vein, in rat interlobar arterial EC, and in microvascular EC
(114, 429). These currents are also present in primary
cultures of microvascular EC, however at a rather small
density (2.1 pA/pF at 0 mV). At the normal resting potential of EC, ⬃50% or more of these channels will be inactivated. Stimulation of PKC increases this current without
any change in its voltage dependence (429).
Voltage-gated Ca2⫹ channels, which share some similarities with the classical L- and T-type Ca2⫹ channels,
have been described in freshly dissociated capillary EC
from bovine adrenal glands (34 –36, 417). Their voltage
dependence, kinetics, responses to BAY K 8644, nifedipine, amiloride, and Cd2⫹, and their different selectivity
for Ba2⫹ and Ca2⫹ indicate that the observed currents are
typical T and L channels resembling those of endocrine
secretory cells (417). The “T-type” Ca2⫹ channels have a
conductance of ⬃8 pS and are involved in depolarizationinduced Ca2⫹ transients, which may suggest a role in
endothelial Ca2⫹ signaling. L-type Ca2⫹ channels in these
EC have a single-channel conductance of ⬃20 pS and are
sensitive to dihydropyridines. In addition to these two
voltage-dependent Ca2⫹ channels, a tiny conductance
channel (SB Ca2⫹ channel, 2.8 pS in 110 mM Ba2⫹) has
been observed in capillary EC. It is sensitive to the Ca2⫹
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
A multitude of Cl⫺ channel types has been characterized in EC, but until recently, no clear evidence for
cAMP-stimulated CFTR channels had been presented.
Tousson et al. (388) reported expression of CFTR in EC
from umbilical vein and lung microvasculature at both the
messenger and protein level, as well as functional expression from Cl⫺ efflux and whole cell patch-clamp data.
cAMP-stimulated Cl⫺ currents in these cells were not
blocked by DIDS, but sensitive to glibenclamide indicative of CFTR Cl⫺ channel activity.
CFTR channels have so far not been observed in
cultured BPAEC cells (294), but a functional interaction
with other Cl⫺ channels has been observed if they are
heterologously expressed in these cells. Overexpression
of CFTR downregulates VRAC in an activity-independent
manner, but expression of the nonmaturating mutant
⌬F508-CFTR does not. Because neither the single-channel
conductance nor the kinetics of macroscopic currents are
affected, CFTR probably downregulates the number of
functional VRAC channels (410). Also CaCC is downregulated by CFTR, but this effect is potentiated by a cAMPmediated activation of CFTR (439). A functional role for
CFTR in EC biology is not clear, but like in epithelial cells,
it could be involved in transendothelial transport and act
as a regulator of other channels or transporters. Interestingly, CFTR has been associated with ATP transport (169,
367, 368). ATP and its metabolites, such as adenosine, are
important autacoid and paracrine substances that affect
many functions of the vessel wall (273).
They regulate the driving force for Ca2⫹ entry via several
pathways and add positive and negative feedback mechanisms to the numerous regulation mechanisms inherent
to EC for controlling Ca2⫹ influx. 2) They might be the
main players in transducing electrical signals such as
flow-dependent hyperpolarization to neighboring EC and
smooth muscle cells (SMC), thereby regulating the functional state in blood vessels upstream and downstream of
sites of EC activation. 3) They control the driving force
for several electrogenic transporters such as NCX. 4) An
increasing number of events in EC are likely dependent
on the polarization state of EC without interference of
Ca2⫹, e.g., depolarization-induced superoxide formation
(see sect. VIIIF). 5) Some channels, such as VRAC, might
be important, in addition to regulation of electrogenesis,
for the transport of amino acids, organic osmolytes, and
HCO⫺
3 , which is important for regulation of the intracellular pH in EC.
ION CHANNELS IN VASCULAR ENDOTHELIUM
VI. MECHANOSENSITIVE CHANNELS
IN ENDOTHELIUM
EC are notorious for their ability to sense mechanical
forces, such as biaxial tensile stress (stretch, pressure), a
force component mainly vertical to the EC surface, and
shear stress, a tangential force due to blood flow (for
detailed reviews, see Ref. 211). Many endothelial responses are modulated by changes in blood flow and
blood pressure: 1) the secretion of prostacyclin (PGI2)
and endothelium-derived relaxing factor (EDRF; and NO);
2) the expression of genes and proteins, such as tPA,
plasminogen-activator inhibitor (PAI-1), tissue factor
(TF), several adhesion molecules (VCAM-1, the intercellular adhesion protein-1), growth inhibitors (heparin),
growth factors (platelet-derived growth factor, PDGF),
endothelin, monocyte-chemoattractant protein (MCP-1),
NO synthase, and activation of early response genes and
small G proteins; 3) cell cycle entry; and 4) cytoskeletal
rearrangement, long-term responses, such as adaptive
changes in cytoskeleton, vessel remodeling, and others
(64, 66, 232).
Shear stress also upregulates expression of the connexin (Cx) 43 (70, 69, 104, 182, 205).
Physiol Rev • VOL
Some mechanisms, such as the mechanically induced
stimulation of DNA binding activities of nuclear factor ␬B
(NF␬B) and the nuclear factor activator protein-1 (AP-1,
AP-2) (206), have been elucidated. Shear stress can directly activate cis-elements of different genes, e.g., the
shear stress response element in the PDGF-B gene and
the tPA-responsive element in the MCP-1 gene, and can
stimulate the GC-rich transcription-initiation site of the
specificity protein 1 (Sp-1) in the TF gene.
Protein kinases appear to be involved in an early step
of mechanosensing. Shear stress induces a fast tyrosine
phosphorylation at the luminal side of EC, primarily in
caveolae and also supports translocation of signaling proteins to caveolae (345). Gene activation is modulated by a
complex network of protein kinases (JKN, ERK, focal
adhesion kinases FAK, c-src), which might be affected by
changes in the ionic conditions and [Ca2⫹]i. (23, 53–56).
Changes in [Ca2⫹]i are also important for the regulation of gene expression (189, 217), since the shear stressmediated Ca2⫹ responses activate protein kinases, e.g.,
the functionally important upregulation of eNOS in response to shear stress depends on an initial Ca2⫹-dependent phosphorylation step (93). The initial fast release of
NO in response to mechanical forces is therefore Ca2⫹
dependent, whereas the sustained NO production is Ca2⫹
independent (42, 71, 93, 263).
Receptor tyrosine kinases and integrins can serve as
mechanosensors (e.g., the vitronectin receptor avb3) by
acting through several kinases including FAK. Interestingly, shear stress induces the translocation of Cdc42 and
Rho from the cytosol to the plasma membrane, a mechanism that might be also involved in VRAC gating (see sect.
IVC1 and Refs. 53, 216). An intriguing example for the role
of tyrosine phosphorylation and the interference of
[Ca2⫹]i is the role of the p130 Crk-associated substrate
(Cas), a c-Src substrate, which has been identified as a
highly phosphorylated protein localized to focal adhesions and acting as an adapter protein. Cas is important
for actin filament assembly and is tyrosine-phosphorylated by shear stress via activation of the Ca2⫹-dependent
c-Src. This involvement of Ca2⫹ in activation of c-Src and
tyrosine phosphorylation of Cas defines a novel mechanosensing mechanism (309).
Ion channels may serve as effective mechanosensors
to convert mechanical forces into electrical responses. It
is not clear how ion channels may be directly involved in
the responses to mechanical forces. Obviously, changes
in [Ca2⫹]i might again be an important transmitter of the
mechanical signal. We therefore first discuss the mechanically induced changes in [Ca2⫹]i and afterward the channels responding to mechanical forces. VRAC or VRAC-like
channels may also belong to this class of channels because they can be obviously activated by shear stress and
membrane stretch, induced by positive pressure in the
patch pipette.
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
agonist BAY K 8644, but not to the Ca2⫹ antagonist nicardipine (35). This channel shows long openings at negative
potentials and might provide a low-threshold Ca2⫹ entry
pathway. A novel type of voltage-dependent Ca2⫹ channel, the R-type Ca2⫹ channel, has been proposed to be
important during activation of EC by PAF (30). This channel, which is activated by a long-lasting depolarization
and does not inactivate at depolarized potentials, has also
been proposed as a possible candidate for a sustained
Ca2⫹ influx.
A hyperpolarization-activated, nonselective cation
channel that carries K⫹ and Na⫹ and is modulated by NO
has been described in vascular brain EC (165), but its
physiological role is completely unknown. A 4-aminopyridine-sensitive, voltage-dependent, transient outward current (A-type K⫹ channel) has been described in cultured
EC (173, 382). In freshly isolated EC from resistance
vessels, a delayed rectifier K⫹ current has been observed
that is probably Kv1.5 and might be involved in release of
vasoactive compounds (143). Dittrich et al. (72) describe
a voltage-dependent current in freshly isolated EC from
heart capillaries that activates at potentials positive to
⫺20 mV and inactivates much slower than A currents at
positive potentials. This current is blocked by TEA but is
insensitive to charybdotoxin and apamin. This 12-pS
channel is different from HERG channels or Shaker-type
K⫹ channels. The functional role of this channel, supposed to be involved in the generation of oscillations in
membrane potential and [Ca2⫹]i, remains hypothetical.
1437
1438
BERND NILIUS AND GUY DROOGMANS
A. Mechanically Induced Ca2ⴙ Entry
1. Channels activated by tensile stress
Membrane stretch induced by negative pressure applied via the patch pipette to cell-attached patches on EC
activates a channel that is permeable for monovalent
cations (⬃50 pS) and Ca2⫹ (19 pS) (209). Stretch-activated NSC with a conductance of 20 –30 pS for monovalent cations and 10 –20 pS for Ca2⫹ and Ba2⫹ have also
been described in endocardial endothelium and microvascular EC (147, 335). Activation of these channels induces
an increase in [Ca2⫹]i that is sufficient to activate BKCa
and to hyperpolarize the membrane. Interestingly, BKCa
itself is sensitized to [Ca2⫹]i when stretch (negative pressure) is applied to inside-out patches (148, 195, 233).
Hoyer et al. (149) have described a K⫹-selective stretchactivated channel in rat intact aortic endothelium and
isolated aortic EC with a similar permeability for K⫹ and
Na⫹. Its single-channel conductance is 22 pS for K⫹ and
Na⫹ and 4 pS for Ca2⫹. Channel activity is clearly pressure dependent in a physiological range and disappears
during application of 20 ␮M gadolinium. Opening of the
Physiol Rev • VOL
2. Channels activated by shear stress
Shear stress-induced activation of endothelial ion
channels represents one of the earliest responses in
mechanosignal transduction, which is important for the
regulation of vascular tone. Several examples of ion channels directly activated by shear stress have been reported,
although most experimental conditions cannot exclude a
stretch component. It is therefore sometimes difficult to
distinguish between these two forces and their role in
channel activation.
HAEC respond to flow with Ca2⫹ entry, activation of
NSC, Cl⫺ channels, and Ca2⫹-activated K⫹ channels. Conversely, similar flow rates do not affect human capillary
EC. The increase in [Ca2⫹]i in macrovascular EC depends
on the presence of extracellular Ca2⫹ (171), but it is not
clear which entry channels are responsible for these
changes in [Ca2⫹]i. A possible candidate is a shear stressactivated NSC, which is slightly more permeable for Ca2⫹
than Na⫹, and reversibly blocked by La3⫹. It is insensitive
to activators of protein kinase and inhibited by nonsteroidal anti-inflammatory drugs, such as mefenamic acid (266,
364, 365).
Other channels, modulated by shear stress, may regulate the driving force for Ca2⫹. However, these channels
can only be effective if at the same time a Ca2⫹ pathway
is activated, e.g., if mechanical stimulation opens CCE
channels (305, 306). Changes in [Ca2⫹]i in human aorta EC
are modulated by shear stress-activated Cl⫺ channels,
most likely VRAC. In BAEC, flow induces an initial hyperpolarization, which is followed by a sustained depolarization, caused by activation of Cl⫺ channels. Thus shear
stress-dependent Cl⫺ channels may be important modulators of endothelial mechanosensitivity (11, 266).
Changes in shear stress due to pulsatile flow or by varying
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
Mechanostimulation of isolated EC by shear stress,
stretch, or swelling in Ca2⫹-free solutions induces an
increase in [Ca2⫹]i (266, 305). Acute changes in flow also
induce Ca2⫹ release from Ins(1,4,5)P3-sensitive pools and
a subsequent NO release (153). Pretreatment of EC with
thapsigargin attenuates this Ca2⫹ release, indicating that
the mechanical stimulus induces a release of Ca2⫹ from
Ins(1,4,5)P3-sensitive stores. However, neither the PLC
pathway nor Ins(1,4,5)P3 are involved, since the PLC inhibitor neomycin and the Ins(1,4,5)P3 receptor antagonist
heparin do not block this release. Also the PKA inhibitory
peptide as well as cyclooxygenase and lipoxygenase inhibition is ineffective (305). Because PLA2 blockers inhibit
the release and arachidonic acid mimics the effect of
mechanostimulation even in the presence of heparin and
PLA2 blockers, it has been proposed that the mechanically induced release of Ca2⫹ is mediated via production
of arachidonic acid.
The depletion of endothelial Ins(1,4,5)P3-sensitive
Ca2⫹ stores by mechanical stimulation may also activate
Ca2⫹ entry via store-operated Ca2⫹ entry channels
(269, 305).
Recently, a mechanosensitive Ca2⫹-permeable channel with a relative permeability of PCa:PNa:PK ⫽ 5:1:1 has
been described in freshly isolated EC from rat aorta and
also in the ECV304 cell line (451). This channel is activated by stretch (negative pressure in inside-out patches),
but its activity correlates with a shear stress-induced Ca2⫹
signal. Single-channel conductance is 32 pS for monovalent cations and 9 pS for Ca2⫹. This channel is inhibited by
PKG, initiating a negative feedback by activation of eNOS.
pressure-activated cation channel (PAC) is followed by
the opening of a Ca2⫹-dependent NSC, indicating that
Ca2⫹ influx through PAC may be sufficient to increase
intracellular Ca2⫹ and to activate thereby neighboring
NSCs (see sect. IIIA). This channel is upregulated in renovascular hypertension and in spontaneous hypertensive
rats (195). Marchenko and Sage (242, 243) described a
similar pressure-activated NSC with a conductance of 34
pS for monovalent cations and 6 pS for Ca2⫹, which
provides an influx pathway for Ca2⫹. Indirect evidence for
a functional role of these stretch-activated channels has
been obtained from experiments in which Gd3⫹, an efficient blocker of mechanosensitive channels, has been
used to inhibit NO synthesis (379).
Although not directly activated by biaxial tension,
voltage-dependent K⫹ channels are upregulated by application of static and cyclic stretch, indicating a role of
these channels in mechanotransduction of cardiac microvascular EC (86).
ION CHANNELS IN VASCULAR ENDOTHELIUM
B. Conclusion
A variety of mechanosensitive ion channels have
been identified at the functional level in various types of
EC. However, none of them has been identified at the
molecular level. It is also difficult to identify such mechanosensitive channels with already described types of ion
channels. Mechanosensitivity is inherent to several types
of ion channels among which Ca2⫹-permeable cation
channels that provide Ca2⫹ entry pathways in EC, different types of K⫹ channels, and also Cl⫺ channels. Therefore, mechanical forces modulate not only Ca2⫹ entry, but
the membrane potential is also a target of the mechanosensing machinery of EC. The signal transduction bePhysiol Rev • VOL
tween mechanical stimulus and gating or modulation of
the respective ion channels is not yet understood.
VII. GAP JUNCTIONAL CHANNELS
Gap junctions are agglomerates of cell-to-cell channels that allow direct electrical and metabolic communication between endothelial cells, between EC and SMC,
and also between EC and lymphocytes/monocytes (67).
An extensive description of EC-EC, EC-SMC, and ECblood cell coupling is beyond the scope of this review. In
short, coupling of EC consists of clublike protrusions,
which contact neighboring smooth muscle cells through
the basement membrane (344). The large variability of
functional low-resistance cell-cell connections may depend on the presence of various isoforms of connexins. At
least three isoforms (Cx37, -40, -43) are expressed in EC
and may give rise to heterogeneous expression patterns.
Cx43 is more abundant in macrovascular than in microvascular cells. Aortic and pulmonary arterial endothelia
express all three types of connexin, whereas coronary
artery endothelium expresses Cx40 and Cx37 but lacks
Cx43 (68, 221, 453). Functional channels can be formed
between the same or different isoforms. Interaction does
not occur between the Cx40 and Cx43 isoforms, but possibly between Cx40 or Cx43 and Cx37 (40). Cx40 is prominent in cultured EC. The single gap junctional channel
conductance in HUVEC is in the range of 19 –75 pS, but in
cultured human aorta EC, an additional conductance of
80 –200 pS has been observed. The latter may possibly
reflect the activity of Cx40 isoform channels, which are
more abundant in cultured arterial endothelial cells (405).
Expression of connexins depends on the functional state
of EC and is modulated by growth factors, by factors that
are present during inflammation (TNF-␣), and by mechanical forces (68, 326, 327, 443). Connexins play an important role in the regulation of inflammatory responses with
a main target on EC, including cell extravasation and
changes in vessel permeability. The normal expression
pattern of connexins is disturbed in the presence of
TNF-␣, a major regulator of inflammatory reactions,
which downregulates Cx37 and Cx40 expression, but
does not alter that of Cx43, and attenuates EC coupling
(406). The electrical coupling between EC is the morphological substrate for the electrotonic spread of electrical
signals along the vessel wall. Electrical coupling via highconductance gap junctional channels between EC and
smooth muscle cells may be functionally important in EC
of small-terminal arterioles where a large total EC surface
contacts a much smaller smooth muscle surface and allows an efficient modulation of the smooth muscle membrane potential by the EC (20, 63, 67). In contrast, other
results show that the agonist-induced hyperpolarization
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
the viscosity activate SKCa and BKCa channels (64, 65,
152). Opening of these K⫹ channels induces hyperpolarization, which may increase Ca2⫹ influx and NO release,
and also modulate the membrane potential in smooth
muscle cells electrically coupled to the endothelium (63,
64, 295). An inwardly rectifying, 30-pS K⫹ channel has
been described as a possible mechanosensor of shear
stress (163, 312). Its single-channel conductance, its degree of rectification, and its Ca2⫹ sensitivity in excised
patches suggest that this channel may belong to the class
of SKCa channels. These channels share some properties
with ROMK-like channels, and the previously described
Ca2⫹- and GTP-modulated inwardly rectifying K⫹ channels (301, 302, 311, 401), but clearly differ from the classical IRK channels (see sect. IVA1). PTX-sensitive mechanisms, cGMP, and NO seem to be involved in the
activation of shear stress-dependent K⫹ channels (133,
302). The observation that various blockers of K⫹ channels reduce shear stress-mediated release of NO provides
indirect evidence for the involvement of these channels
(5, 70, 376).
Another mechanosensitive ion channel participating
in EC responses to shear stress is surprisingly a voltagegated Na⫹ channel (see sect. V). The activation of the
shear stress-mediated extracellular signal-regulated kinase (ERK1/2) is potentiated by substituting extracellular
[Na⫹] with N-methyl-D-glucamine (NMDG⫹) and by the
voltage-gated Na⫹ channel antagonist tetrodotoxin. Evidence for the expression of the Na⫹ channel genes SCN4a
and SCN8a ␣-subunit genes in these EC has been obtained from RT-PCR analysis and is supported by Western
blotting of a 250-kDa protein with Na⫹ channel antibodies
(390). These results may indicate that Na⫹ channels are
involved in regulation of shear stress-mediated ERK1/2
activation. Because of the low membrane potential and
the slow changes in membrane polarization, it is difficult
to reconcile this interpretation with activation of voltagedependent Na⫹ channels.
1439
1440
BERND NILIUS AND GUY DROOGMANS
of EC does not spread electrotonically to smooth muscle
cells, but rather the opposite happens, i.e., an electrotonic
spread from smooth muscle to the lining EC (18 –20).
VIII. FUNCTIONAL ROLE OF ION CHANNELS
IN ENDOTHELIUM
A. Ion Channels and Electrogenesis in EC
FIG. 11. Electrogenesis in a nonstimulated (resting)
BPAEC. Current-voltage relationships from linear voltage ramps (from ⫺150 to ⫹100 mV) under control conditions (1), in the presence of 1 mM Ba2⫹ to inhibit IRK
(2), and after shrinking the cell in the presence of Ba2⫹
by adding of 100 mM mannitol to inhibit VRAC (3; see
text). The remaining current represents a nonselective
cation current (for detailed discussion see Ref. 416). The
difference current (1–2) is an inwardly rectifying K⫹
current (IRK) through the recently identified channel Kir
2.1 (95, 179). The VRAC Cl⫺ current (276, 416) is reconstructed from the difference current (2–3). The resting
membrane potential reflects the respective contributions
of these three conductances. Membrane potentials in
this cell type show a bimodal distribution with a peak at
⫺80 mV and another around ⫺25 mV (416). Mibefradil
(10 ␮M) induces a fast and reversible block of VRAC.
Therefore, the K⫹ conductance becomes dominant and
the membrane hyperpolarizes (281). [Modified from
Voets et al. (419).]
Physiol Rev • VOL
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
The membrane potential of vascular endothelial cells
is negative compared with the blood and tissue compartment. Its value, as well as that of cell capacitance and
input resistance, varies considerably between different
cells types and conditions of cell isolation and culturing.
Resting membrane potentials between 0 and ⫺80 mV,
membrane capacitances between 30 and 80 pF, and input
resistances between 1 and 10 G⍀ have been reported for
isolated, nonconfluent EC. Confluent cells have a higher
membrane capacitance (up to 160 pF) and a lower input
resistance 0.01– 0.4 G⍀. Although these latter values are
uncertain, they are consistent with the existence of intercellular electrical coupling via gap junctions. The membrane potential also depends on the EC type and is in
general more negative in macrovascular than in microvascular EC (63, 295, 449, 462).
The main determinant of the resting potential in most
cell types is a basal K⫹ conductance. However, the expression of K⫹ channels varies greatly between different
EC types and even within the same strain of cultured EC.
For example, inwardly rectifying K⫹ channels (IRK) are
functional in some macrovascular EC (137, 138, 180, 290,
295) but seem to be preferentially expressed in cultured
cells and absent in freshly isolated cells (349, 381). They
have also been reported to be either present (427) or
absent in freshly isolated coronary microvascular EC
(72). This heterogeneous expression may contribute to
the highly variable resting potential of EC.
The resting membrane potential in several endothelial cell types has a bimodal distribution, consisting of
cells with a resting potential between ⫺70 and ⫺60 mV
that is mainly controlled by IRK (K⫹-type EC) and cells in
which the Cl⫺ conductance is dominating (Cl⫺-type EC)
with a resting potential between ⫺40 and ⫺10 mV. The
latter values are close to ECl, suggesting that a Cl⫺ conductance is activated under resting conditions. This conductance, which is suppressed by cell shrinkage, apparently represents basal activity of swelling-induced Cl⫺
channels, as described above. This bimodal distribution
may be explained by the N-shaped current-voltage relationship observed in these cells (274, 419). In addition, the
hyperpolarization induced by decreased extracellular
Na⫹ is consistent with a contribution of either a Na⫹selective or a nonselective cation conductance to the
resting permeability. The relative contributions of the
Na⫹, K⫹, and Cl⫺ conductance to the resting potential
range from 3 to 30%, 27 to 95%, and 9 to 35% (138, 240, 295,
407, 419). Figure 11 gives an example for the electrogenesis of the membrane potential in a “resting,” nonstimulated macrovascular BPAEC.
The hyperpolarization, induced by vasoactive stimuli,
such as acetylcholine, bradykinin, and histamine, in cells
that do not express inwardly rectifying K⫹ channels is
mediated by the activation of Ca2⫹-dependent K⫹ currents and is often followed by a sustained depolarization
due to activation of NSC (63, 177, 179, 225, 239, 250, 274).
Figure 12 shows an example of stimulation-dependent
changes in membrane potential that are a mirror image of
the concomitant changes in [Ca2⫹]i. The electrogenic
Na⫹-K⫹-ATPase contributes approximately ⫺8 mV to the
resting potential of EC (63, 303, 370). Although a NCX has
ION CHANNELS IN VASCULAR ENDOTHELIUM
1441
1. Ca2⫹ pattern, waves, and release sites
been identified in EC (75, 385), no evidence for its electrogenic contribution to the membrane potential has been
presented so far.
B. Ca2ⴙ Signaling in EC
Various extracellular signals involved in the regulation of many of the described EC responses via specific second messengers are integrated at the EC surface. Between these second messengers, cytosolic Ca2⫹
is one of the most important signals, which is very well
conserved throughout the phylogenetic tree (318). Regulation of intracellular Ca2⫹ signals is probably the
most important functional task of ion channels in EC.
We will thus first describe the phenomenology of Ca2⫹
signals in EC before discussing in detail the role of ion
channels for Ca2⫹ entry and regulation of the inwardly
driving force for Ca2⫹.
Physiol Rev • VOL
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
2⫹
FIG. 12. Changes in membrane potential and [Ca ]i by stimulating
umbilical vein-derived endothelial cells with the P2U agonists UTP and
ATP. A: changes in [Ca2⫹]i in a fura 2-AM-loaded EC during stimulation
with 0.5, 2, and 20 ␮M UTP. B: concomitant changes in membrane
potential induced by agonist stimulation. (From F. Viana and B. Nilius,
unpublished data.) C and D: changes in [Ca2⫹]i in a cell stimulated with
10 ␮M ATP are modulated by changing the membrane potential of a
voltage-clamped cell. (From M. Kamouchi and B. Nilius, unpublished
data.)
Two phenotypes of Ca2⫹ signals can be distinguished, i.e., [Ca2⫹]i oscillations or a biphasic increase in
[Ca2⫹]i, consisting of a fast peak followed by a longlasting plateau (see Figs. 1 and 12 and Refs. 161, 162, 272,
295, 296). Low concentrations of various agonists, such as
acetylcholine, ATP, histamine, and bradykinin, often induce oscillations of [Ca2⫹]i. These oscillations appear
only in a small window of agonist concentrations (162,
304), whereas a biphasic rise in [Ca2⫹]i prevails at higher
concentrations (144, 271, 272, 291, 295, 296). The fast
transient Ca2⫹ peak represents release of Ca2⫹ from
InsP3-sensitive intracellular Ca2⫹ stores, whereas Ca2⫹
influx from the extracellular space activated by store
depletion accounts for the plateaulike phase. Plasmalemmal ion channels provide an influx route for Ca2⫹ and
tune the inwardly driving force for Ca2⫹ influx (271, 272,
295, 318). The sustained plateau level can be further modulated at a constant driving force for Ca2⫹ by mitochondrial Ca2⫹ buffering, Na⫹/Ca2⫹ exchange, PMCA, and
SERCA (predominantly SERCA-3; Refs. 222, 262).
Application of vasoactive agonists can also induce
Ca2⫹ waves that travel through the cell at a speed of 5– 60
␮m/s depending on the agonist concentration and cellular
region (151, 255). Ca2⫹ waves are regenerative, often arise
at peripheral cytoplasmic processes, and propagate from
the cell in which they originate to neighboring cells in the
cell cluster without decrement of amplitude, kinetics, and
speed of propagation (74, 151). These waves spread
through gap junctions, mainly consisting of Cx40 and -43
(221). Regenerative propagation depends on PLC and
likely also on the presence of calcium-induced calcium
release (CICR). Extracellular Ca2⫹ is required, which
points to a role of Ca2⫹ entry channels in wave propagation (74). Activation of a Ca2⫹ transient and wave is
preceded by small highly localized increases in [Ca2⫹]i
that originate from clusters of elementary Ca2⫹ release
events (blips). The amplitude of these blips of ⬃23 nM
corresponds to a Ca2⫹ current of 0.3 pA and is consistent
with a current through a single or a small number of
InsP3-sensitive release channels (151). The expression
pattern of SERCA and inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in the endothelial endoplasmic
reticulum varies between different cell types. Most EC
express only SERCA-3. During cell differentiation, the
expression of InsP3R-1 decreases, whereas that of
InsP3R-3 increases (262). Isshiki et al. (160) reported that
the initial rise in [Ca2⫹]i and Ca2⫹ wave in EC stimulated
with agonists originate at peripheral loci enriched in
caveolin, suggesting that caveolae may be involved in the
initiation of Ca2⫹ waves. In addition, Ca2⫹ stores in EC
might also be compartmentalized, since Ca2⫹ signals during
submaximal stimulation were restricted to a subplasmalemmal region, which probably reflects functional units consist-
1442
BERND NILIUS AND GUY DROOGMANS
2. Ca2⫹ oscillations
EC exhibit oscillations in [Ca2⫹]i if stimulated with low
agonist concentrations in a narrow concentration window
(see Fig. 12 and Refs. 162, 272, 295, 296, 304). Synchronization of these Ca2⫹ oscillations in confluent EC depends on
cell-cell coupling (268). These oscillations are frequently
accompanied by oscillatory changes in membrane potential
due to activation of BKCa channels (272, 295, 296, 314, 415).
These oscillations are mainly due to periodic discharges of
intracellular Ca2⫹ stores, since they are sustained for a long
time in cells exposed to Ca2⫹-free media (162, 272) and
apparently not via activation of a Ca2⫹-influx pathway since
they are not affected by changes in the driving force for Ca2⫹
(272). It is therefore likely that low agonist concentrations
do not activate Ca2⫹ entry channels and that these channels
are not involved in Ca2⫹ oscillations. However, [Ca2⫹]i oscillations in EA.hy926 cells seem to depend on Na⫹ loading
Physiol Rev • VOL
via NSC and Ca2⫹ entry via the endothelial NCX, a mechanism that also depends on membrane hyperpolarization via
activation of BKCa channels (314). However, in the same cell
type, similar Ca2⫹ oscillations were observed in voltageclamped cells (272). Activation of Ca2⫹ entry channels at
higher agonist concentrations is important for refilling of
depleted intracellular stores. Activation of PKC, PLC blockers, and inhibitors of SERCA pumps strongly suppress Ca2⫹
oscillations (49, 272). Under pathological conditions, glucose overload and superoxide anions also impair Ca2⫹ release and Ca2⫹ oscillations (191).
3. Structural organization of Ca2⫹ signaling
in endothelium: a role for caveolae?
Caveolae, abundantly present in EC, are specialized
plasmalemmal microdomains enriched in glycosphingolipids, cholesterol, sphingolipds, and lipid-anchored membrane proteins (321). They are omega-shaped invaginations with a diameter of 50 –100 nm. Caveolae originate
from “lipid raft domains” (LRD) in which sphingolipids
and cholesterol are highly accumulated. Specific proteins
attach to these LRD (101–103, 130). The major protein
component of endothelial caveolar structures is caveolin-1 (CAV-1). It consists of 178 amino acid residues and
has a molecular mass of 22 kDa. A Tyr residue at position
14 provides a target for protein tyrosine kinase (PTK).
The stretch of residues 81–101 is the scaffolding region
(CSD), which binds and “deactivates” several other proteins. Membrane insertion of CAV-1 occurs via interaction
with the hydrophobic hairpin (residue 102–134). Caveolar
structures contain a variety of signal transduction molecules, including GPCRs, G proteins, and adenylyl cyclase,
molecules involved in the regulation of intracellular Ca2⫹
homeostasis, such as the plasma membrane Ca2⫹-ATPase
and InsP3R-3 (361), eNOS, multiple components of the
tyrosine kinase mitogen-activated protein kinase pathway, Ca2⫹-sensitive activated PKC-␣, and numerous lipid
signaling molecules (3, 371). Various membrane receptors, such as the BK2 receptor and the muscarinic m2AchR, are targeted to caveolae upon agonist binding (21,
89). Vesicular transport systems in EC are in close contact
with caveolae, which contain key proteins that mediate
different aspects of exo- and endocytotic vesicle formation, docking, and/or fusion, including the vSNARE
VAMP-2, monomeric and trimeric GTPases, annexins II
and VI, and the NEM-sensitive fusion factor NSF with its
attachment protein SNAP. VAMP is also sensitive to cleavage by botulinum B and tetanus neurotoxins. Caveolae in
endothelium may therefore be considered as complex
signaling and transport units (246, 360).
Mechanostimulation by shear stress induces rapid tyrosine phosphorylation of proteins located in caveolae, e.g.,
ERK activation (320, 345). Tyrosine phosphorylation influences Ca2⫹ entry (94) and is necessary for gating of VRAC
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
ing of superficial endoplasmic reticulum in close contact
with the plasma membrane, such as caveolae and membrane near mitochondria. These restricted Ca2⫹ signals induce local subplasmalemmal Ca2⫹ gradients. According to
Graier et al. (118), release from ryanodine-sensitive Ca2⫹
stores contributes to these local gradients.
The most important release sites in EC are
Ins(1,4,5)P3-sensitive Ca2⫹ stores, and the release channel
is most likely InsP3R-3 (262). Other release sites, clearly
distinct from mitochondria and from stores sensitive to
ryanodine or Ins(1,4,5)P3, seem to respond to mechanical
forces, such as stretch and shear stress (167). The intracellular messenger responsible for this mechanosensitive
release may be an arachidonic derivative (305). The pharmacological properties of the ryanodine receptors mediating CICR in EC (213, 432) are clearly different from the
CICR mechanism in other cell types. Ruthenium red inhibits and caffeine induces only a small release. The
ryanodine-induced Ca2⫹ release is also different from the
ruthenium red-blocked release, suggesting the existence
of a ruthenium red-sensitive but ryanodine-insensitive
CICR mechanism. CICR may operate to amplify the magnitude of the Ins(1,4,5)P3 response (313, 315, 350, 432,
454). A mitochondrial CICR mechanism, insensitive to
ryanodine and caffeine and blocked by cyclosporin A, an
inhibitor of the mitochondrial permeability transition
pore, has been described recently (440).
Equally important for Ca2⫹ signaling is the refilling of
2⫹
Ca stores. It probably occurs via the SERCA-3 isoform
of the endoplasmic Ca2⫹-ATPase, which is abundantly
expressed in EC. Because the acetylcholine-induced endothelium-dependent relaxation of aorta precontracted
with phenylephrine was attenuated in SERCA-3-deficient
mouse, it has been concluded that SERCA-3 plays a role in
EC Ca2⫹ signaling involved in NO-mediated relaxation of
vascular smooth muscle (222).
ION CHANNELS IN VASCULAR ENDOTHELIUM
entry channels (STRPCs), channels regulating the driving
force for Ca2⫹ entry (e.g., VRAC) connected with Ca2⫹
release sites (InsP3R), the responding elements for agonist stimulation (GPCR and G proteins) and NCX in a
close vicinity to caveolin-bound eNOS would provide a
powerful signaling unit in EC (Fig. 13).
C. Secretion of Vasoactive Compounds: A Potential
Role for Ion Channels
Endothelial function and its role in hypertension,
arteriosclerosis, and coronary diseases depend on a balance between the release of various factors (32). These
factors promote or inhibit 1) vasodilatation and vasoconstriction of preferentially small vessels, 2) blood coagulation and fibrinolysis, 3) thrombogenesis and thromboly-
FIG. 13. Scheme of the cholesteroland sphingolipid-rich caveolar compartments in EC. This compartment contains
various important proteins, such as signaling molecules and tools for the exocytotic
machinery. VAMP-2, vesicle-associated
membrane protein is a vesicular SNAP receptor similar to synaptobrevin; NSF, the
N-ethylmaleimide-sensitive fusion factor
with its soluble attachment protein SNAP.
Caveolae are formed from “lipid raft domains.” It is depicted that possibly a functional unit comprising receptors for the
agonist response (GPRC), Ca2⫹ entry
channels (TRPs), channels for regulating
the driving force for Ca2⫹ and being involved in electrogenesis (e.g., the volumeregulated anion channel, VRAC) and possible also the Na⫹/Ca2⫹ exchanger (NCX)
are components of such units (for a discussion see text). Probably TRPs could be
coupled to the InsP3 receptor (InsP3R) in
subplasmalemmal store. Coupling domain
has been identified in the NH2 terminus of
the InsP3R downstream of the InsP3 binding site and the COOH terminus of TRPs.
TRPs might couple to caveolin-1 (cav-1) as
well as VRAC and GPRC (for discussion
see text). The main structural component
of the endothelial caveolae, caveolin-1␣, is
shown at the bottom. CSD marks the
caveolar scaffolding domain. The FEDVIA
sequence is conserved in all known caveolins. The putative PTK phosphorylation
site at position 14 is present in caveolin-1␣
but not in caveolin-1␤. The hydrophobic
hairpin region links this protein to the
caveolar membrane (for detailed description, see text).
Physiol Rev • VOL
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
channels, which are probably associated with CAV-1 (391).
Ca2⫹ waves are triggered from caveolin-rich edges of EC
(160), and Ca2⫹ entry pathways are probably colocalized
with caveolar sites in EC (210). Recently, TRP1 has been
identified in caveolae and LRD in human submandibular
glands and found to be associated with CAV-1, G␣q/11 proteins, and the InsP3 receptor type 3 (InsP3R3). The scaffolding domain of CAV-1 probably binds to NH2- and COOHterminal sites of TRP1 (224). An NH2-terminal site
downstream of the InsP3 binding site of the InsP3R associates with several COOH-terminal sites of TRP3 (37).
An NCX protein has been detected in caveolin-rich
membrane fractions containing both eNOS and CAV-1. It
is interesting to speculate that also transmembrane Na⫹
gradients may be involved in regulation of eNOS activity.
This would provide a tight functional interaction between
endothelial NCX and eNOS (385). A complex of Ca2⫹
1443
1444
BERND NILIUS AND GUY DROOGMANS
TABLE
3. Factors released from endothelial cells
Vasoactive factors
Vasodilators
Nitric oxide
Endothelium-derived
hyperpolarizing factor
Prostacyclins, PGI2
Natriuretic peptide
Vasoconstrictors
Endothelin-1
Angiotensin II
Thromboxane A2
Prostaglandin H2
Superoxide radicals
Peroxynitrite, ONOO⫺
Hydroxyls, OH⫺
Hemostasis and thrombolysis
Procoagulant, prothrombotic
factors
von Willebrand factor
Plasminogen activator inhibitor
Platelet activator
Anticoagulant, thrombolytic
factors
Tissue factor plasminogen
activator
Tissue factor pathway
inhibitor
Nitric oxide
PGI2
Protein S
EC growth modulators
Promoters
Tumor necrosis factor-␣
Superoxide radicals
Inhibitors
Nitric oxide
PGI2
Natriuretic peptide
Inflammatory agents
Promoters
Tumor necrosis factor-␣
Superoxide radicals
Peroxynitrite, ONOO⫺
Inhibitors
Nitric oxide
[Modified from Born et al. (32).]
Physiol Rev • VOL
blood on the endothelial layer. This basal release seems to
be Ca2⫹ independent. The serine/threonine protein kinase
Akt/PKB mediates the activation of eNOS, leading to increased NO production and might be the target of shear
stress probably involving a sensor function of platelet
endothelial cell adhesion molecule-1 (PECAM-1 or CD31,
71). NO can also be released via activation of a Ca2⫹/CaMdependent pathway which requires sustained influx of
extracellular Ca2⫹ (116, 157, 210). In EC, eNOS is localized in signal-transducing domains of the plasmalemma,
the so-called caveolae (321). Acetylation of eNOS by myristic and palmitoyl acid enables targeting of the enzyme to
the caveolae. Under resting conditions, eNOS is inactivated due to its binding to the caveolar scaffolding protein
CAV-1. An increase in [Ca2⫹]i promotes binding of calmodulin to eNOS and its dissociation from caveolin. A
more prolonged exposure to increased levels of [Ca2⫹]i
redistributes eNOS to the cytosol and is accompanied by
depalmitoylation and phosphorylation (for reviews, see
Refs. 90, 252, 253, 336). Lin et al. (219) showed in a recent
elegant study that NO production is mainly controlled by
myristoylated, membrane-associated eNOS that senses
CCE, and that it is less sensitive to intracellular Ca2⫹
release.
The rapid synthesis and release of the anticoagulant
prostacyclin (PGI2) requires a rise in [Ca2⫹]i and phosphorylation of PLA2 (323). PGI2 is released “on demand”.
An increase in [Ca2⫹]i translocates cytosolic PLA2 from
the cytosol to phospholipid membranes, especially to the
nuclear membrane and the plasmalemma. PLA2 is supposed to be colocalized in these regions with cyclooxygenase and lipoxygenase, which are necessary to convert
arachidonic acid to vasoactive compounds, such as PGI2
and EET products. This translocation occurs via a Ca2⫹dependent phospholipid-binding domain at the NH2 terminus of PLA2 (CaLB; Refs. 59, 97). An additional phosphorylation of cPLA2 is required for the synthesis of PGI2.
This phosphorylation is mediated by a p42/p44 MAP kinase, which is activated by PKC and probably also by an
increase in [Ca2⫹]i. Stimulation of PGI2 synthesis by vasoactive compounds (ACh, bradykinin, and ATP) is supported by Ca2⫹ influx via Ca2⫹ entry channels, including
SOC (115, 181, 261). PGI2 supports the vasodilating action
of NO and is also produced in response to shear stress.
PAF is also produced on demand. Its synthesis is
triggered by an increase in [Ca2⫹]i which precedes activation of PLA2. Importantly, released PAF also binds to
EC and mediates Ca2⫹ influx into its generator cells (200).
Another factor that is synthesized and released in a
Ca2⫹-dependent way is the EDHF, which might be 5,6EET. Another recent hypothesis suggests that EDHF, or at
least one of its components, is K⫹ released from EC by
activation of BKCa channels into the space between EC
and smooth muscle cells (82). This effect would also
require a sustained elevation of [Ca2⫹]i in EC.
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
sis, 4) EC growth and remodeling of the vessel wall, and
5) mediate inflammation. Table 3 gives an overview of
various factors released from EC.
It is generally agreed that synthesis and/or release of
vasoactive compounds depends on or can be modulated
by changes in [Ca2⫹]i (41, 50, 154, 273, 295). Sources for
an increase in [Ca2⫹]i are primarily release of Ca2⫹ from
intracellular stores via an Ins(1,4,5)P3-dependent mechanism and influx of extracellular Ca2⫹. Ion channels play a
role in the latter process by either providing an influx
pathway for extracellular Ca2⫹ or by regulating the driving force for this influx. We first discuss some EC functions in which Ca2⫹ plays a regulatory role.
The release of NO is involved in the control of relaxation of vascular SMC, but also inhibits platelet aggregation, proliferation of SMC (an anti-atherosclerotic function), the migration of monocytes and leukocytes, and the
expression of adhesion molecules. NO produced by eNOS
is a fundamental determinant of cardiovascular homeostasis and regulates systemic blood pressure, vascular remodeling, and angiogenesis. The most important
physiological stimulus for the continuous formation of
NO is viscous drag (shear stress) exerted by streaming
ION CHANNELS IN VASCULAR ENDOTHELIUM
Physiol Rev • VOL
ological factors that elevate [Ca2⫹]i, such as thrombin and
histamine, but also by endothelins and cytokines. The
regulation of tPA release and that of PAI-1 release are not
necessarily coupled, and the exact role of [Ca2⫹]i has to
be elucidated (324, 446).
Another stored factor in the regulation of coagulation
is protein S, a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C that has a strong anticoagulant function. An increase in [Ca2⫹]i releases protein S from cell organelles
with a diameter of 0.1 ␮m (375).
Vasoconstrictor responses of EC are mainly mediated by the release of ET-1, which is produced during
stimulating of EC with thrombin, transforming growth
factor-␤ (TGF-␤), interleukin-1, angiotensin II, and arginine vasopressin. It acts via ETA receptors on subendothelial vascular SMC, but it can also act on ETB receptors
on EC and provide a negative feedback by stimulating NO
release. ET-1 and its precursor big ET-1 are colocalized in
granules with a diameter of ⬃0.1 ␮m, suggesting that they
are not only release sites but also important subcellular
compartments for ET processing (131). Small amounts of
ET-3 increase NO release (436). Ca2⫹ is involved in this
regulation probably via activation of PKC and an
Ins(1,4,5)P3-mediated increase in [Ca2⫹]i. Both ET-1 and
ET-3 mobilize stored Ca2⫹ and activate Ca2⫹ entry pathways (4). The release of ETs is, however, not transient but
persistent, and the relationship between [Ca2⫹]i and ET
release seems to be contradictory, since thrombin increases but bradykinin decreases ET release although
both elevate [Ca2⫹]i (120). The role of Ca2⫹ for ET release
remains unclear.
All substances stored in granules are released by
regulated exocytosis, e.g., vWF, tPA, TFPI, protein S, and
ET-1 and ET-3. Exocytosis, probably except for the release of ET-1, depends on an increase in [Ca2⫹]i. Vesicular
secretion of vasoactive mediators has been assessed from
changes in membrane capacitance. Intracellular Ca2⫹ increases induced by flash photolysis of caged Ca2⫹ or
Ins(1,4,5)P3 exert a biphasic effect on membrane capacitance, consisting of an initial transient increase followed
by a much larger delayed component and which both
depend on [Ca2⫹]i (51). Probably, the late and large
changes in membrane capacitance reflect the exocytotic
release of vWF, whereas the faster events might be related to tPA and TFPI release. NO release and vWF release clearly depend on the presence of extracellular
Ca2⫹, indicating that Ca2⫹ influx is necessary to support
this EC function (51), whereas prostacyclin, tPA, and
TFPI depend to a larger extent on release of intracellular
Ca2⫹ (50). In summary, several EC functions including the
production of vasoactive compounds on demand and the
release of stored compounds by exocytosis depend on an
increase in [Ca2⫹]i. Two sources of Ca2⫹ are involved: an
Ins(1,4,5)P3-mediated Ca2⫹ release from intracellular
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
In addition to synthesis and release on demand, several stored vasoactive compounds are secreted during EC
stimulation. The best-studied example is the vWF, a large
polymeric protein that binds coagulation factor VIII and
protects it from cleavage, and which also contains binding
sites for the platelet glycoproteins GPIb and GPIIb/IIIa to
collagen, heparin, and vitronectin. It is stored in secretory
granules, Weibel-Palade bodies, which are large vesicular
organelles of ⬃200 nm in diameter and up to 4,000 nm in
length. vWF is released by CaM-dependent exocytosis
triggered by an increase in [Ca2⫹]i (28). Weibel-Palade
bodies also contain the leukocyte adhesion protein
P-selectin (GMP-140), which appears at the cell surface
as a result of exocytosis (428).
tPA, which activates fibrinolysis and thrombolysis in
blood vessels, is another example of a stored anticoagulant molecule that is released in a Ca2⫹-dependent way.
The mechanisms involved in the secretion of vWF and tPA
differ considerably. Thrombin stimulation of EC induces
tPA secretion from small vesicles morphologically different from the larger Weibel-Palade bodies and from caveolae (85). Release of tPA from EC is regulated by histamine, which like thrombin elevates [Ca2⫹]i, but also by
endothelins and cytokines. In addition, tPA is constitutively released.
TFPI is an important antithrombotic factor that inhibits complex “tissue factor (TF) VIIa,” which in turn
activates factors IXa and Xa and starts coagulation.
TFPI-1, a 36-kDa glycoprotein, is a Kunitz-type serine
protease inhibitor with an obvious role in the prevention
of thrombosis. TFPI is released upon heparin administration. The heparin-binding site of TFPI (HBS-1) is located
in its COOH-terminal basic portion. This Kunitz-type
serine proteinase inhibitor contains three tandem KPI
domains. TFPI-2 is another heparin-releasable 33-kDa
serine-protease inhibitor. TFPIs are located in well-defined granules evenly spread over the cell surface and in
the apical part of the cytoplasm. These granules are distinct from Weibel-Palade bodies and have a smaller diameter of ⬃200 nm. TFPI colocalizes in EC with caveolin. It
is bound by a specific glycosylphosphatidylinositol-anchorage mechanism that depends on the presence of cholesterol (229, 230). TPFI containing vesicles might be
docked on caveolae and can therefore be rapidly translocated to the exocytotic machinery (360). TFPI binds in a
Ca2⫹-independent way to a phosphatidylserine phospholipid surface depending on the presence of the TFPI
COOH-terminal Kunitz domain. The interaction of TFPI
with phosphatidylserine is decreased when [Ca2⫹]i is increased, a mechanism important for TFPI release (402).
PAI-1, an inhibitor of fibrinolysis, is also released in a
Ca2⫹-dependent manner. However, the increase in [Ca2⫹]i
induces only a modest increase of PAI-1 but suppresses
the action of much stronger release stimuli, such as
TNF-␣ (324). The release of PAI-1 is regulated by physi-
1445
1446
BERND NILIUS AND GUY DROOGMANS
stores, which in a long term must be replenished from
extracellular sources, and Ca2⫹ entry from the extracellular side. Although not many details are known about the
mechanisms that regulate exocytosis in endothelium, it is
obvious that ion channels play an important role in these
processes by modulating the changes in [Ca2⫹]i.
D. Ion Channels and Control of Vessel Permeation,
Leukocyte Migration, and Cell-Cell Contacts
Physiol Rev • VOL
E. Ion Channels: A Role in EC Growth
and Angiogenesis?
Extracellular angiogenesis inducers (VEGF, basic fibroblast growth factor, acidic fibroblast growth factor,
PDGF, IL-8, PAF, etc.) that stimulate proliferation and
migration of EC regulate the formation of new blood
vessels (angiogenesis). Angiogenesis inhibitors, such as
tumor suppressor genes, angiostatin, and endostatin,
counteract these processes (for a review, see Refs. 43,
48). Surprisingly, blockers of VRAC, such as tamoxifen,
clomiphene, ␤-estradiol, NPPB, IAA-94, DIDS, quinine,
quinidine, mibefradil, and flufenamic acid, also suppress
the growth of EC (238, 278, 283, 425). It is well known that
ion channels modulate the progression through the cell
cycle. Block of K⫹ channels and cell depolarization induce G1 arrest in several cells types (275). The cyclindependent kinase inhibitors p27(Kip1) and p21(CIP1)
might be involved in this mechanism (112). In addition,
angiogenesis is inhibited by various blockers of VRAC
with unrelated chemical structure, such as NPPB, mibefradil, and the antiestrogens tamoxifen and clomiphene,
as assessed in four assays of angiogenesis: the Matrigel
and fibrin gel assays of tube formation by endothelial cells
in vitro, the rat aorta-ring assay of spontaneous microvessel formation ex vivo, and the chick chorioallantoic membrane assay of neovascularization in vivo. These results
suggest that anion channels may play a role in angiogen-
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
A major activity of EC is the regulation of exchange
of solutes, proteins, and endocytosed particles between
blood and tissue. Part of this transport is mediated by
changes in the paracellular permeability of the endothelial layer. An increase in endothelial [Ca2⫹]i enhances the
permeability of microvessels, which is abolished in Ca2⫹free solution, but also when the cells are depolarized.
Agonists like thrombin or histamine, which all transiently
increase [Ca2⫹]i, will therefore induce a short increase in
vascular permeability, whereas others, such as cytokines
and growth factors, induce a more sustained change. This
increase in permeability is due to changes in the cytoskeleton, cellular contraction, and cell-cell coupling (68), processes that are all controlled by myosin light-chain phosphorylation. EC contraction and regulation of hydraulic
permeability depend on influx of Ca2⫹ (174) and involve a
Ca2⫹/CaM-dependent nonmuscle MLCK isoform that has
recently been cloned in human endothelium (see below
and Refs. 412, 413).
The regulation of permeability in macrovessels is
more complex. An ionomycin-induced increase in [Ca2⫹]i
enhances the permeability of porcine aorta endothelium,
whereas a comparable increase in [Ca2⫹]i by ATP reduces
its permeability. This increase is attenuated by the PLC
inhibitor U-73122 (299).
Shrinkage of EC increases microvessel permeability
if perfused with the Gly-Arg-Gly Asp-Thr-Pro (GRGDTP)
peptide to disrupt integrin-dependent attachment of EC to
the extracellular matrix (174, 175). In general, spreading
of EC on their matrix and contact formation with their
basement membrane has a selective requirement for Ca2⫹
influx (2).
[Ca2⫹]i and cAMP are two interactive signals that
control vessel permeability. Increased [Ca2⫹]i promotes
disruption of the macrovascular EC barrier but not the
microvascular barrier. Increased cAMP enhances endothelial barrier function. During inflammation, elevated
[Ca2⫹]i decreases cAMP to increase intercellular permeability (260). Changes in [Ca2⫹]i that regulate the permeability of the EC barrier possibly require activation of
store-operated Ca2⫹ entry channels (259).
We have shown (see sect. IVC1) that the VRAC regulates cell volume and provides a pathway for organic
osmolytes and amino acids. Interestingly, its gating in-
volves activation of RhoA (288, 298). Because inactivation
of RhoA also enhances the endothelial barrier function
due to enhanced myosin ribbon and stress fiber-focal
adhesion formation (47), VRAC may also be involved in
the control of endothelial barrier function. Also, the control of EC permeability via MLCK is tightly coupled to
CCE-mediated Ca2⫹ entry (300).
EC permeability and neutrophilic leukocyte diapedesis through paracellular gaps are cardinal features of
acute inflammation, which are mediated by myosin lightchain phosphorylation. This phosphorylation results from
a Ca2⫹/CaM-dependent activation of an EC-specific, 214kDa, nonmuscle MLCK distinct from the smooth muscle
MLCK isoforms (130 –150 kDa) (106). This EC MLCK isoform binds biotinylated CaM in a Ca2⫹-dependent manner
and colocalizes with myosin, actin, and CaM. Thus MLCK
in EC is a further target for changes in [Ca2⫹]i. Inhibition
of MLCK activity by activating cAMP-PKA reduced leukocyte transmigration. The myosin-associated phosphatase
inhibitor calyculin induces accumulation of phosphorylated myosin light chains, EC contraction, and significantly enhances leukocyte migration. Ca2⫹-dependent EC
MLCK is therefore a key determinant of vascular permeability and transendothelial leukocyte migration
(107, 412, 413).
ION CHANNELS IN VASCULAR ENDOTHELIUM
F. Role of Ion Channels in Dysfunction
of Endothelium?
Several disorders, among which hypertension, arteriosclerosis and other cardiovascular diseases, diabetes,
and hypercholesterolemia, are associated with a dysfunctional endothelium (33). Endothelial-dependent relaxation is impaired in essential hypertension because of a
deficient release of NO. Release of ET-1 exacerbates hypertension. Obviously, defective Ca2⫹ signaling can be the
cause of this deficient NO production. CCE is significantly
reduced in aorta EC of trp4-deficient mice, and the NOmediated vasorelaxation of aortic rings is impaired (99),
indicating that a sustained influx of Ca2⫹ is important for
endothelial NO production. It is well known that BKCa
channels modulate NO production (256), indicating that
channels that control the driving force for Ca2⫹ entry are
also essential for the control of NO synthesis.
Changes in membrane potential per se also induce
significant changes in NO-mediated EC responses. An
intriguing example is the depolarization-induced superoxPhysiol Rev • VOL
ide formation in HUVEC (374). Superoxide anions not
only impair NO-mediated responses but are also involved
in the development of hypertensive vascular hypertrophy.
EC depolarization also enhanced tyrosine phosphorylation of as yet unidentified membrane proteins and induced membrane association of the small G protein Rac.
Cation and anion channels are the main candidates for
inducing depolarization and are therefore probably involved in this important pathogenic mechanism.
Hypoxic and ischemic conditions also mediate ECdependent vasoconstriction by release of arachidonic
acid, PGH2, thromboxane A2, and endoperoxides. The
enhanced NO production under these conditions increases the amount of NO-derived peroxydenitrite
(ONOO⫺), which becomes peroxinitrous acid and forms
hydroxyl ions. These vasoconstrictor superoxide anions
are highly reactive, damage the cell membrane, and finally
reduce NO production (32). A possible mechanism for the
hypoxia-induced autacoid release, which seems to depend on extracellular Ca2⫹, could be an increase in
[Ca2⫹]i (6, 10, 42, 93, 178, 348). It has been recently
described that metabolic inhibition stimulates Ca2⫹ entry
in EC probably via activation of TRP3, a NSC that is
a member of the STRPC subfamily (see sect. IIIC and
Ref. 380).
G. Concluding Remarks
EC form a multifunctional signal-transducing surface
that regulates many fundamental processes in the vascular system, such as the control of blood flow and blood
pressure, coagulation, platelet aggregation, vessel permeability, wound healing, and angiogenesis. It is rather surprising that these cells express such a huge variety of ion
channels. We are still in the very beginning of identifying
these channels at the molecular level, although some of
the main players, such as trp gene-encoded channels, are
distinctly emerging. One obvious functional target of EC
ion channels is Ca2⫹ signaling. It is firmly established that
many EC functions depend on Ca2⫹, but we do not understand at all how this signal, an increase in [Ca2⫹]i,
allows for specific EC responses on demand. The Ca2⫹
signal in EC is shaped by release of Ca2⫹ from intracellular pools, entry of Ca2⫹ via plasmalemmal channels, and
the concerted action of several Ca2⫹ sequestration mechanisms, such as activation of PMCA and SERCA Ca2⫹
pumps and probably NCX. However, several EC functions, e.g., release of vasoactive compounds including NO,
vWF, PAF, tPA, and TFPI, probably require activation of
exocytosis and need a high and a long-lasting plateaulike
increase in [Ca2⫹]i that mainly depends on Ca2⫹ entry.
The molecular nature of Ca2⫹ entry channels is not yet
known. However, there is increasing evidence that SOC
and nonselective Ca2⫹-permeable channels are the main
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
esis and have a potential therapeutic action in tumor
growth and other angiogenesis-dependent pathophysiological processes (238). The mechanism of action is unknown, but it may be linked to the role of VRAC in volume
and pH regulation or fine-tuning the driving force for Ca2⫹
entry, which is important at the Ca2⫹-dependent cell cycle
control point, i.e., between G1 and S. It has also been
shown that the functional expression of VRAC changes
when cells switch from proliferation to differentiation,
further supporting a role of ion channels in the regulation
of cell growth (237, 278, 295, 426).
Recently, clear evidence has been presented for a
role of Ca2⫹ in the regulation of angiogenesis in vivo.
VEGF-A, a member of the family of endothelial growth
factors (VEGFs), plays a dominant role in the regulation
of angiogenesis and vascular permeability (91). The endothelial vascular growth factor receptor, VEGFR-2,
binds VEGF-A and VEGF-E and activates a signaling cascade involving autophosphorylation, activation of PLC-␥
and elevation of [Ca2⫹]i, and a VEGF-dependent Ca2⫹
influx (135, 341, 430). Activation of VEGFR-2 induces
store depletion and triggers activation of CCE (CRAC)
channels, resulting in Ca2⫹ influx that might be important
for the control of angiogenesis. This example clearly demonstrates that ion channels are involved in a complex
phenomenon like angiogenesis. Furthermore, CAI, a
blocker of ligand-stimulated Ca2⫹ influx, inhibits EC proliferation, adhesion, and invasion into the basement membrane of EC. Likely, Ca2⫹-regulated events are important
in native and FGF2-stimulated EC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events (197).
1447
1448
BERND NILIUS AND GUY DROOGMANS
We thank Drs. S. H. Suh (Seoul), M. Kamouchi (Fukuoka),
and F. Viana (Alicante) for providing unpublished material.
This work was supported by the Belgian Federal GovernPhysiol Rev • VOL
ment; the Flemish Government and the Onderzoeksraad KU
Leuven Grants GOA 99/07, F.W.O.G. 0237.95, F.W.O.G. 0214.99,
F.W.O.G. 0136.00; Interuniversity Poles of Attraction Program,
Prime Minister’s Office IUAP Nr.3P4/23, and C.O.F./96/22-A069;
“Levenslijn” Grant 7.0021.99; “Alphonse and Jean Forton-Koning
Boudewijn Stichting” Grant R7115 B0; and European Commission Grant BMH4-CT96 – 0602.
Address for reprint requests and other correspondence: B.
Nilius, Laboratorium voor Fysiologie, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium (E-mail:
[email protected]).
REFERENCES
1. ADAMS DJ, RUSKO J, AND VAN SLOOTEN G. Calcium signalling in
vascular endothelial cells: Ca2⫹ entry and release. In: Ion Flux in
Pulmonary Vascular Control, edited by Weir EK, Hume JR, and
Reeves JT. New York: Plenum, 1993, p. 259 –275.
2. ALESSANDRO R, MASIERO L, LAPIDOS K, SPOONSTER J, AND KOHN EC.
Endothelial cell spreading on type IV collagen and spreading-induced FAK phosphorylation is regulated by Ca2⫹ influx. Biochem
Biophys Res Commun 248: 635– 640, 1998.
3. ANDERSON RG. The caveolae membrane system. Annu Rev Biochem
67: 199 –225, 1998.
4. AOKI H, KOBAYASHI S, NISHIMURA J, YAMAMOTO H, AND KANAIDE H.
Endothelin induces the Ca2⫹-transient in endothelial cells in situ.
Biochem Biophys Res Commun 181: 1352–1357, 1991.
5. ARCHER SL, TOLINS JP, RAIJ L, AND WEIR EK. Hypoxic pulmonary
vasoconstriction is enhanced by inhibition of the synthesis of an
endothelium derived relaxing factor. Biochem Biophys Res Commun 164: 1198 –1205, 1989.
6. ARNOULD T, MICHIELS C, ALEXANDRE I, AND REMACLE J. Effect of
hypoxia upon intracellular calcium concentration of human endothelial cells. J Cell Physiol 152: 215–221, 1992.
7. ARREOLA J, MELVIN JE, AND BEGENISICH T. Activation of calciumdependent chloride channels in rat parotid acinar cells. J Gen
Physiol 108: 35– 47, 1996.
8. BAHNSON TD, PANDOL SJ, AND DIONNE VE. Cyclic GMP modulates
depletion-activated Ca2⫹ entry in pancreatic acinar cells. J Biol
Chem 268: 10808 –10812, 1993.
9. BAI C, FUKUDA N, SONG Y, MA T, MATTHAY MA, AND VERKMAN AS. Lung
fluid transport in aquaporin-1 and aquaporin-4 knockout mice.
J Clin Invest 103: 555–561, 1999.
10. BALZER M, LINTSCHINGER B, AND GROSCHNER K. Evidence for a role of
Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells. Cardiovasc Res 42: 543–
549, 1999.
11. BARAKAT AI, LEAVER EV, PAPPONE PA, AND DAVIES PF. A flow-activated chloride-selective membrane current in vascular endothelial
cells. Circ Res 85: 820 – 828, 1999.
12. BARON A, FRIEDEN M, AND BÉNY JL. Epoxyeicosatrienoic acids activate a high-conductance, Ca2⫹-dependent K⫹ channel on pig coronary artery endothelial cells. J Physiol (Lond) 504: 537–543, 1997.
13. BARON A, FRIEDEN M, CHABAUD F, AND BÉNY JL. Ca2⫹-dependent
non-selective cation and potassium channels activated by bradykinin in pig coronary artery endothelial cell. J Physiol Lond 493:
691–706, 1996.
14. BARRITT GJ. Does a decrease in subplasmalemmal Ca2⫹ explain
how store-operated Ca2⫹ channels are opened? Cell Calcium 23:
65–75, 1998.
15. BEHRENS R, NOLTING A, REIMANN F, SCHWARZ M, WALDSCHÜTZ R, AND
PONGS O. hKCNMB3 and hKCNMB4, cloning, characterization of
two members of the large-conductance calcium-activated potassium channel ␤ subunit family. FEBS Lett 474: 99 –106, 2000.
16. BENNETT DL, PETERSEN CC, AND CHEEK TR. Calcium signalling.
Cracking ICRAC in the eye. Curr Biol 5: 1225–1228, 1995.
17. BENOS DJ AND STANTON BA. Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion
channels. J Physiol (Lond) 520: 631– 644, 1999.
18. BÉNY JL. Endothelial and smooth muscle cells hyperpolarized by
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
players for this entry. Probably, members of the STRPC
family are involved in functionally structuring this Ca2⫹
entry.
We have also shown that it is mandatory for EC not
only to activate Ca2⫹ entry channels upon various stimuli,
but also to provide a sufficiently large inwardly driving
force for Ca2⫹. Among the channels that influence electrogenesis in EC are various types of K⫹ channels. Especially Ca2⫹-activated K⫹ channels, mainly belonging to
the slo family, are important targets for regulating Ca2⫹
entry and Ca2⫹-dependent EC functions. Surprisingly, Cl⫺
channels are also abundantly present in EC. Among these
channels, especially the VRAC might be multifunctional.
This channel can counteract the depolarizing action of
other channels and contribute to a stabilization of the
inwardly driving force for Ca2⫹. Interestingly, these channels might be localized in the caveolar compartment together with eNOS, signaling proteins, including receptors
such as GPCRs, ATPases, small G proteins, kinases, the
endo- and exocytotic machinery, and other ion channels,
such as STRPCs, forming a complex that seems to play an
important role in signal transduction in EC. Future work
must focus on the functional role of these important
cellular compartments.
Obviously, ion channels are also important players in
other EC functions. VRAC channels are involved in transport of organic molecules including amino acids, and
various channels influence cell proliferation and angiogenesis as well as control EC permeation and cell-cell
coupling.
Another intriguing topic in EC biology is its role as a
mechanosensor. Irrefutably, mechanosensing properties
of EC are directly or indirectly coupled with ion channels,
although clear mechanosensor channels are, after several
preliminary candidates have been proposed, elusive. We
discuss here the possibility that CCE channels are indirectly involved in mechanosensing and discuss channels
with obvious mechanosensing properties, VRAC and inwardly rectifying K⫹ channels.
We are obviously at the verge of understanding the
role of ion channels in EC and to appreciate that these
channels might be important targets for the pharmacological modulation of fundamental EC functions. Indeed, we
are at the very beginning of considering EC ion channels
as targets of novel therapeutic compounds to control
Ca2⫹ entry and its driving force. In addition, ion channels
might be the cause for EC dysfunction linked to human
disorders. To catalyze a further understanding of EC
physiology and pathophysiology, it is necessary to include
also the expanding field of ion channels expressed in EC
in the general interest of vascular cell biologists.
ION CHANNELS IN VASCULAR ENDOTHELIUM
19.
20.
21.
22.
23.
24.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
Physiol Rev • VOL
40. BRUZZONE R, HAEFLIGER JA, GIMLICH RL, AND PAUL DL. Connexin40, a
component of gap junctions in vascular endothelium, is restricted
in its ability to interact with other connexins. Mol Biol Cell 4: 7–20,
1993.
41. BUSSE R, LÜCKHOFF A, AND MULSCH A. Cellular mechanisms controlling EDRF/NO formation in endothelial cells. Basic Res Cardiol 86
Suppl 2: 7–16, 1991.
42. BUSSE R, MULSCH A, FLEMING I, AND HECKER M. Mechanisms of nitric
oxide release from the vascular endothelium. Circulation 87: 18 –
25, 1993.
43. BUSSOLINO F, MANTOVANI A, AND PERSICO G. Molecular mechanisms
of blood vessel formation. Trends Biochem Sci 22: 251–256, 1997.
44. CAI S, GARNEAU L, AND SAUVÉ R. Single-channel characterization of
the pharmacological properties of the KCa channel of intermediate
conductance in bovine aortic endothelial cells. J Membr Biol 163:
147–158, 1998.
45. CAI S AND SAUVÉ R. Effects of thiol-modifying agents on a KCa
channel of intermediate conductance in bovine aortic endothelial
cells. J Membr Biol 158: 147–158, 1997.
46. CAMPBELL DL, STRAUSS HC, AND WHORTON AR. Voltage dependence of
bovine pulmonary artery endothelial cell function. J Mol Cell Cardiol 23 Suppl 1: 133–144, 1991.
47. CARBAJAL JM AND SCHAEFFER RC. RhoA inactivation enhances endothelial barrier function. Am J Physiol Cell Physiol 277: C955–C964,
1999.
48. CARMELIET P AND COLLEN D. Molecular analysis of blood vessel
formation and disease. Am J Physiol Heart Circ Physiol 273:
H2091–H2104, 1997.
49. CARTER TD, BOGLE RG, AND BJAALAND T. Spiking of intracellular
calcium ion concentration in single cultured pig aortic endothelial
cells stimulated with ATP or bradykinin. Biochem J 278: 697–704,
1991.
50. CARTER TD AND PEARSON JD. Regulation of prostacyclin synthesis in
endothelial cells. News Physiol Sci 7: 64 – 69, 1992.
51. CARTER TD, ZUPANCIC G, SMITH SM, WHEELER JONES C, AND OGDEN D.
Membrane capacitance changes induced by thrombin and calcium
in single endothelial cells cultured from human umbilical vein.
J Physiol (Lond) 513: 845– 855, 1998.
52. CHANG AS, CHANG SM, GARCIA RL, AND SCHILLING WP. Concomitant
and hormonally regulated expression of trp genes in bovine aortic
endothelial cells. FEBS Lett 415: 335–340, 1997.
53. CHEN KD, LI YS, KIM M, LI S, YUAN S, CHIEN S, AND SHYY JY.
Mechanotransduction in response to shear stress. Roles of receptor
tyrosine kinases, integrins, and Shc. J Biol Chem 274: 18393–18400,
1999.
54. CHIEN S, LI S, AND SHYY YJ. Effects of mechanical forces on signal
transduction and gene expression in endothelial cells. Hypertension 31: 162–169, 1998.
55. CHIEN S AND SHYY JY. Effects of hemodynamic forces on gene
expression and signal transduction in endothelial cells. Biol Bull
194: 390 –391, 1998.
56. CHIU JJ, WANG DL, CHIEN S, SKALAK R, AND USAMI S. Effects of
disturbed flow on endothelial cells. J Biomech Eng 120: 2– 8, 1998.
57. CHOI S-Y AND KIM K-T. Capsaicin inhibits phospholipase C-mediated
Ca2⫹ increase by blocking thapsigargin-sensitive store operated
Ca2⫹ entry in PC12 cells. J Pharmacol Exp Ther 291: 107–114, 1999.
58. CHYB S, RAGHU P, AND HARDIE RC. Polyunsaturated fatty acids
activate the Drosophila light-sensitive channels TRP and TRPL.
Nature 397: 255–259, 1999.
59. CLARK JD, SCHIEVELLA AR, NALEFSKI EA, AND LIN LL. Cytosolic phospholipase A2. J Lipid Mediat Cell Signal 12: 83–117, 1995.
60. COLDEN STANFIELD M, CRAMER EB, AND GALLIN EK. Comparison of
apical and basal surfaces of confluent endothelial cells: patchclamp and viral studies. Am J Physiol Cell Physiol 263: C573–C583,
1992.
61. COLDEN STANFIELD M, SCHILLING WP, POSSANI LD, AND KUNZE DL.
Bradykinin-induced potassium current in cultured bovine aortic
endothelial cells. J Membr Biol 116: 227–238, 1990.
62. CORDA S, SPURGEON HA, LAKATTA EG, CAPOGROSSI MC, AND ZIE2⫹
GELSTEIN RC. Endoplasmic reticulum Ca
depletion unmasks a
caffeine-induced Ca2⫹ influx in human aortic endothelial cells. Circ
Res 77: 927–935, 1995.
63. DAUT J, STANDEN NB, AND NELSON MT. The role of the membrane
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
25.
bradykinin are not dye coupled. Am J Physiol Heart Circ Physiol
258: H836 –H841, 1990.
BÉNY JL AND CONNAT JL. An electron-microscopic study of smooth
muscle cell dye coupling in the pig coronary arteries. Role of gap
junctions. Circ Res 70: 49 –55, 1992.
BÉNY JL AND PACICCA C. Bidirectional electrical communication
between smooth muscle and endothelial cells in the pig coronary
artery. Am J Physiol Heart Circ Physiol 266: H1465–H1472, 1994.
BENZING T, FLEMING I, BLAUKAT A, MULLER ESTERL W, AND BUSSE R.
Angiotensin-converting enzyme inhibitor ramiprilat interferes with
the sequestration of the B2 kinin receptor within the plasma membrane of native endothelial cells. Circulation 99: 2034 –2040, 1999.
BERRIDGE MJ. Capacitative calcium entry. Biochem J 312: 1–11,
1995.
BHULLAR IS, LI YS, MIAO H, ZANDI E, KIM M, SHYY JY, AND CHIEN S.
Fluid shear stress activation of IkappaB kinase is integrin-dependent. J Biol Chem 273: 30544 –30549, 1998.
BIEL M, LUDWIG A, ZONG X, AND HOFMANN F. Hyperpolarizationactivated cation channels: a multi-gene family. Rev Physiol Biochem Pharmacol 136: 165–181, 1999.
BIEL M, SAUTTER A, LUDWIG A, HOFMANN F, AND ZONG X. Cyclic
nucleotide-gated channels mediators of NO:cGMP-regulated processes. Naunyn-Schmiedebergs Arch Pharmacol 358: 140 –144,
1998.
BIEL M, ZONG X, AND HOFMANN F. Cyclic nucleotide gated channels.
Adv Second Messenger Phosphoprotein Res 33: 231–250, 1999.
BIEL M, ZONG X, LUDWIG A, SAUTTER A, AND HOFMANN F. Structure and
function of cyclic nucleotide-gated channels. Rev Physiol Biochem
Pharmacol 135: 151–171, 1999.
BIRCH KA, POBER JS, ZAVOICO GB, MEANS AR, AND EWENSTEIN BM.
Calcium/calmodulin transduces thrombin-stimulated secretion:
studies in intact and minimally permeabilized human umbilical vein
endothelial cells. J Cell Biol 118: 1501–1510, 1992.
BIRNBAUMER L, ZHU X, JIANG M, BOULAY G, PEYTON M, VANNIER B,
BROWN D, PLATANO D, SADEGHI H, STEFANI E, AND BIRNBAUMER M. On
the molecular basis and regulation of cellular capacitative calcium
entry: roles for Trp proteins. Proc Natl Acad Sci USA 93: 15195–
15202, 1996.
BKAILY G, DORLEANSJUSTE P, NAIK R, PERODIN J, STANKOVA J, ABDULNOUR E, AND ROLAPLESZCZYNSKI M. Paf Activation of a voltage-gated
R-type Ca2⫹ channel in human and canine aortic endothelial cells.
Br J Pharmacol 110: 519 –520, 1993.
BOLOTINA VM, NAJIBI S, PALACINO JJ, PAGANO PJ, AND COHEN RA.
Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850 – 853, 1994.
BORN G, RABELINK T, AND SMITH T. Endothelium and Cardiovascular Disease. London: Science Press, 1998.
BORN GVR AND SCHWARTZ CJ. Vascular Endothelium: Physiology,
Pathology and Therapeutics. Stuttgart, Germany: Schatthauer Verlag, 1997.
BOSSU JL, ELHAMDANI A, AND FELTZ A. Voltage-dependent calcium
entry in confluent bovine capillary endothelial cells. FEBS Lett 299:
239 –242, 1992.
BOSSU JL, ELHAMDANI A, FELTZ A, TANZI F, AUNIS D, AND THIERSE D.
Voltage-gated Ca entry in isolated bovine capillary endothelial
cells: evidence of a new type of BAY-K 8644-sensitive channel.
Pflügers Arch 420: 200 –207, 1992.
BOSSU JL, FELTZ A, RODEAU JL, AND TANZI F. Voltage-dependent
transient calcium currents in freshly dissociated capillary endothelial cells. FEBS Lett 255: 377–380, 1989.
BOULAY G, BROWN DM, QIN N, JIANG M, DIETRICH A, ZHU MX, CHEN Z,
BIRNBAUMER M, MIKOSHIBA K, AND BIRNBAUMER L. Modulation of Ca2⫹
entry by polypeptides of the inositol 1,4,5-trisphosphate receptor
(IP3R) that bind transient receptor potential (TRP): evidence for
roles of TRP and IP3R in store depletion-activated Ca2⫹ entry. Proc
Natl Acad Sci USA 96: 14955–14960, 1999.
BRAUNEIS U, GATMAITAN Z, AND ARIAS IM. Serotonin stimulates a Ca2⫹
permeant nonspecific cation channel in hepatic endothelial cells.
Biochem Biophys Res Commun 186: 1560 –1566, 1992.
BREGESTOVSKI P, BAKHRAMOV A, DANILOV S, MOLDOBAEVA A, AND
TAKEDA K. Histamine-induced inward currents in cultured endothelial cells from human umbilical vein. Br J Pharmacol 95: 429 – 436,
1988.
1449
1450
64.
65.
66.
67.
68.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
potential of endothelial and smooth muscle cells in the regulation
of coronary blood flow. J Cardiovasc Electrophysiol 5: 154 –181,
1994.
DAVIES PF. Flow-mediated endothelial mechanotransduction.
Physiol Rev 75: 519 –560, 1995.
DAVIES PF AND BARBEE KA. Endothelial cell surface imaging: insights into hemodynamic force transduction. News Physiol Sci 9:
153–157, 1994.
DAVIES PF, BARBEE KA, VOLIN MV, ROBOTEWSKYJ A, CHEN J, JOSEPH L,
GRIEM ML, WERNICK MN, JACOBS E, POLACEK DC, DEPAOLA N, AND
BARAKAT AI. Spatial relationships in early signaling events of flowmediated endothelial mechanotransduction. Annu Rev Physiol 59:
527–549, 1997.
DAVIES PF, OLESEN SP, CLAPHAM DE, MORREL EM, AND SCHOEN FJ.
Endothelial communication. State of the art lecture. Hypertension
11: 563–572, 1988.
DEJANA E, CORADA M, AND LAMPUGNANI MG. Endothelial cell-to-cell
junctions. FASEB J 9: 910 –918, 1995.
DEPAOLA N, DAVIES PF, PRITCHARD WF JR, FLOREZ L, HARBECK N, AND
POLACEK DC. Spatial and temporal regulation of gap junction connexin43 in vascular endothelial cells exposed to controlled disturbed flows in vitro. Proc Natl Acad Sci USA 96: 3154 –3159, 1999.
DE WIT C, SCHAFER C, VON BISMARCK P, BOLZ SS, AND POHL U.
Elevation of plasma viscosity induces sustained NO-mediated dilation in the hamster cremaster microcirculation in vivo. Pflügers
Arch 434: 354 –361, 1997.
DIMMELER S, FLEMING I, FISSLTHALER B, HERMANN C, BUSSE R, AND
ZEIHER AM. Activation of nitric oxide synthase in endothelial cells
by Akt-dependent phosphorylation. Nature 399: 601– 605, 1999.
DITTRICH M AND DAUT J. Voltage-dependent K⫹ current in capillary
endothelial cells isolated from guinea pig heart. Am J Physiol
Heart Circ Physiol 277: H119 –H127, 1999.
DOLOR RJ, HURWITZ LM, MIRZA Z, STRAUSS HC, AND WHORTON AR.
Regulation of extracellular calcium entry in endothelial cells: role
of intracellular calcium pool. Am J Physiol Cell Physiol 262: C171–
C181, 1992.
DOMENIGHETTI AA, BÉNY JL, CHABAUD F, AND FRIEDEN M. An intercellular regenerative calcium wave in porcine coronary artery endothelial cells in primary culture. J Physiol (Lond) 513: 103–116,
1998.
DOMOTOR E, ABBOTT NJ, AND ADAM VIZI V. Na⫹-Ca2⫹ exchange and its
implications for calcium homeostasis in primary cultured rat brain
microvascular endothelial cells. J Physiol (Lond) 515: 147–155,
1999.
DROOGMANS G, MAERTENS C, PRENEN J, AND NILIUS B. Sulphonic acid
derivatives as probes of pore properties of volume-regulated anion
channels in endothelial cells. Br J Pharmacol 128: 35– 40, 1999.
DROOGMANS G, PRENEN J, EGGERMONT J, VOETS T, AND NILIUS B.
Voltage-dependent block of endothelial volume-regulated anion
channels by calix[4]arenes. Am J Physiol Cell Physiol 275: C646 –
C652, 1998.
DUAN D, COWLEY S, HOROWITZ B, AND HUME JR. A serine residue in
ClC-3 links phosphorylation-dephosphorylation to chloride channel
regulation by cell volume. J Gen Physiol 113: 57–70, 1999.
DUAN D, WINTER C, COWLEY S, HUME JR, AND HOROWITZ B. Molecular
identification of a volume-regulated chloride channel. Nature 390:
417– 421, 1997.
DWORETZKY SI, TROJNACKI JT, AND GRIBKOFF VK. Cloning and expression of a human large-conductance calcium-activated potassium
channel. Brain Res 27: 189 –193, 1994.
EDGELL CJ, MCDONALD CC, AND GRAHAM JB. Permanent cell line
expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci USA 80: 3734 –3737, 1983.
EDWARDS G, DORA KA, GARDENER MJ, GARLAND CJ, AND WESTON AH.
K⫹ is an endothelium-derived hyperpolarizing factor in rat arteries.
Nature 396: 269 –272, 1998.
EISENMANN G. Cation selective glass electrodes and their mode of
operation. Biophys J 2: 259 –323, 1962.
ELAM TR AND LANSMAN JB. The role of Mg2⫹ in the inactivation of
inwardly rectifying K⫹ channels in aortic endothelial cells. J Gen
Physiol 105: 463– 484, 1995.
EMEIS JJ, VAN DEN EIJNDEN SCHRAUWEN Y, VAN DEN HOOGEN CM, DE
PRIESTER W, WESTMUCKETT A, AND LUPU F. An endothelial storage
Physiol Rev • VOL
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
granule for tissue-type plasminogen activator. J Cell Biol 139:
245–256, 1997.
FAN JP AND WALSH KB. Mechanical stimulation regulates voltagegated potassium currents in cardiac microvascular endothelial
cells. Circ Res 84: 451– 457, 1999.
FANGER CM, GHANSHANI S, LOGSDON NJ, RAUER H, KALMAN K, ZHOU J,
BECKINGHAM K, CHANDY KG, CAHALAN MD, AND AIYAR J. Calmodulin
mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1. J Biol Chem 274: 5746 –5754, 1999.
FASOLATO C AND NILIUS B. Store depletion triggers the calcium
release-activated calcium current (I-CRAC) in macrovascular endothelial cells: a comparison with Jurkat and embryonic kidney cell
lines. Pflügers Arch 436: 69 –74, 1998.
FERON O, HAN X, AND KELLY RA. Muscarinic cholinergic signaling in
cardiac myocytes: dynamic targeting of M2AChR to sarcolemmal
caveolae and eNOS activation. Life Sci 64: 471– 477, 1999.
FERON O, SMITH TW, MICHEL T, AND KELLY RA. Dynamic targeting of
the agonist-stimulated m2 muscarinic acetylcholine receptor to
caveolae in cardiac myocytes. J Biol Chem 272: 17744 –17748, 1997.
FERRARA N AND SMYTH TD. The biology of vascular endothelial
growth factor. Endocr Rev 18: 4 –25, 1997.
FISSLTHALER B, POPP R, KISS L, POTENTE M, HARDER DR, FLEMING I,
AND BUSSE R. Cytochrome P-4502C is an EDHF synthase in coronary arteries. Nature 401: 493– 497, 1999.
FLEMING I, BAUERSACHS J, AND BUSSE R. Calcium-dependent and
calcium-independent activation of the endothelial NO synthase. J
Vasc Res 34: 165–174, 1997.
FLEMING I, FISSLTHALER B, AND BUSSE R. Calcium signaling in endothelial cells involves activation of tyrosine kinases and leads to
activation of mitogen-activated protein kinases. Circ Res 76: 522–
529, 1995.
FORSYTH SE, HOGER A, AND HOGER JH. Molecular cloning and expression of a bovine endothelial inward rectifier potassium channel. FEBS Lett 409: 277–282, 1997.
FRANSEN PF, DEMOLDER MJ, AND BRUTSAERT DL. Whole cell membrane currents in cultured pig endocardial endothelial cells. Am J
Physiol Heart Circ Physiol 268: H2036 –H2047, 1995.
FREARSON JA, HARRISON P, SCRUTTON MC, AND PEARSON JD. Differential regulation of von Willebrand factor exocytosis and prostacyclin
synthesis in electropermeabilized endothelial cell monolayers. Biochem J 309: 473– 479, 1995.
FREICHEL M, SCHWEIG U, STAUFFENBERGER S, FREISE D, SCHORB W, AND
FLOCKERZI V. Store-operated cation channels in the heart and cells
of the cardiovascular system. Cell Physiol Biochem 9: 270 –283,
1999.
FREICHEL M, SUH SH, PFEIFER A, SCHWEIG U, TROST C, WEISSGERBER P,
PHILLIP S, FREISE D, DROOGMANS G, HOFMANN F, FLOCKERZI V, AND
NILIUS B. Lack of an endothelial store-operated Ca2⫹-current impairs agonist-dependent Ca2⫹ entry and vasorelaxation in TRP4
(CCE1) ⫺/⫺ mice. Nature Cell Biol. In press.
FRIEDEN M AND GRAIER WF. Subplasmalemmal ryanodine-sensitive
Ca2⫹ release contributes to Ca2⫹-dependent K⫹ channel activation
in a human umbilical vein endothelial cell line. J Physiol (Lond)
524: 715–724, 2000.
FUJIMOTO T. Calcium pump of the plasma membrane is localized in
caveolae. J Cell Biol 120: 1147–1157, 1993.
FUJIMOTO T, MIYAWAKI A, AND MIKOSHIBA K. Inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae is linked to
actin filaments. J Cell Sci 108: 7–15, 1995.
FUJIMOTO T, NAKADE S, MIYAWAKI A, MIKOSHIBA K, AND OGAWA K.
Localization of inositol 1,4,5-trisphosphate receptor-like protein in
plasmalemmal caveolae. J Cell Biol 119: 1507–1513, 1992.
GALBRAITH CG, SKALAK R, AND CHIEN S. Shear stress induces spatial
reorganization of the endothelial cell cytoskeleton. Cell Motil Cytoskeleton 40: 317–330, 1998.
GANDHI R, ELBLE RC, GRUBER AD, SCHREUR KD, JI HL, FULLER CM,
AND PAULI BU. Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung. J Biol Chem 273:
32096 –32101, 1998.
GARCIA JG, LAZAR V, GILBERT MCCLAIN LI, GALLAGHER PJ, AND VERIN
AD. Myosin light chain kinase in endothelium: molecular cloning
and regulation. Am J Respir Cell Mol Biol 16: 489 – 494, 1997.
GARCIA JGN, VERIN AD, HERENYIOVA M, AND ENGLISH D. Adherent
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
69.
BERND NILIUS AND GUY DROOGMANS
ION CHANNELS IN VASCULAR ENDOTHELIUM
108.
109.
110.
111.
112.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
Physiol Rev • VOL
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
149.
family of Ca2⫹-activated Cl⫺ channels. Biochim Biophys Acta
1444: 418 – 423, 1999.
GRUBER AD, SCHREUR KD, JI HL, FULLER CM, AND PAULI BU. Molecular cloning and transmembrane structure of hCLCA2 from human
lung, trachea, and mammary gland. Am J Physiol Cell Physiol 276:
C1261–C1270, 1999.
HABURCÁK M, WEI L, VIANA F, PRENEN J, DROOGMANS G, AND NILIUS B.
calcium activated potassium channels in cultured human endothelial cells are not directly modulated by nitric oxide. Cell Calcium
21: 291–300, 1997.
HARDER T AND SIMONS K. Caveolae, DIGs, and the dynamics of
sphingolipid-cholesterol microdomains. Curr Opin Cell Biol 9:
534 –542, 1997.
HARRISON VJ, BARNES K, TURNER AJ, WOOD E, CORDER R, AND VANE JR.
Identification of endothelin 1 and big endothelin 1 in secretory
vesicles isolated from bovine aortic endothelial cells. Proc Natl
Acad Sci USA 92: 6344 – 6348, 1995.
HARTNECK C, PLANT TD, AND SCHULTZ G. From worm to man: three
subfamilies of TRP channels. Trends Neurosci 23: 159 –166, 2000.
HASSESSIAN H, BODIN P, AND BURNSTOCK G. Blockade by glibenclamide of the flow-evoked endothelial release of ATP that contributes to vasodilatation in the pulmonary vascular bed of the rat. Br J
Pharmacol 109: 466 – 472, 1993.
HEINKE S, SZUCS G, NORRIS A, DROOGMANS G, AND NILIUS B. Inhibition
of volume-activated chloride currents in endothelial cells by
chromones. Br J Pharmacol 115: 1393–1398, 1995.
HELDIN CH. Dimerization of cell surface receptors in signal transduction. Cell 80: 213–223, 1995.
HEWETT PW, MURRAY JC, PRICE EA, WATTS ME, AND WOODCOCK M.
Isolation and characterization of microvessel endothelial cells from
human mammary adipose tissue. In Vitro Cell Dev Biol 29: 325–331,
1993.
HIMMEL HM, RASMUSSON RL, AND STRAUSS HC. Agonist-induced
changes of [Ca2⫹]i and membrane currents in single bovine aortic
endothelial cells. Am J Physiol Cell Physiol 267: C1338 –C1350,
1994.
HIMMEL HM, WHORTON AR, AND STRAUSS HC. Intracellular calcium,
currents, and stimulus response coupling in endothelial cells. Hypertension 21: 112–127, 1993.
HOEBEL BG, KOSTNER GM, AND GRAIER WF. Activation of microsomal
cytochrome P-450 mono-oxygenase by Ca2⫹ store depletion and its
contribution to Ca2⫹ entry in porcine aortic endothelial cells. Br J
Pharmacol 121: 1579 –1588, 1997.
HOENDEROP JG, VAN DER KEMP AW, HARTOG A, VAN DE GRAAF SF, VAN
OS CH, WILLEMS PH, AND BINDELS RJ. Molecular identification of the
apical Ca2⫹ channel in 1,25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem 274: 8375– 8378, 1999.
HOFMANN T, OBUKHOV AG, SCHAEFER M, HARTENECK C, GUDERMANN T,
AND SCHULTZ G. Direct activation of human TRPC6 and TRPC3
channels by diacylglycerol. Nature 397: 259 –263, 1999.
HOFMANN T, SCHAEFER M, SCHULTZ G, AND GUDERMANN T. Transient
receptor potential channels as molecular substrates of receptormediated cation entry. J Mol Med 78: 14 –25, 2000.
HOGG DS, ALBARWANI S, DAVIES ARL, AND KOZLOWSKI RZ. Endothelial
cells freshly isolated from resistance-sized pulmonary arteries possess a unique K⫹ current profile. Biochem Biophys Res Commun
263: 405– 409, 1999.
HOLDA JR, KLISHIN A, SEDOVA M, HÜSER J, AND BLATTER LA. Capacitative calcium entry. News Physiol Sci 13: 157–163, 1998.
HOTH M, FASOLATO C, AND PENNER R. Ion channels and calcium
signaling in mast cells. Ann NY Acad Sci 707: 198 –209, 1993.
HOTH M AND PENNER R. Depletion of intracellular calcium stores
activates a calcium current in mast cells. Nature 355: 353–356,
1992.
HOYER J, DISTLER A, HAASE W, AND GÖGELEIN H. Ca2⫹ influx through
stretch-activated cation channels activates maxi K⫹ channels in
porcine endocardial endothelium. Proc Natl Acad Sci USA 91:
2367–2371, 1994.
HOYER J, KÖHLER R, AND DISTLER A. Mechanosensitive cation channels in aortic endothelium of normotensive and hypertensive rats.
Hypertension 30: 112–119, 1997.
HOYER J, KÖHLER R, HAASE W, AND DISTLER A. Up-regulation of
pressure-activated Ca2⫹-permeable cation channel in intact vascu-
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
113.
neutrophils activate endothelial myosin light chain kinase: role in
transendothelial migration. J Appl Physiol 84: 1817–1821, 1998.
GARCIA RL AND SCHILLING WP. Differential expression of mammalian
TRP homologues across tissues and cell lines. Biochem Biophys
Res Commun 239: 279 –283, 1997.
GERICKE M, DROOGMANS G, AND NILIUS B. Thapsigargin discharges
intracellular calcium stores and induces transmembrane currents
in human endothelial cells. Pflügers Arch 422: 552–557, 1993.
GERICKE M, DROOGMANS G, AND NILIUS B. thimerosal induced changes
of intracellular calcium in human endothelial cells. Cell Calcium
14: 201–207, 1993.
GERICKE M, OIKE M, DROOGMANS G, AND NILIUS B. Inhibition of
capacitative Ca2⫹ entry by a Cl⫺ channel blocker in human endothelial cells. Eur J Pharmacol 269: 381–384, 1994.
GHIANI CA, YUAN XQ, EISEN AM, KNUTSON PL, DEPINHO RA, MCBAIN
CJ, AND GALLO V. Voltage-activated K⫹ channels and membrane
depolarization regulate accumulation of the cyclin-dependent kinase inhibitors p277(Kip1) and p21(CIP1) in glial progenitor cells.
J Neurosci 19: 5380 –5392, 1999.
GIANGIACOMO KM, KAMASSAH A, HARRIS G, AND MCMANUS OB. Mechanism of maxi-K channel activation by dehydrosoyasaponin-I.
J Gen Physiol 112: 485–501, 1998.
GORDIENKO DV AND TSUKAHARA H. Tetrodotoxin-blockable depolarization-activated Na⫹ currents in a cultured endothelial cell line
derived from rat interlobar artery and human umbilical vein.
Pflügers Arch 428: 91–93, 1994.
GOSINK EC AND FORSBERG EJ. Effects of ATP and bradykinin on
endothelial cell Ca2⫹ homeostasis and formation of cGMP and
prostacyclin. Am J Physiol Cell Physiol 265: C1620 –C1629, 1993.
GRAIER WF, GROSCHNER K, SCHMIDT K, AND KUKOVETZ WR. SK&F
96365 inhibits histamine-induced formation of endothelium-derived
relaxing factor in human endothelial cells. Biochem Biophys Res
Commun 186: 1539 –1545, 1992.
GRAIER WF, KUKOVETZ WR, AND GROSCHNER K. Cyclic AMP enhances
agonist-induced Ca2⫹ entry into endothelial cells by activation of
potassium channels and membrane hyperpolarization. Biochem J
291: 263–267, 1993.
GRAIER WF, PALTAUF DOBURZYNSKA J, HILL BJ, FLEISCHHACKER E,
HOEBEL BG, KOSTNER GM, AND STUREK M. Submaximal stimulation of
porcine endothelial cells causes focal Ca2⫹ elevation beneath the
cell membrane. J Physiol (Lond) 506: 109 –125, 1998.
GRAIER WF, SIMECEK S, AND STUREK M. Cytochrome P-450 monooxygenase-regulated signalling of Ca2⫹ entry in human and bovine
endothelial cells. J Physiol (Lond) 482: 259 –274, 1995.
GRAIER WF, STUREK M, AND KUKOVETZ WR. Ca2⫹ regulation and
endothelial vascular function. Endothelium 1: 223–236, 1994.
GRIBKOFF VK, LUM RAGAN JT, BOISSARD CG, POST MUNSON DJ, MEANWELL NA, STARRETT JE JR, KOZLOWSKI ES, ROMINE JL, TROJNACKI JT,
MCKAY MC, ZHONG J, AND DWORETZKY SI. Effects of channel modulators on cloned large-conductance calcium-activated potassium
channels. Mol Pharmacol 50: 206 –217, 1996.
GROSCHNER K, GRAIER WF, AND KUKOVETZ WR. Activation of a smallconductance Ca2⫹-dependent K⫹ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells. Biochim Biophys Acta 1137: 162–170, 1992.
GROSCHNER K, GRAIER WF, AND KUKOVETZ WR. Histamine induces K⫹,
Ca2⫹, and Cl⫺ currents in human vascular endothelial cells: role of
ionic currents in stimulation of nitric oxide biosynthesis. Circ Res
75: 304 –314, 1994.
GROSCHNER K, HINGEL S, LINTSCHINGER B, BALZER M, ROMANIN C, ZHU
X, AND SCHREIBMAYER W. Trp proteins form store-operated cation
channels in human vascular endothelial cells. FEBS Lett 437: 101–
106, 1998.
GROSCHNER K AND KUKOVETZ WR. Voltage-sensitive chloride channels of large conductance in the membrane of pig aortic endothelial
cells. Pflügers Arch 421: 209 –217, 1992.
GRUBER AD, ELBLE RC, JI HL, SCHREUR KD, FULLER CM, AND PAULI
BU. Genomic cloning, molecular characterization, and functional
analysis of human CLCA1, the first human member of the family of
Ca2⫹-activated Cl⫺ channel proteins. Genomics 54: 200 –214, 1998.
GRUBER AD AND PAULI BU. Molecular cloning and biochemical
characterization of a truncated, secreted member of the human
1451
1452
150.
151.
152.
153.
154.
155.
157.
158.
159.
160.
161.
162.
163.
164.
165.
166.
167.
168.
169.
170.
lar endothelium of hypertensive rats. Proc Natl Acad Sci USA 93:
11253–11258, 1996.
HOYER J, POPP R, MEYER J, GALLA HJ, AND GÖGELEIN H. Angiotensin
II, vasopressin and GTP␥S inhibit inward-rectifying K⫹ channels in
porcine cerebral capillary endothelial cells. J Membr Biol 123:
55– 62, 1991.
HUSER J AND BLATTER LA. Elementary events of agonist-induced
Ca2⫹ release in vascular endothelial cells. Am J Physiol Cell
Physiol 273: C1775–C1782, 1997.
HUTCHESON IR AND GRIFFITH TM. Heterogeneous populations of K⫹
channels mediate EDRF release to flow but not agonists in rabbit
aorta. Am J Physiol Heart Circ Physiol 266: H590 –H596, 1994.
HUTCHESON IR AND GRIFFITH TM. Central role of intracellular calcium stores in acute flow- and agonist-evoked endothelial nitric
oxide release. Br J Pharmacol 122: 117–125, 1997.
INAGAMI T, NARUSE M, AND HOOVER R. Endothelium: as an endocrine
organ. Annu Rev Physiol 57: 171–189, 1995.
INAZU M, ZHANG H, AND DANIEL EE. Lp-805, a releaser of endothelium-derived nitric oxide, activates an endothelial calcium permeable non-specific cation channel. Life Sci 53: 315–320, 1993.
INAZU M, ZHANG H, AND DANIEL EE. Properties of the Lp-805-induced
potassium currents in cultured bovine pulmonary artery endothelial cells. J Pharmacol Exp Ther 268: 403– 408, 1994.
IOUZALEN L, LANTOINE F, PERNOLLET MG, MILLANVOYE-VAN-BRUSSEL E,
DEVYNCK MA, AND DAVID-DUFILHO M. Sk&F 96365 inhibits intracellular Ca2⫹ pumps and raises cytosolic Ca2⫹ concentration without
production of nitric oxide and von Willebrand factor. Cell Calcium
20: 501–508, 1996.
ISHII TM, SILVIA C, HIRSCHBERG B, BOND CT, ADELMAN JP, AND MAYLIE
J. A human intermediate conductance calcium activated potassium
channel. Proc Natl Acad Sci USA 94: 11651–11656, 1997.
ISHIZAKA H AND KUO L. Endothelial ATP-sensitive potassium channels mediate coronary microvascular dilation to hyperosmolarity.
Am J Physiol Heart Circ Physiol 273: H104 –H112, 1997.
ISSHIKI M, ANDO J, KORENAGA R, KOGO H, FUJIMOTO T, FUJITA T, AND
KAMIYA A. Endothelial Ca2⫹ waves preferentially originate at specific loci in caveolin-rich cell edges. Proc Natl Acad Sci USA 95:
5009 –5014, 1998.
JACOB R. Agonist-stimulated divalent cation entry into single cultured human umbilical vein endothelial cells. J Physiol (Lond) 421:
55–77, 1990.
JACOB R, MERRITT JE, HALLAM TJ, AND RINK TJ. Repetitive spikes in
cytoplasmic calcium evoked by histamine in human endothelial
cells. Nature 335: 40 – 45, 1988.
JACOBS ER, CHELIAKINE C, GEBREMEDHIN D, BIRKS EK, DAVIES PF, AND
HARDER DR. Shear activated channels in cell-attached patches of
cultured bovine aortic endothelial cells. Pflügers Arch 431: 129 –
131, 1995.
JANIGRO D, WEST GA, GORDON EL, AND WINN HR. ATP-sensitive K⫹
channels in rat aorta and brain microvascular endothelial cells.
Am J Physiol Cell Physiol 265: C812–C821, 1993.
JANIGRO D, WEST GA, NGUYEN TS, AND WINN HR. Regulation of
blood-brain barrier endothelial cells by nitric oxide. Circ Res 75:
528 –538, 1994.
JANSSEN LJ, PREMJI M, LU-CHAO H, COX G, AND KESHAVJEE S. NO⫹ but
not NO radical relaxes airway smooth muscle via a cGMP-independent release of internal Ca2⫹. Am J Physiol 278: L899 –L905, 2000.
JENA M, MINORE JF, AND O’NEILL WC. A volume-sensitive, IP3-insensitive Ca2⫹ store in vascular endothelial cells. Am J Physiol Cell
Physiol 273: C316 –C322, 1997.
JENSEN BS, STRØBRÆK D, CHRISTOPHERSEN P, TDJØRGENSEN HANSEN C,
SILAHTAROGLU A, OLESEN S-P, AND AHRING PK. Characterization of the
cloned human intermediate-conductance Ca2⫹-activated K⫹ channel. Am J Physiol Cell Physiol 275: C848 –C856, 1998.
JIANG Q, MAK D, DEVIDAS S, SCHWIEBERT EM, BRAGIN A, ZHANG Y,
SKACH WR, GUGGINO WB, FOSKETT JK, AND ENGELHARDT JF. Cystic
fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor. J Cell Biol 143: 645– 657,
1998.
JOHNS A, LATEGAN TW, LODGE NJ, RYAN US, VAN BREEMEN C, AND
ADAMS DJ. Calcium entry through receptor-operated channels in
bovine pulmonary artery endothelial cells. Tissue Cell 19: 733–745,
1987.
Physiol Rev • VOL
171. JOW F AND NUMANN R. Fluid flow modulates calcium entry and
activates membrane currents in cultured human aortic endothelial
cells. J Membr Biol 171: 127–139, 1999.
172. JOW F AND NUMANN R. Histamine increases [Ca2⫹]in and activates
Ca-K and nonselective cation currents in cultured human capillary
endothelial cells. J Membr Biol 173: 107–116, 2000.
173. JOW F, SULLIVAN K, SOKOL P, AND NUMANN R. Induction of Ca2⫹
activated K⫹ current and transient outward currents in human
capillary endothelial cells. J Membr Biol 167: 53– 64, 1999.
174. KAJIMURA M AND CURRY FE. Endothelial cell shrinkage increases
permeability through a Ca2⫹-dependent pathway in single frog
mesenteric microvessels. J Physiol (Lond) 518: 227–238, 1999.
175. KAJIMURA M, O’DONNELL ME, AND CURRY FE. Effect of cell shrinkage
on permeability of cultured bovine aortic endothelia and frog mesenteric capillaries. J Physiol (Lond) 503: 413– 425, 1997.
176. KAMOUCHI M, DROOGMANS G, AND NILIUS B. Membrane potential as a
modulator of the free intracellular Ca2⫹ concentration in agonistactivated endothelial cells. Gen Physiol Biophys 18: 199 –208, 1999.
177. KAMOUCHI M, MAMIN A, DROOGMANS G, AND NILIUS B. Nonselective
cation channels in endothelial cells derived from human umbilical
vein. J Membr Biol 169: 29 –38, 1999.
178. KAMOUCHI M, PHILIPP S, FLOCKERZI V, WISSENBACH U, MAMIN A, RAEYMAEKERS L, EGGERMONT J, DROOGMANS G, AND NILIUS B. Properties of
heterologously expressed hTRP3 channels in bovine pulmonary
artery endothelial cells. J Physiol (Lond) 518: 345–358, 1999.
179. KAMOUCHI M, TROUET D, DEGREEF C, DROOGMANS G, EGGERMONT J,
2⫹
AND NILIUS B. Functional effects of expression of hslo Ca
activated K⫹ channels in cultured macrovascular endothelial cells. Cell
Calcium 22: 497–506, 1997.
180. KAMOUCHI M, VANDENBREMT K, EGGERMONT J, DROOGMANS G, AND
NILIUS B. Modulation of inwardly rectifying potassium channels in
cultured bovine pulmonary artery endothelial cells. J Physiol
(Lond) 504: 545–556, 1997.
181. KAN H, RUAN Y, AND MALIK KU. Signal transduction mechanism(s)
involved in prostacyclin production elicited by acetylcholine in
coronary endothelial cells of rabbit heart. J Pharmacol Exp Ther
282: 113–122, 1997.
182. KANDA K, HAYMAN GT, SILVERMAN MD, AND LELKES PI. Comparison of
ICAM-1 and VCAM-1 expression in various human endothelial cell
types and smooth muscle cells. Endothelium 6: 33– 44, 1998.
183. KANZAKI M, NAGASAWA M, KOJIMA I, SATO C, NARUSE K, SOKABE M, AND
IIDA H. Molecular identification of a eukaryotic, stretch-activated
nonselective cation channel. Science 285: 882– 886, 1999.
184. KATNIK C AND ADAMS DJ. An ATP-sensitive potassium conductance
in rabbit arterial endothelial cells. J Physiol (Lond) 485: 595– 606,
1995.
185. KATNIK C AND ADAMS DJ. Characterization of ATP-sensitive potassium channels in freshly dissociated rabbit aortic endothelial cells.
Am J Physiol Heart Circ Physiol 272: H2507–H2511, 1997.
186. KAWASAKI J, HIRANO K, HIRANO M, NISHIMURA J, FUJISHIMA M, AND
KANAIDE H. Troglitazone inhibits the capacitative Ca2⫹ entry in
endothelial cells. Eur J Pharmacol 373: 111–120, 1999.
187. KEEN JE, KHAWALED R, FARRENS DL, NEELANDS T, RIVARD A, BOND CT,
JANOWSKY A, FAKLER B, ADELMAN JP, AND MAYLIE J. Domains responsible for constitutive and Ca2⫹-dependent interactions between
calmodulin and small conductance Ca2⫹-activated potassium channels. J Neurosci 19: 8830 – 8838, 1999.
188. KERSCHBAUM HH AND CAHALAN MD. Single-channel recording of a
store-operated Ca2⫹ channel in Jurkat T lymphocytes. Science 283:
836 – 839, 1999.
189. KERSCHBAUM HH, NEGULESCU PA, AND CAHALAN MD. Ion channels,
Ca2⫹ signaling, and reporter gene expression in antigen-specific
mouse T cells. J Immunol 159: 1628 –1638, 1997.
190. KHANNA R, CHANG MC, JOINER WJ, KACZMAREK LK, AND SCHLICHTER
LC. hSK4/hIK1, a calmodulin-binding KCa channel in human T
lymphocytes. Roles in proliferation and volume regulation. J Biol
Chem 274: 14838 –14849, 1999.
191. KIMURA C, OIKE M, AND ITO Y. Acute glucose overload abolishes Ca2⫹
oscillation in cultured endothelial cells from bovine aorta: a possible role of superoxide anion. Circ Res 82: 677– 685, 1998.
192. KISELYOV K AND MUALLEM S. Fatty acids, diacylglycerol, Ins(1,4,5)P3
receptors and Ca2⫹ influx. Trends Neurosci 22: 334 –337, 1999.
193. KISELYOV K, XU X, MOZHAYEVA G, KUO T, PESSAH I, MIGNERY G, ZHU X,
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
156.
BERND NILIUS AND GUY DROOGMANS
ION CHANNELS IN VASCULAR ENDOTHELIUM
194.
195.
196.
197.
198.
200.
201.
202.
203.
204.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
Physiol Rev • VOL
216.
217.
218.
219.
220.
221.
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
234.
235.
WL. Inaf, a protein required for transient receptor potential Ca2⫹
channel function. Proc Natl Acad Sci USA 96: 13474 –13479, 1999.
LI S, CHEN BP, AZUMA N, HU YL, WU SZ, SUMPIO BE, SHYY JY, AND
CHIEN S. Distinct roles for the small GTPases Cdc42 and Rho in
endothelial responses to shear stress. J Clin Invest 103: 1141–1150,
1999.
LI W, LLOPIS J, WHITNEY M, ZLOKARNIK G, AND TSIEN RY. Cell-permeant caged InsP3 ester shows that Ca2⫹ spike frequency can
optimize gene expression. Nature 392: 936 –941, 1998.
LI Z, NIWA Y, SAKAMOTO S, CHEN X, AND NAKAYA Y. Estrogen modulates a large conductance chloride channel in cultured porcine
aortic endothelial cells. J Cardiovasc Pharmacol 35: 506 –510, 2000.
LIN S, FAGAN KA, LI K-X, SHAUL PW, COOPER DMF, AND RODMAN DM.
Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2⫹ entry. J Biol Chem 275: 17979 –17985, 2000.
LING BN AND O’NEILL WC. Ca2⫹-dependent and Ca2⫹-permeable ion
channels in aortic endothelial cells. Am J Physiol Heart Circ
Physiol 263: H1827–H1838, 1992.
LITTLE TL, BEYER EC, AND DULING BR. Connexin 43 and connexin 40
gap junctional proteins are present in arteriolar smooth muscle and
endothelium in vivo. Am J Physiol Heart Circ Physiol 268: H729 –
H739, 1995.
LIU LH, PAUL RJ, SUTLIFF RL, MILLER ML, LORENZ JN, PUN RY, DUFFY
JJ, DOETSCHMAN T, KIMURA Y, MACLENNAN DH, HOYING JB, AND SHULL
GE. Defective endothelium-dependent relaxation of vascular
smooth muscle and endothelial cell Ca2⫹ signaling in mice lacking
sarco(endo)plasmic reticulum Ca2⫹-ATPase isoform 3. J Biol
Chem 272: 30538 –30545, 1997.
LIU X, WANG W, SINGH BB, LOCKWICH T, JADLOWIEC J, OCB, WELLNER
R, ZHU MX, AND AMBUDKAR IS. Trp1, a candidate protein for the
store-operated Ca2⫹ influx mechanism in salivary gland cells.
J Biol Chem 275: 3403–3411, 2000.
LOCKWICH TP, LIU XB, SINGH BB, JADLOWIEC J, WEILAND S, AND AMBUDKAR IS. Assembly of Trp1 in a signaling complex associated with
caveolin-scaffolding lipid raft domains. J Biol Chem 275: 11934 –
11942, 2000.
LÜCKHOFF A AND BUSSE R. Activators of potassium channels enhance
calcium influx into endothelial cells as a consequence of potassium
currents. Naunyn-Schmiedebergs Arch Pharmacol 342: 94 –99,
1990.
LÜCKHOFF A AND BUSSE R. Calcium influx into endothelial cells and
formation of endothelium-derived relaxing factor is controlled by
the membrane potential. Pflügers Arch 416: 305–311, 1990.
LÜCKHOFF A AND CLAPHAM DE. Inositol 1,3,4,5-tetrakisphosphate
activates an endothelial Ca2⫹-permeable channel. Nature 355: 356 –
358, 1992.
LUDWIG A, ZONG X, JEGLITSCH M, HOFMANN F, AND BIEL M. A family of
hyperpolarization-activated mammalian cation channels. Nature
393: 587–591, 1998.
LUPU C, GOODWIN CA, WESTMUCKETT AD, EMEIS JJ, SCULLY MF, KAKKAR VV, AND LUPU F. Tissue factor pathway inhibitor in endothelial
cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium. Arterioscler Thromb Vasc Biol 17: 2964 –2974, 1997.
LUPU C, LUPU F, DENNEHY U, KAKKAR VV, AND SCULLY MF. Thrombin
induces the redistribution and acute release of tissue factor pathway inhibitor from specific granules within human endothelial cells
in culture. Arterioscler Thromb Vasc Biol 15: 2055–2062, 1995.
MAERTENS C, WEI L, VOETS T, DROOGMANS G, AND NILIUS B. Block by
fluoxetine of volume-regulated anion channels. Br J Pharmacol
126: 508 –514, 1999.
MALEK AM AND IZUMO S. Molecular aspects of signal transduction of
shear stress in the endothelial cell. J Hypertens 12: 989 –999, 1994.
MALLOUK N AND ALLARD B. Stretch-induced activation of Ca2⫹-activated K⫹ channels in mouse skeletal muscle fibers. Am J Physiol
Cell Physiol 278: C473–C479, 2000.
MANABE K, ITO H, MATSUDA H, AND NOMA A. Hyperpolarization induced by vasoactive substances in intact guinea-pig endocardial
endothelial cells. J Physiol (Lond) 484: 25– 40, 1995.
MANABE K, ITO H, MATSUDA H, NOMA A, AND SHIBATA Y. Classification
of ion channels in the luminal and abluminal membranes of guineapig endocardial endothelial cells. J Physiol (Lond) 484: 41–52,
1995.
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
199.
BIRNBAUMER L, AND MUALLEM S. Functional interaction between
InsP3 receptors and store-operated Htrp3 channels. Nature 396:
478 – 482, 1998.
KLISHIN A, SEDOVA M, AND BLATTER LA. Time-dependent modulation
of capacitative Ca2⫹ entry signals by plasma membrane Ca2⫹ pump
in endothelium. Am J Physiol Cell Physiol 274: C1117–C1128, 1998.
KÖHLER R, DISTLER A, AND HOYER J. Pressure-activated cation channel in intact rat endocardial endothelium. Cardiovasc Res 38: 433–
440, 1998.
KÖHLER R, PAUL M, DISTLER A, AND HOYER J. Expression of trp
channels and Ca2⫹ activated K⫹ channels in intact human endothelium (Abstract). FASEB J 13: A862, 1999.
KOHN EC, ALESSANDRO R, SPOONSTER J, WERSTO RP, AND LIOTTA LA.
Angiogenesis: role of calcium-mediated signal transduction. Proc
Natl Acad Sci USA 92: 1307–1311, 1995.
KOLIWAD SK, ELLIOTT SJ, AND KUNZE DL. Oxidized glutathione mediates cation channel activation in calf vascular endothelial cells
during oxidant stress. J Physiol (Lond) 495: 37– 49, 1996.
KOLIWAD SK, KUNZE DL, AND ELLIOTT SJ. Oxidant stress activates a
non-selective cation channel responsible for membrane depolarization in calf vascular endothelial cells. J Physiol (Lond) 491: 1–12,
1996.
KORTH RM, HIRAFUJI M, BENVENISTE J, AND RUSSO MARIE F. Human
umbilical vein endothelial cells: specific binding of platelet-activating factor and cytosolic calcium flux. Biochem Pharmacol 49:
1793–1799, 1995.
KRAPIVINSKY G, PU W, WICKMAN K, KRAPIVINSKY L, AND CLAPHAM DE.
pICln binds to a mammalian homolog of a yeast protein involved in
regulation of cell morphology. J Biol Chem 273: 10811–10814, 1998.
KUMAR S, WEST DC, AND AGER A. Heterogeneity in endothelial cells
from large vessels and microvessels. Differentiation 36: 57–70,
1987.
KUO L AND CHANCELLOR JD. Adenosine potentiates flow-induced
dilation of coronary arterioles by activating KATP channels in endothelium. Am J Physiol Heart Circ Physiol 269: H541–H549, 1995.
KWAN HY, HUANG Y, AND YAO X. Store-operated calcium entry in
vascular endothelial cells is inhibited by cGMP via a protein kinase
G-dependent mechanism. J Biol Chem 275: 6758 – 6763, 2000.
LAMONTAGNE D, POHL U, AND BUSSE R. Mechanical deformation of
vessel wall and shear stress determine the basal release of endothelium-derived relaxing factor in the intact rabbit coronary vascular bed. Circ Res 70: 123–130, 1992.
LAN Q, MERCURIUS KO, AND DAVIES PF. Stimulation of transcription
factors NF kappa B and AP1 in endothelial cells subjected to shear
stress. Biochem Biophys Res Commun 201: 950 –956, 1994.
LANGHEINRICH U AND DAUT J. Hyperpolarization of isolated capillaries from guinea-pig heart induced by K⫹ channel openers and
glucose deprivation. J Physiol (Lond) 502: 397– 408, 1997.
LANGHEINRICH U, MEDEROS Y SCHNITZLER M, AND DAUT J. Ca2⫹-transients induced by K⫹ channel openers in isolated coronary capillaries. Pflügers Arch 435: 435– 438, 1998.
LANSMAN JB, HALLAM TJ, AND RINK TJ. Single stretch-activated ion
channels in vascular endothelial cells as mechanotransducers? Nature 325: 811– 813, 1987.
LANTOINE F, IOUZALEN L, DEVYNCK MA, MILLANVOYE-VAN-BRUSSEL E,
AND DAVID-DUFILHO M. Nitric oxide production in human endothelial
cells stimulated by histamine requires Ca2⫹ influx. Biochem J 330:
695– 699, 1998.
LELKES PF (Editor). Mechanical Forces and the Endothelium. London: Harvard Academic, 1999, p. 33–54.
LEPPLE WIENHUES A, SZABO I, LAUN T, KABA NK, GULBINS E, AND LANG
F. The tyrosine kinase p56lck mediates activation of swellinginduced chloride channels in lymphocytes. J Cell Biol 141: 281–286,
1998.
LESH RE, MARKS AR, SOMLYO AV, FLEISCHER S, AND SOMLYO AP.
Anti-ryanodine receptor antibody binding sites in vascular and
endocardial endothelium. Circ Res 72: 481– 488, 1993.
LEUNG YM, KWAN CY, AND DANIEL EE. Block of inwardly rectifying
K⫹ currents by extracellular Mg2⫹ and Ba2⫹ in bovine pulmonary
artery endothelial cells. Can J Physiol Pharmacol 78: 751–756,
2000.
LI C, GENG C, LEUNG HT, HONG YS, STRONG LL, SCHNEUWLY S, AND PAK
1453
1454
BERND NILIUS AND GUY DROOGMANS
Physiol Rev • VOL
258.
259.
260.
261.
262.
263.
264.
265.
266.
267.
268.
269.
270.
271.
272.
273.
274.
275.
276.
277.
278.
279.
280.
through nitric oxide production in bovine coronary artery. FEBS
Lett 457: 375–380, 1999.
MONTELL C. Trp trapped in fly signaling web. Curr Opin Neurobiol
8: 389 –397, 1998.
MOORE TM, BROUGH GH, BABAL P, KELLY JJ, LI M, AND STEVENS T.
Store-operated calcium entry promotes shape change in pulmonary
endothelial cells expressing Trp1. Am J Physiol Lung Cell Mol
Physiol 275: L574 –L582, 1998.
MOORE TM, CHETHAM PM, KELLY JJ, AND STEVENS T. Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP. Am J Physiol Lung Cell Mol
Physiol 275: L203–L222, 1998.
MOTTE S, COMMUNI D, PIROTTON S, AND BOEYNAEMS JM. Involvement
of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells. Int J Biochem Cell Biol 27: 1–7,
1995.
MOUNTIAN I, MANOLOPOULOS VG, DE SMEDT H, PARYS JB, MISSIAEN L,
AND WUYTACK F. Expression patterns of sarco/endoplasmic reticulum Ca2⫹-ATPase and inositol 1,4,5-trisphosphate receptor isoforms in vascular endothelial cells. Cell Calcium 25: 371–380, 1999.
MULLER JM, DAVIS MJ, KUO L, AND CHILIAN WM. Changes in coronary
endothelial cell Ca2⫹ concentration during shear stress- and agonist-induced vasodilation. Am J Physiol Heart Circ Physiol 276:
H1706 –H1714, 1999.
MURAI T, MURAKI K, IMAIZUMI Y, AND WATANABE M. Levcromakalim
causes indirect endothelial hyperpolarization via a myo-endothelial
pathway. Br J Pharmacol 128: 1491–1496, 1999.
MURAKI K, IMAIZUMI Y, OHYA S, SATO K, TAKII T, ONOZAKI K, AND
WATANABE M. Apamin-sensitive Ca2⫹-dependent K⫹ current and
hyperpolarization in human endothelial cells. Biochem Biophys
Res Commun 236: 340 –343, 1997.
NAKAO M, ONO K, FUJISAWA S, AND IIJIMA T. Mechanical stressinduced Ca2⫹ entry and Cl⫺ current in cultured human aortic
endothelial cells. Am J Physiol 45: C238 –C249, 1999.
NELSON MT. Regulation of arterial tone by potassium channels. Jpn
J Pharmacol 58: P238 –P242, 1992.
NEYLON CB AND IRVINE RF. Synchronized repetitive spikes in cytoplasmic calcium in confluent monolayers of human umbilical vein
endothelial cells. FEBS Lett 275: 173–176, 1990.
NIGGEL J, SIGURDSON W, AND SACHS F. Mechanically induced calcium
movements in astrocytes, bovine aortic endothelial cells and C6
glioma cells. J Membr Biol 174: 121–134, 2000.
NILIUS B. Permeation properties of a non-selective cation channel in
human vascular endothelial cells. Pflügers Arch 416: 609 – 611,
1990.
NILIUS B. Regulation of transmembrane calcium fluxes in endothelium. News Physiol Sci 6: 110 –114, 1991.
NILIUS B. Signal transduction in vascular endothelium: the role of
ion channels and calcium. Verhandelingen Koninklijk Academie
voor Geneeskunde 60: 215–250, 1998.
NILIUS B AND CASTEELS R. Biology of the vascular wall and its
interaction with migratory and blood cells. In: Comprehensive
Human Physiology, edited by Greger R and Windhorst U. Berlin:
Springer-Verlag, 1996, p. 1981–1994.
NILIUS B AND DROOGMANS G. Ion channels of endothelial cells. In:
Physiology and Pathophysiology of the Heart, edited by Sperelakis
N. London: Kluwer Academic, 1995, p. 961–973.
NILIUS B AND DROOGMANS G. A role for K⫹ channels in cell proliferation. News Physiol Sci 9: 105–110, 1994.
NILIUS B, DROOGMANS G, GERICKE M, AND SCHWARTZ G. Non-selective
ion pathways in human endothelial cells. In: Nonselective Cation
Channels, Physiology and Biophysics, edited by Siemen D and
Hescheler JD. Basel: Birkhäuser Verlag, 1993, p. 269 –280.
NILIUS B, EGGERMONT J, AND DROOGMANS G. Chloride channels in
endothelium: the role of mechano-stimulation and changes in cell
volume. In: Mechanical Forces and the Endothelium, edited by
Lelkes PF. London: Harvard Academic, 1999, p. 33–54.
NILIUS B, EGGERMONT J, VOETS T, BUYSE G, MANOLOPOULOS VG, AND
DROOGMANS G. Properties of volume-regulated anion channels in
mammalian cells. Prog Biophys Mol Biol 68: 69 –119, 1997.
NILIUS B, EGGERMONT J, VOETS T, AND DROOGMANS G. Volume-activated Cl⫺-channels. Gen Pharmacol 27: 67–77, 1996.
NILIUS B, GERKE V, PRENEN J, SZÜCS G, HEINKE S, WEBER K, AND
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
236. MANABE K, TAKANO M, AND NOMA A. Non-selective cation current of
guinea-pig endocardial endothelial cells. J Physiol (Lond) 487:
407– 419, 1995.
237. MANOLOPOULOS VG, DROOGMANS G, AND NILIUS B. Hypotonicity and
thrombin activate taurine efflux in BC3H1 and C2C12 myoblasts that
is down regulated during differentiation. Biochem Biophys Res
Commun 232: 74 –79, 1997.
238. MANOLOPOULOS VG, LIEKENS S, KOOLWIJK P, PETERS E, DROOGMANS G,
LELKES P, DE CLERQ E, AND NILIUS B. Inhibition of angiogenesis by
blockers of volume regulated anion channels. Gen Physiol. In
press.
239. MARCHENKO SM AND SAGE SO. Electrical properties of resting and
acetylcholine-stimulated endothelium in intact rat aorta. J Physiol
(Lond) 462: 735–751, 1993.
240. MARCHENKO SM AND SAGE SO. Mechanism of acetylcholine action on
membrane potential of endothelium of intact rat aorta. Am J
Physiol Heart Circ Physiol 266: H2388 –H2395, 1994.
241. MARCHENKO SM AND SAGE SO. Calcium-activated potassium channels in the endothelium of intact rat aorta. J Physiol (Lond) 492:
53– 60, 1996.
242. MARCHENKO SM AND SAGE SO. Mechanosensitive cation channels
from endothelium of excised intact rat aorta (Abstract). Biophys J
70: A365, 1996.
243. MARCHENKO SM AND SAGE SO. A novel mechanosensitive cationic
channel from the endothelium of rat aorta. J Physiol (Lond) 498:
419 – 425, 1997.
244. MATHES C, FLEIG A, AND PENNER R. Calcium release-activated calcium current (ICRAC) is a direct target for sphingosine. J Biol Chem
273: 25020 –25030, 1998.
245. MCCARTHY SA, KUZU I, GATTER KC, AND BICKNELL R. Heterogeneity of
the endothelial cell and its role in organ preference of tumour
metastasis. Trends Pharmacol Sci 12: 462– 467, 1991.
246. MCINTOSH DP AND SCHNITZER JE. Caveolae require intact VAMP for
targeted transport in vascular endothelium. Am J Physiol Heart
Circ Physiol 277: H2222–H2232, 1999.
247. MCMANUS OB, HARRIS GH, GIANGIACOMO KM, FEIGENBAUM P, REUBEN
JP, ADDY ME, BURKA JF, KACZOROWSKI GJ, AND GARCIA ML. An
activator of calcium-dependent potassium channels isolated from a
medicinal herb. Biochemistry 32: 6128 – 6133, 1993.
248. MCMANUS OB, HELMS LM, PALLANCK L, GANETZKY B, SWANSON R, AND
LEONARD RJ. Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14: 645– 650,
1995.
249. MEDEROS Y SCHNITZLER M, DERST C, DAUT J, AND PREISIG-MÜLLER R.
ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart. J Physiol (Lond) 525: 307–317, 2000.
250. MEHRKE G, POHL U, AND DAUT J. Effects of vasoactive agonists on
the membrane potential of cultured bovine aortic and guinea-pig
coronary endothelium. J Physiol (Lond) 439: 277–299, 1991.
251. MENDELOWITZ D, BACAL K, AND KUNZE DL. Bradykinin-activated calcium influx pathway in bovine aortic endothelial cells. Am J
Physiol Heart Circ Physiol 262: H942–H948, 1992.
252. MICHEL JB, FERON O, SASE K, PRABHAKAR P, AND MICHEL T. Caveolin
versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase. J Biol Chem 272: 25907–25912,
1997.
253. MICHEL T AND FERON O. Nitric oxide synthases: which, where, how,
and why? J Clin Invest 100: 2146 –2152, 1997.
254. MIRSHAHI M, NICOLAS C, MIRSHAHI S, GOLESTANEH N, D’ HERMIES F, AND
AGARWAL M. Immunochemical analysis of the sodium channel in
rodent and human eye. Exp Eye Res 69: 21–32, 1999.
255. MISSIAEN L, LEMAIRE FX, PARYS JB, DE SMEDT H, SIENAERT I, AND
CASTEELS R. Initiation sites for Ca2⫹ signals in endothelial cells.
Pflügers Arch 431: 318 –324, 1996.
256. MIURA H, LIU Y, AND GUTTERMAN DD. Human coronary arteriolar
dilation to bradykinin depends on membrane hyperpolarization:
contribution of nitric oxide and Ca2⫹-activated K⫹ channels. Circulation 99: 3132–3138, 1999.
257. MOGAMI K, MIZUKAMI Y, TODOROKI IKEDA N, OHMURA M, YOSHIDA K,
MIWA S, MATSUZAKI M, MATSUDA M, AND KOBAYASHI S. Sphingosylphosphorylcholine induces cytosolic Ca2⫹ elevation in endothelial cells in situ and causes endothelium-dependent relaxation
ION CHANNELS IN VASCULAR ENDOTHELIUM
281.
282.
283.
284.
285.
287.
288.
289.
290.
291.
292.
293.
294.
295.
296.
297.
298.
299.
300.
301.
Physiol Rev • VOL
302.
303.
304.
305.
306.
307.
308.
309.
310.
311.
312.
313.
314.
315.
316.
317.
318.
319.
320.
321.
induces endothelial transforming growth factor beta-1 transcription and production. Modulation by potassium channel blockade.
J Clin Invest 95: 1363–1369, 1995.
OHNO M, GIBBONS GH, DZAU VJ, AND COOKE JP. Shear stress elevates
endothelial cGMP. Role of a potassium channel and G protein
coupling. Circulation 88: 193–197, 1993.
OIKE M, DROOGMANS G, CASTEELS R, AND NILIUS B. Electrogenic
Na⫹/K⫹-transport in human endothelial cells. Pflügers Arch 424:
301–307, 1993.
OIKE M, DROOGMANS G, AND NILIUS B. Amplitude modulation of Ca2⫹
signals induced by histamine in human endothelial cells. Biochim
Biophys Acta 1222: 287–291, 1994.
OIKE M, DROOGMANS G, AND NILIUS B. Mechanosensitive Ca2⫹ transients in endothelial cells from human umbilical vein. Proc Natl
Acad Sci USA 91: 2940 –2944, 1994.
OIKE M, GERICKE M, DROOGMANS G, AND NILIUS B. Calcium entry
activated by store depletion in human umbilical vein endothelial
cells. Cell Calcium 16: 367–376, 1994.
OKADA T, INOUE R, YAMAZAKI K, MAEDA A, KUROSAKI T, YAMAKUNI T,
TANAKA I, SHIMIZU S, IKENAKA K, IMOTO K, AND MORI Y. Molecular and
functional characterization of a novel mouse transient receptor
potential protein homologue TRP7. Ca2⫹-permeable cation channel
that is constitutively activated and enhanced by stimulation of G
protein-coupled receptor. J Biol Chem 274: 27359 –27370, 1999.
OKADA Y. Volume expansion-sensing outward-rectifier Cl⫺ channel:
fresh start to the molecular identity and volume sensor. Am J
Physiol Cell Physiol 273: C755–C789, 1997.
OKUDA M, TAKAHASHI M, SUERO J, MURRY CE, TRAUB O, KAWAKATSU H,
AND BERK BC. Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation. J Biol
Chem 274: 26803–26809, 1999.
OLESEN SP AND BUNDGAARD M. Chloride-selective channels of large
conductance in bovine aortic endothelial cells. Acta Physiol Scand
144: 191–198, 1992.
OLESEN SP AND BUNDGAARD M. ATP-dependent closure and reactivation of inward rectifier K⫹ channels in endothelial cells. Circ Res
73: 492– 495, 1993.
OLESEN SP, CLAPHAM DE, AND DAVIES PF. Haemodynamic shear
stress activates a K⫹ current in vascular endothelial cells. Nature
331: 168 –170, 1988.
OTUN H, AIDULIS DM, YANG JM, AND GILLESPIE JI. Interactions between inositol trisphosphate and Ca2⫹ dependent Ca2⫹ release
mechanisms on the endoplasmic reticulum of permeabilised bovine aortic endothelial cells. Cell Calcium 19: 315–325, 1996.
PALTAUF DOBURZYNSKA J, FRIEDEN M, SPITALER M, AND GRAIER WF.
Histamine-induced Ca2⫹ oscillations in a human endothelial cell
line depend on transmembrane ion flux, ryanodine receptors and
endoplasmic reticulum Ca2⫹-ATPase. J Physiol (Lond) 524: 701–
713, 2000.
PALTAUF DOBURZYNSKA J, POSCH K, PALTAUF G, AND GRAIER WF.
Stealth ryanodine-sensitive Ca2⫹ release contributes to activity of
capacitative Ca2⫹ entry and nitric oxide synthase in bovine endothelial cells. J Physiol (Lond) 513: 369 –379, 1998.
PAPASSOTIRIOU J, KOHLER R, PRENEN J, KRAUSE H, AKBAR M, EGGER⫹
MONT J, PAUL M, DISTLER A, NILIUS B, AND HOYER J. Endothelial K
2⫹
channel lacks the Ca sensitivity-regulating beta subunit. FASEB
J 14: 885– 894, 2000.
PAPASSOTIRIOU J, PRENEN J, KÖHLER R, KRAUSE H, EGGERMONT J, PAUL
M, DISTLER A, NILIUS B, AND HOYER J. Increased Ca2⫹-sensitivity of a
human Ca2⫹-activated K⫹ channels (BK) by expression of the
modulatory ␤-subunit in endothelial cells. Pflügers Arch 437: R65,
1999.
PAREKH AB AND PENNER R. Store depletion and calcium influx.
Physiol Rev 77: 901–930, 1997.
PARFENOVA H, HAFFNER J, AND LEFFLER CW. Phosphorylation-dependent stimulation of prostanoid synthesis by nigericin in cerebral
endothelial cells. Am J Physiol Cell Physiol 277: C728 –C738, 1999.
PARK H, GO YM, ST JOHN PL, MALAND MC, LISANTI MP, ABRAHAMSON
DR, AND JO H. Plasma membrane cholesterol is a key molecule in
shear stress-dependent activation of extracellular signal-regulated
kinase. J Biol Chem 273: 32304 –32311, 1998.
PARTON RG. Caveolae and caveolins. Curr Opin Cell Biol 8: 542–
548, 1996.
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
286.
DROOGMANS G. Annexin II modulates volume-activated chloride
currents in vascular endothelial cells. J Biol Chem 271: 30631–
30636, 1996.
NILIUS B, OIKE M, ZAHRADNIK I, AND DROOGMANS G. Activation of a Cl⫺
current by hypotonic volume increase in human endothelial cells.
J Gen Physiol 103: 787– 805, 1994.
NILIUS B, PRENEN J, AND DROOGMANS G. Modulation of volumeregulated anion channels by extra- and intracellular pH. Pflügers
Arch 436: 742–748, 1998.
NILIUS B, PRENEN J, KAMOUCHI M, VIANA F, VOETS T, AND DROOGMANS
G. Inhibition by mibefradil, a novel calcium channel antagonist, of
Ca2⫹- and volume-activated Cl⫺ channels in macrovascular endothelial cells. Br J Pharmacol 121: 547–555, 1997.
NILIUS B, PRENEN J, SZUCS G, WEI L, TANZI F, VOETS T, AND DROOGMANS
G. Calcium-activated chloride channels in bovine pulmonary artery
endothelial cells. J Physiol (Lond) 498: 381–396, 1997.
NILIUS B, PRENEN J, VOETS T, EGGERMONT J, BRUZIK KS, SHEARS SB,
AND DROOGMANS G. Inhibition by inositol tetrakisphosphates of
calcium- and volume-activated Cl⫺ currents in macrovascular endothelial cells. Pflügers Arch 435: 637– 644, 1998.
NILIUS B, PRENEN J, VOETS T, EGGERMONT J, AND DROOGMANS G.
Reduction of intracellular ionic strength activates the volume-regulated chloride current in cultured bovine pulmonary endothelial
cells. J Physiol (Lond) 506: 353–361, 1998.
NILIUS B, PRENEN J, VOETS T, VANDENBREMT K, EGGERMONT J, AND
DROOGMANS G. Kinetic and pharmacological properties of the calcium activated chloride current in macrovascular endothelial cells.
Cell Calcium 22: 53– 63, 1997.
NILIUS B, PRENEN J, WALSH M, BOLLEN M, DROOGMANS G, AND EGGERMONT J. Myosin light chain dependent modulation of volume-regulated anion channels (VRAC) in macrovascular endothelium. FEBS
Lett 466: 346 –350, 2000.
NILIUS B AND RIEMANN D. Ion channels in human endothelial cells.
Gen Physiol Biophys 9: 89 –112, 1990.
NILIUS B, SCHWARZ G, AND DROOGMANS G. Modulation by histamine of
an inwardly rectifying potassium channel in human endothelial
cells. J Physiol (Lond) 472: 359 –371, 1993.
NILIUS B, SCHWARZ G, OIKE M, AND DROOGMANS G. Histamine-activated, non-selective cation currents and Ca2⫹ transients in endothelial cells from human umbilical vein. Pflügers Arch 424: 285–293,
1993.
NILIUS B, SEHRER J, AND DROOGMANS G. Permeation properties and
modulation of volume-activated Cl⫺-currents in human endothelial
cells. Br J Pharmacol 112: 1049 –1056, 1994.
NILIUS B, SEHRER J, VIANA F, DEGREEF C, RAEYMAEKERS L, EGGERMONT
J, AND DROOGMANS G. Volume-activated Cl⫺ currents in different
mammalian non-excitable cell types. Pflügers Arch 428: 364 –371,
1994.
NILIUS B, SZÜCS G, HEINKE S, VOETS T, AND DROOGMANS G. Multiple
types of chloride channels in bovine pulmonary artery endothelial
cells. J Vasc Res 34: 220 –228, 1997.
NILIUS B, VIANA F, AND DROOGMANS G. Ion channels in vascular
endothelium. Annu Rev Physiol 59: 145–170, 1997.
NILIUS B, VIANA F, KAMOUCHI M, FASOLATO C, EGGERMONT J, AND
DROOGMANS G. Ca2⫹ signalling in endothelial cells: role of ion
channels. Kor J Physiol 2: 133–145, 1998.
NILIUS B, VOETS T, EGGERMONT J, AND DROOGMANS G. Vrac: a multifunctional volume-regulated anion channel in vascular endothelium. In: Chloride Channels, edited by Kozlowski R. Oxford, UK:
Isis Medical Media, 1999, p. 47– 63.
NILIUS B, VOETS T, PRENEN J, BARTH H, AKTORIES K, KAIBUCHI K,
DROOGMANS G, AND EGGERMONT J. Role of Rho and Rho kinase in the
activation of volume-regulated anion channels in bovine endothelial cells. J Physiol (Lond) 516: 67–74, 1999.
NOLL T, HOLSCHERMANN H, KOPREK K, GUNDUZ D, HABERBOSCH W,
TILLMANNS H, AND PIPER HM. ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2⫹]i. Am J
Physiol Heart Circ Physiol 276: H1892–H1901, 1999.
NORWOOD N, MOORE TM, DEAN DA, BHATTACHARJEE R, LI M, AND
STEVENS T. Store-operated calcium entry and increased endothelial
cell permeability. Am J Physiol Lung Cell Mol Physiol 279: L815–
L824, 2000.
OHNO M, COOKE JP, DZAU VJ, AND GIBBONS GH. Fluid shear stress
1455
1456
BERND NILIUS AND GUY DROOGMANS
Physiol Rev • VOL
343.
344.
345.
346.
347.
348.
349.
350.
351.
352.
353.
354.
355.
356.
357.
358.
359.
360.
361.
362.
363.
364.
stores releases a novel small messenger that stimulates Ca2⫹ influx.
Nature 364: 809 – 814, 1993.
REVEST PA AND ABBOTT NJ. Membrane ion channels of endothelial
cells. Trends Pharmacol Sci 13: 404 – 407, 1992.
RHODIN JAG. Architecture of the vessel wall. In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. Bethesda, MD: Am Physiol Soc, 1980, sect. 2, vol. II, chapt. 1, p. 1–31.
RIZZO V, SUNG A, OH P, AND SCHNITZER JE. Rapid mechanotransduction in situ at the luminal cell surface of vascular endothelium and
its caveolae. J Biol Chem 273: 26323–26329, 1998.
ROBERTSON BE, SCHUBERT R, HESCHELER J, AND NELSON MT. cGMPdependent protein kinase activates Ca-activated K channels in
cerebral artery smooth muscle cells. Am J Physiol Cell Physiol 265:
C299 –C303, 1993.
ROY G, BERNATCHEZ G, AND SAUVÉ R. Halide and alkyl phenols block
volume-sensitive chloride channels in human glial cells (U-138MG).
J Membr Biol 162: 191–200, 1998.
RUBANYI GM AND VANHOUTTE PM. Calcium and activation of the
release of endothelium-derived relaxing factor. Ann NY Acad Sci
522: 226 –233, 1988.
RUSKO J, TANZI F, VAN BREEMEN C, AND ADAMS DJ. Calcium-activated
potassium channels in native endothelial cells from rabbit aorta:
conductance, Ca2⫹ sensitivity and block. J Physiol (Lond) 455:
601– 621, 1992.
RUSKO J, VANSLOOTEN G, AND ADAMS DJ. Caffeine-evoked, calciumsensitive membrane currents in rabbit aortic endothelial cells. Br J
Pharmacol 115: 133–141, 1995.
SABIROV RZ, PRENEN J, DROOGMANS G, AND NILIUS B. Extra- and
intracellular proton-binding sites of volume-regulated anion channels. J Membr Biol. In press.
SAGE SO, VAN BREEMEN C, AND CANNELL MB. Sodium-calcium exchange in cultured bovine pulmonary artery endothelial cells.
J Physiol (Lond) 440: 569 –580, 1991.
SAKAI T. Acetylcholine induces Ca-dependent K currents in rabbit
endothelial cells. Jpn J Pharmacol 53: 235–246, 1990.
SASAJIMA H, WANG X, AND VAN BREEMEN C. Fractional Ca2⫹ release
from the endoplasmic reticulum activates Ca2⫹ entry in freshly
isolated rabbit aortic endothelial cells. Biochem Biophys Res Commun 241: 471– 475, 1997.
SAUVÉ R, CHAHINE M, TREMBLAY J, AND HAMET P. Single-channel
analysis of the electrical response of bovine aortic endothelial cells
to bradykinin stimulation: contribution of a Ca2⫹-dependent K⫹
channel. J Hypertens 8 Suppl: S193–S201, 1990.
SAUVÉ R, PARENT L, SIMONEAU C, AND ROY G. External ATP triggers a
biphasic activation process of a calcium-dependent K⫹ channel in
cultured bovine aortic endothelial cells. Pflügers Arch 412: 469 –
481, 1988.
SCHAEFER M, PLANT TD, OBUKHOV AG, HOFMANN T, GUDERMANN T, AND
SCHULTZ G. Receptor-mediated regulation of the nonselective cation
channels TRPC4 and TRPC5. J Biol Chem 275: 17517–17526, 2000.
SCHILLING WP, CABELLO OA, AND RAJAN L. Depletion of the inositol
1,4,5-trisphosphate-sensitive intracellular Ca2⫹ store in vascular
endothelial cells activates the agonist-sensitive Ca2⫹-influx pathway. Biochem J 284: 521–530, 1992.
SCHILLING WP, RAJAN L, AND STROBL JAGER E. Characterization of the
bradykinin-stimulated calcium influx pathway of cultured vascular
endothelial cells. Saturability, selectivity, and kinetics. J Biol Chem
264: 12838 –12848, 1989.
SCHNITZER JE, LIU J, AND OH P. Endothelial caveolae have the
molecular transport machinery for vesicle budding, docking, and
fusion including VAMP, NSF, SNAP, annexins, and GTPases. J Biol
Chem 270: 14399 –14404, 1995.
SCHNITZER JE, OH P, JACOBSON BS, AND DVORAK AM. Caveolae from
luminal plasmalemma of rat lung endothelium: microdomains enriched in caveolin, Ca2⫹-ATPase, and inositol trisphosphate receptor. Proc Natl Acad Sci USA 92: 1759 –1763, 1995.
SCHREIBER M AND SALKOFF L. A novel calcium-sensing domain in the
BK channel. Biophys J 73: 1355–1363, 1997.
SCHWARTZ MA, BROWN EJ, AND FAZELI B. A 50-kDa integrin-associated protein is required for integrin-regulated calcium entry in
endothelial cells. J Biol Chem 268: 19931–19934, 1993.
SCHWARZ G, CALLEWAERT G, DROOGMANS G, AND NILIUS B. Shear
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
322. PASYK E, MAO YK, AHMAD S, SHEN SH, AND DANIEL EE. An endothelial
cell-line contains functional vasoactive intestinal polypeptide receptors: they control inwardly rectifying K⫹ channels. Eur J Pharmacol 212: 209 –214, 1992.
323. PATEL V, BROWN C, GOODWIN A, WILKIE N, AND BOARDER MR. Phosphorylation and activation of p42 and p44 mitogen-activated protein kinase are required for the P2 purinoceptor stimulation of
endothelial prostacyclin production. Biochem J 320: 221–226, 1996.
324. PEIRETTI F, ALESSI MC, HENRY M, ANFOSSO F, JUHAN VAGUE I, AND
NALBONE G. Intracellular calcium mobilization suppresses the TNFalpha-stimulated synthesis of PAI-1 in human endothelial cells.
Indications that calcium acts at a translational level. Arterioscler
Thromb Vasc Biol 17: 1550 –1560, 1997.
325. PENNEFATHER PS AND DECOURSEY TE. A scheme to account for the
effects of Rb⫹ and K⫹ on inward rectifier K channels of bovine
artery endothelial cells. J Gen Physiol 103: 549 –581, 1994.
326. PEPPER MS AND MEDA P. Basic fibroblast growth factor increases
junctional communication and connexin 43 expression in microvascular endothelial cells. J Cell Physiol 153: 196 –205, 1992.
327. PEPPER MS, MONTESANO R, EL AOUMARI A, GROS D, ORCI L, AND MEDA
P. Coupling and connexin 43 expression in microvascular and large
vessel endothelial cells. Am J Physiol Cell Physiol 262: C1246 –
C1257, 1992.
328. PFAU S, LEITENBERG D, RINDER H, SMITH BR, PARDI R, AND BENDER JR.
Lymphocyte adhesion-dependent calcium signaling in human endothelial cells. J Cell Biol 128: 969 –978, 1995.
329. PHILIPP S, CAVALIÉ A, FREICHEL M, WISSENBACH U, ZIMMER S, TROST C,
MARQUART A, MURAKAMI M, AND FLOCKERZI V. A mammalian capacitative calcium entry channel homologous to Drosophila TRP and
TRPL. EMBO J 15: 6166 – 6171, 1996.
330. PHILIPP S AND FLOCKERZI V. Molecular characterization of a novel
human PDZ domain protein with homology to INAD from Drosophila melanogaster. FEBS Lett 413: 243–248, 1997.
331. PHILIPP S, HAMBRECHT J, BRASLAVSKI L, SCHROTH G, FREICHEL M,
MURAKAMI M, CAVALIE A, AND FLOCKERZI V. A novel capacitative
calcium entry channel expressed in excitable cells. EMBO J 17:
4274 – 4282, 1998.
332. PHILIPP S, TROST C, WARNAT J, RAUTMANN J, HIMMERKUS N, SCHROTH G,
KRETZ O, NASTAINCZYK W, CAVALIÉ A, HOTH M, AND FLOCKERZI V. Trp4
(CCe1) protein is part of native CRAC-like channels in adrenal
cells. J Biol Chem. In press.
333. PHILIPP S, WISSENBACH U, AND FLOCKERZI V. Calcium signaling. In:
Molecular Biology of Calcium Channels, edited by Putney JW Jr.
Boca Raton, FL: CRC, 2000, p. 321–342.
334. POPP R, BAUERSACHS J, HECKER M, FLEMING I, AND BUSSE R. A transferable, ␤-naphthoflavone-inducible, hyperpolarizing factor is synthesized by native and cultured porcine coronary endothelial cells.
J Physiol (Lond) 497: 699 –709, 1996.
335. POPP R, HOYER J, MEYER J, GALLA HJ, AND GÖGELEIN H. Stretchactivated non-selective cation channels in the antiluminal membrane of porcine cerebral capillaries. J Physiol (Lond) 454: 435–
449, 1992.
336. PRABHAKAR P, THATTE HS, GOETZ RM, CHO MR, GOLAN DE, AND
MICHEL T. Receptor-regulated translocation of endothelial nitricoxide synthase. J Biol Chem 273: 27383–27388, 1998.
337. PREISIG MULLER R, MEDEROS Y SCHNITZLER M, DERST C, AND DAUT J.
Separation of cardiomyocytes and coronary endothelial cells for
cell-specific RT-PCR. Am J Physiol Heart Circ Physiol 277: H413–
H416, 1999.
338. PU WT, KRAPIVINSKY GB, KRAPIVINSKY L, AND CLAPHAM DE. pICln
inhibits snRNP biogenesis by binding core spliceosomal proteins.
Mol Cell Biol 19: 4113– 4120, 1999.
339. PUTNEY JW. Trp, inositol 1,4,5-triphosphate receptors, and capacitative calcium entry. Proc Natl Acad Sci USA 96: 14669 –14671,
1999.
340. QUEYROY A AND VERDETTI J. Cooperative gating of chloride channel
subunits in endothelial cells. Biochim Biophys Acta 1108: 159 –168,
1992.
341. QUINN TP, PETERS KG, DE VRIES C, FERRARA N, AND WILLIAMS LT.
Fetal liver kinase 1 is a receptor for vascular endothelial growth
factor and is selectively expressed in vascular endothelium. Proc
Natl Acad Sci USA 90: 7533–7537, 1993.
342. RANDRIAMAMPITA C AND TSIEN RY. Emptying of intracellular Ca2⫹
ION CHANNELS IN VASCULAR ENDOTHELIUM
365.
366.
367.
368.
369.
370.
372.
373.
374.
375.
376.
377.
378.
379.
380.
381.
382.
383.
384.
385.
386.
Physiol Rev • VOL
387.
388.
389.
390.
391.
392.
393.
394.
395.
396.
397.
398.
399.
400.
401.
402.
403.
404.
405.
406.
407.
Human vascular endothelium heterogeneity. A comparative study
of cerebral and peripheral cultured vascular endothelial cells.
Stroke 28: 375–381, 1997.
TOMITA Y, KANEKO S, FUNAYAMA M, KONDO H, SATOH M, AND AKAIKE A.
Intracellular Ca2⫹ store-operated influx of Ca2⫹ through TRP-R, a
rat homolog of TRP, expressed in Xenopus oocytes. Neurosci Lett
248: 195–198, 1998.
TOUSSON A, VAN TINE BA, NAREN AP, SHAW GM, AND SCHWIEBERT LM.
Characterization of CFTR expression and chloride channel activity
in human endothelia. Am J Physiol Cell Physiol 275: C1555–C1564,
1998.
TRACEY WR AND PEACH MJ. Differential muscarinic receptor mRNA
expression by freshly isolated and cultured bovine aortic endothelial cells. Circ Res 70: 234 –240, 1992.
TRAUB O, ISHIDA T, ISHIDA M, TUPPER JC, AND BERK BC. Shear stressmediated extracellular signal-regulated kinase activation is regulated by sodium in endothelial cells: potential role for a voltagedependent sodium channel. J Biol Chem 274: 20144 –20150, 1999.
TROUET D, NILIUS B, JACOBS A, REMACLE C, DROOGMANS G, AND EGGERMONT J. Caveolin-1 modulates the activity of volume-regulated
chloride channels. J Physiol (Lond) 520: 113–119, 1999.
TSENG CRANK J, FOSTER CD, KRAUSE JD, MERTZ R, GODINOT N, DICHIARA TJ, AND REINHART PH. Cloning, expression, and distribution of
functionally distinct Ca2⫹-activated K⫹ channel isoforms from human brain. Neuron 13: 1315–1330, 1994.
UMEDA F, MASAKADO M, TAKEI A, YAMAUCHI T, SEKIGUCHI N, HASHIMOTO T, AND NAWATA H. Difference in serum-induced prostacyclin
production by cultured aortic and capillary endothelial cells. Prostaglandins Leukotrienes Essent Fatty Acids 56: 51–55, 1997.
VACA L. Calmodulin inhibits calcium influx current in vascular
endothelium. FEBS Lett 390: 289 –293, 1996.
VACA L. SITS blockade induces multiple subconductance states in a
large conductance chloride channel. J Membr Biol 169: 65–73,
1999.
VACA L, GURROLA GB, POSSANI LD, AND KUNZE DL. Blockade of a KCa
channel with synthetic peptides from noxiustoxin: a K⫹ channel
blocker. J Membr Biol 134: 123–129, 1993.
VACA L AND KUNZE DL. cAMP-dependent phosphorylation modulates
voltage gating in an endothelial Cl⫺ channel. Am J Physiol Cell
Physiol 264: C370 –C375, 1993.
VACA L AND KUNZE DL. Depletion and refilling of intracellular Ca2⫹
stores induce oscillations of Ca2⫹ current. Am J Physiol Heart Circ
Physiol 264: H1319 –H1322, 1993.
VACA L AND KUNZE DL. Depletion of intracellular Ca2⫹ stores activates a Ca2⫹-selective channel in vascular endothelium. Am J
Physiol Cell Physiol 3267: C920 –C925, 1994.
VACA L AND KUNZE DL. IP3-activated Ca2⫹ channels in the plasma
membrane of cultured vascular endothelial cells. Am J Physiol Cell
Physiol 269: C733–C738, 1995.
VACA L, SCHILLING WP, AND KUNZE DL. G-protein-mediated regulation
of a Ca2⫹-dependent K⫹ channel in cultured vascular endothelial
cells. Pflügers Arch 422: 66 –74, 1992.
VALENTIN S AND SCHOUSBOE I. Factor Xa enhances the binding of
tissue factor pathway inhibitor to acidic phospholipids. Thromb
Haemost 75: 796 – 800, 1996.
VANDORPE DH, SHMUKLER BE, JIANG L, LIM B, MAYLIE J, ADELMAN JP,
DE FRANCESCHI L, CAPPELLINI MD, BRUGNARA C, AND ALPER SL. cDNA
cloning and functional characterization of the mouse Ca2⫹-gated
K⫹ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation. J Biol Chem 273: 21542–21553, 1998.
VAN RENTERGHEM C, VIGNE P, AND FRELIN C. A charybdotoxin-sensitive, Ca2⫹-activated K⫹ channel with inward rectifying properties
in brain microvascular endothelial cells: properties and activation
by endothelins. J Neurochem 65: 1274 –1281, 1995.
VAN RIJEN H, VAN KEMPEN MJ, ANALBERS LJ, ROOK MB, VAN GINNEKEN
AC, GROS D, AND JONGSMA HJ. Gap junctions in human umbilical
cord endothelial cells contain multiple connexins. Am J Physiol
Cell Physiol 272: C117–C130, 1997.
VAN RIJEN HV, VAN KEMPEN MJ, POSTMA S, AND JONGSMA HJ. Tumour
necrosis factor alpha alters the expression of connexin43, connexin40, and connexin37 in human umbilical vein endothelial cells.
Cytokine 10: 258 –264, 1998.
VARGAS FF, CAVIEDES PF, AND GRANT DS. Electrophysiological char-
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
371.
stress-induced calcium transients in endothelial cells from human
umbilical cord veins. J Physiol (Lond) 458: 527–538, 1992.
SCHWARZ G, DROOGMANS G, AND NILIUS B. Shear stress induced
membrane currents and calcium transients in human vascular endothelial cells. Pflügers Arch 421: 394 –396, 1992.
SCHWARZ G, DROOGMANS G, AND NILIUS B. Multiple effects of SK&F
96365 on ionic currents and intracellular calcium in human endothelial cells. Cell Calcium 15: 45–54, 1994.
SCHWIEBERT EM. ABC transporter-facilitated ATP conductive transport. Am J Physiol Cell Physiol 276: C1–C8, 1999.
SCHWIEBERT EM, BENOS DJ, EGAN ME, STUTTS MJ, AND GUGGINO WB.
CFTR is a conductance regulator as well as a chloride channel.
Physiol Rev 79 Suppl: S145–S166, 1999.
SEDOVA M AND BLATTER LA. Dynamic regulation of [Ca2⫹]i by plasma
membrane Ca2⫹-ATPase and Na⫹/Ca2⫹ exchange during capacitative Ca2⫹ entry in bovine vascular endothelial cells. Cell Calcium
25: 333–343, 1999.
SEIß-GEUDER M, MEHRKE G, AND DAUT J. Sustained hyperpolarization
of cultured guinea-pig endothelila cells induced by adenosine.
J Cardiovasc Pharmacol 20: S97–S100, 1992.
SHAUL PW AND ANDERSON RG. Role of plasmalemmal caveolae in
signal transduction. Am J Physiol Lung Cell Mol Physiol 275:
L843–L851, 1998.
SILVER MR AND DECOURSEY TE. Intrinsic gating of inward rectifier in
bovine pulmonary artery endothelial cells in the presence or absence of internal Mg2⫹. J Gen Physiol 96: 109 –133, 1990.
SILVER MR, SHAPIRO MS, AND DECOURSEY TE. Effects of external Rb⫹
on inward rectifier K⫹ channels of bovine pulmonary artery endothelial cells. J Gen Physiol 103: 519 –548, 1994.
SOHN-Y, KELLER M, GLOE T, MORAWIETZ H, RUECKSCHLOSS U, AND POHL
U. The small G-protein Rac mediates depolarization-induced superoxide formation in human endothelial cells. J Biol Chem 275:
18745–18750, 2000.
STERN D, BRETT J, HARRIS K, AND NAWROTH P. Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the
synthesis and release of protein S. J Cell Biol 102: 1971–1978, 1986.
STORME L, RAIRIGH RL, PARKER TA, CORNFIELD DN, KINSELLA JP, AND
ABMAN SH. K⫹-channel blockade inhibits shear stress-induced pulmonary vasodilation in the ovine fetus. Am J Physiol Lung Cell Mol
Physiol 276: L220 –L228, 1999.
STRANGE K, EMMA F, AND JACKSON PS. Cellular and molecular physiology of volume-sensitive anion channels. Am J Physiol Cell
Physiol 270: C711–C730, 1996.
STUREK M, SMITH P, AND STEHNO-BITTEL L. In vitro models of vascular
endothelial cell calcium regulation. In: Ion Channels in Vascular
Smooth Muscle and Endothelium, edited by Sperelakis HKN. New
York: Elsevier, 1991, p. 349 –364.
SUAREZ J, TORRES C, SANCHEZ L, DEL VALLE L, AND PASTELIN G. Flow
stimulates nitric oxide release in guinea pig heart: role of stretchactivated ion channels. Biochem Biophys Res Commun 261: 6 –9,
1999.
SUH SH, DROOGMANS G, AND NILIUS B. Effects of cyanide and deoxyglucose on Ca2⫹ signalling in macrovascular endothelial cells. Endothelium 7: 155–168, 2000.
SUH SH, VENNEKENS R, MANOLOPOULOS VG, FREICHEL M, SCHWEIG U,
PRENEN J, FLOCKERZI V, DROOGMANS G, AND NILIUS B. Characterisation
of explanted endothelial cells from mouse aorta: electrophysiology
and Ca2⫹ signalling. Pflügers Arch 438: 612– 620, 1999.
TAKEDA K, SCHINI V, AND STOECKEL H. Voltage-activated potassium,
but not calcium currents in cultured bovine aortic endothelial cells.
Pflügers Arch 410: 385–393, 1987.
TANAKA Y, MEERA P, SONG M, KNAUS HG, AND TORO L. Molecular
constituents of maxi KCa channels in human coronary smooth
muscle: predominant alpha ⫹ beta subunit complexes. J Physiol
(Lond) 502: 545–557, 1997.
TANIGUCHI J, FURUKAWA KI, AND SHIGEKAWA M. Maxi K⫹ channels are
stimulated by cyclic guanosine monophosphate-dependent protein
kinase in canine coronary artery smooth muscle cells. Pflügers
Arch 423: 167–172, 1993.
TEUBL M, GROSCHNER K, KOHLWEIN SD, MAYER B, AND SCHMIDT K.
Na⫹/Ca2⫹ exchange facilitates Ca2⫹-dependent activation of endothelial nitric-oxide synthase. J Biol Chem 274: 29529 –29535, 1999.
THORIN E, SHATOS MA, SHREEVE SM, WALTERS CL, AND BEVAN JA.
1457
1458
408.
409.
410.
411.
413.
415.
416.
417.
418.
419.
421.
422.
423.
424.
425.
426.
427.
428.
429.
acteristics of cultured human umbilical vein endothelial cells. Microvasc Res 47: 153–165, 1994.
VENNEKENS R, HOENDEROP JG-J, PRENEN J, STUIVER M, WILLEMS
PHGM, DROOGMANS G, NILIUS B, AND BINDELS RJM. Permeation and
gating properties of the novel epithelial Ca2⫹ channel, ECaC. J Biol
Chem 275: 3963–3969, 2000.
VENNEKENS R, KAMOUCHI M, WISSENBACH U, PHILLIP S, EGGERMONT J,
DROOGMANS G, FLOCKERZI V, AND NILIUS B. Functional expression of
Trp1 and Trp4 in vascular endothelium. Pflügers Arch 437: R42,
1999.
VENNEKENS R, TROUET D, VANKEERBERGHEN A, VOETS T, CUPPENS H,
EGGERMONT J, CASSIMAN JJ, DROOGMANS G, AND NILIUS B. Inhibition of
volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator. J Physiol (Lond) 515:
75– 85, 1999.
VERGARA C, LATORRE R, MARRION NV, AND ADELMAN JP. Calciumactivated potassium channels. Curr Opin Neurobiol 8: 321–329,
1998.
VERIN AD, GILBERT MCCLAIN LI, PATTERSON CE, AND GARCIA JGN.
Biochemical regulation of the nonmuscle myosin light chain kinase
isoform in bovine endothelium. Am J Respir Cell Mol Biol 19:
767–776, 1998.
VERIN AD, LAZAR V, TORRY RJ, LABARRERE CA, PATTERSON CE, AND
GARCIA JG. Expression of a novel high-molecular-weight myosin
light chain kinase in endothelium. Am J Respir Cell Mol Biol 19:
758 –766, 1998.
VIANA V, DE SMEDT H, DROOGMANS G, AND NILIUS B. Calcium signalling through nucleotide receptor P2Y2 in cultured human vascular
endothelium. Cell Calcium 24: 117–127, 1998.
VIGNE P, CHAMPIGNY G, MARSAULT R, BARBRY P, FRELIN C, AND LAZDUNSKI M. A new type of amiloride-sensitive cationic channel in
endothelial cells of brain microvessels. J Biol Chem 264: 7663–
7668, 1989.
VINET R AND VARGAS FF. L- and T-type voltage-gated Ca2⫹ currents
in adrenal medulla endothelial cells. Am J Physiol Heart Circ
Physiol 276: H1313–H1322, 1999.
VOETS T, BUYSE G, DROOGMANS G, EGGERMONT J, AND NILIUS B. The
GXGXG motif in the pICln protein is not important for the nucleotide sensitivity of the pICln-induced Cl⫺ current in Xenopus oocytes. FEBS Lett 426: 171–173, 1998.
VOETS T, DROOGMANS G, AND NILIUS B. Ionic currents in non-stimulated endothelial cells from bovine pulmonary artery. J Physiol
(Lond) 497: 95–107, 1996.
VOETS T, DROOGMANS G, AND NILIUS B. Modulation of voltage-dependent properties of a swelling-activated Cl⫺ current. J Gen Physiol
110: 313–325, 1997.
VOETS T, DROOGMANS G, AND NILIUS B. Potent block of volumeactivated chloride currents in endothelial cells by the uncharged
form of quinine and quinidine. Br J Pharmacol 118: 1869 –1871,
1996.
VOETS T, DROOGMANS G, RASKIN G, EGGERMONT J, AND NILIUS B.
Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels. Proc Natl
Acad Sci USA 96: 5298 –5303, 1999.
VOETS T, MANOLOPOULOS V, EGGERMONT J, ELLORY C, DROOGMANS G,
AND NILIUS B. Regulation of a swelling-activated Cl-current in bovine endothelium by protein tyrosine phosphorylation and G-proteins. J Physiol (Lond) 506: 341–352, 1998.
VOETS T, SZÜCS G, DROOGMANS G, AND NILIUS B. Blockers of volumeactivated Cl⫺ currents inhibit endothelial cell proliferation.
Pflügers Arch 431: 132–134, 1995.
VOETS T, WEI L, DE SMET P, VAN DRIESSCHE W, EGGERMONT J, DROOG⫺
MANS G, AND NILIUS B. Downregulation of volume-activated Cl
currents during muscle differentiation. Am J Physiol Cell Physiol
272: C667–C674, 1997.
VON BECKERATH M, DITTRICH M, KLIEBER H-G, AND DAUT J. Inwardly
rectifying K⫹ channels in freshly dissociated coronary endothelial
cells from guinea-pig heart. J Physiol (Lond) 491: 357–365, 1996.
WAGNER DD. Cell biology of von Willebrand factor. Annu Rev Cell
Biol 6: 217–246, 1990.
WALSH KB, WOLF MB, AND FAN J. Voltage-gated sodium channels in
cardiac microvascular endothelial cells. Am J Physiol Heart Circ
Physiol 274: H506 –H512, 1998.
Physiol Rev • VOL
430. WALTENBERGER J, CLAESSON WELSH L, SIEGBAHN A, SHIBUYA M, AND
HELDIN CH. Different signal transduction properties of KDR and
Flt1, two receptors for vascular endothelial growth factor. J Biol
Chem 269: 26988 –26995, 1994.
431. WANG W, O’CONNELL B, DYKEMAN R, SAKAI T, DELPORTE C, SWAIM W,
ZHU X, BIRNBAUMER L, AND AMBUDKAR IS. Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein. Am J Physiol Cell Physiol 276: C969 –C979,
1999.
432. WANG X, LAU F, LI L, YOSHIKAWA A, AND VAN BREEMEN C. Acetylcholine-sensitive intracellular Ca2⫹ store in fresh endothelial cells and
evidence for ryanodine receptors. Circ Res 77: 37– 42, 1995.
433. WANG XD AND VAN BREEMEN C. Depolarization-mediated inhibition
of Ca2⫹ entry in endothelial cells. Am J Physiol Heart Circ Physiol
277: H1498 –H1504, 1999.
434. WARNAT J, PHILIPP S, ZIMMER S, FLOCKERZI V, AND CAVALIE A. Phenotype of a recombinant store-operated channel: highly selective
permeation of Ca2⫹. J Physiol (Lond) 518: 631– 638, 1999.
436. WARNER TD, SCHMIDT HH, AND MURAD F. Interactions of endothelins
and EDRF in bovine native endothelial cells: selective effects of
endothelin-3. Am J Physiol Heart Circ Physiol 262: H1600 –H1605,
1992.
437. WATANABE H, TAKAHASHI R, TRAN QK, TAKEUCHI K, KOSUGE K, SATOH
H, UEHARA A, TERADA H, HAYASHI H, OHNO R, AND OHASHI K. Increased cytosolic Ca2⫹ concentration in endothelial cells by calmodulin antagonists. Biochem Biophys Res Commun 265: 697–702,
1999.
438. WATANABE M, YUMOTO K, AND OCHI R. Indirect activation by internal
calcium of chloride channels in endothelial cells. Jpn J Physiol 44:
S233–S236, 1994.
439. WEI L, VANKEERBERGHEN A, CUPPENS H, EGGERMONT J, CASSIMAN JJ,
DROOGMANS G, AND NILIUS B. Interaction between calcium-activated
chloride channels and the cystic fibrosis transmembrane conductance regulator. Pflügers Arch 438: 635– 641, 1999.
440. WOOD PG AND GILLESPIE JI. Evidence for mitochondrial Ca2⫹-induced Ca2⫹ release in permeabilised endothelial cells. Biochem
Biophys Res Commun 246: 543–548, 1998.
441. WU SN, MOORE TM, BROUGH GH, WHITT SR, CHINKERS M, LI M, AND
STEVENS T. Cyclic nucleotide gated channels mediate membrane
depolarization following activation of store operated calcium entry
channels in endothelial cells. J Biol Chem 275: 1887–18896, 2000.
442. XIA XM, FAKLER B, RIVARD A, WAYMAN G, JOHNSON PAIS T, KEEN JE,
ISHII T, HIRSCHBERG B, BOND CT, LUTSENKO S, MAYLIE J, AND ADELMAN
JP. Mechanism of calcium gating in small-conductance calciumactivated potassium channels. Nature 395: 503–507, 1998.
443. XIE HQ AND HU VW. Modulation of gap junctions in senescent
endothelial cells. Exp Cell Res 214: 172–176, 1994.
444. XIE W, KAETZEL MA, BRUZIK KS, DEDMAN JR, SHEARS SB, AND NELSON
DJ. Inositol 3,4,5,6-tetrakisphosphate inhibits the calmodulin-dependent protein kinase II-activated chloride conductance in T84
colonic epithelial cells. J Biol Chem 271: 14092–14097, 1996.
445. YAMAKAGE M, HIRSHMAN CA, AND CROXTON TL. Sodium nitroprusside
stimulates Ca2⫹-dependent K⫹ channels in porcine tracheal
smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 270:
L338 –L345, 1996.
446. YAMAMOTO C, KAJI T, SAKAMOTO M, KOZUKA H, AND KOIZUMI F. Calcium
regulation of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial
cells. Thromb Res 74: 163–168, 1994.
447. YAMAMOTO K, KORENAGA R, KAMIYA A, AND ANDO J. Fluid shear stress
activates Ca2⫹ influx into human endothelial cells via P2X4 purinoceptors. Circ Res 87: 385–391, 2000.
448. YAMAMOTO K, KORENAGA R, KAMIYA A, QI Z, SOKABE M, AND ANDO J.
P2x4 receptors mediate ATP-induced calcium influx in human vascular endothelial cells. Am J Physiol Heart Circ Physiol 279:
H285–H292, 2000.
449. YAMAMOTO Y, CHEN G, MIWA K, AND SUZUKI H. Permeability and Mg2⫹
blockade of histamine-operated cation channel in endothelial cells
of rat intrapulmonary artery. J Physiol (Lond) 450: 395– 408, 1992.
450. YAMAZAKI J, DUAN D, JANIAK R, KUENZLI K, HOROWITZ B, AND HUME JR.
Functional and molecular expression of volume-regulated chloride
channels in canine vascular smooth muscle cells. J Physiol (Lond)
507: 729 –736, 1998.
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
412.
BERND NILIUS AND GUY DROOGMANS
ION CHANNELS IN VASCULAR ENDOTHELIUM
Physiol Rev • VOL
457. ZHU X AND BIRNBAUMER L. Calcium channels formed by mammalian
trp homologues. News Physiol Sci 13: 211–271, 1998.
458. ZHU X, CHU PB, PEYTON M, AND BIRNBAUMER L. Molecular cloning of
a widely expressed human homologue for the Drosophila trp gene.
FEBS Lett 373: 193–198, 1995.
459. ZHU X, JIANG M, PEYTON M, BOULAY G, HURST R, STEFANI E, AND
BIRNBAUMER L. trp, a novel mammalian gene family essential for
agonist-activated capacitative Ca2⫹ entry. Cell 85: 661– 671, 1996.
460. ZIEGELSTEIN RC, SPURGEON HA, PILI R, PASSANITI A, CHENG L, CORDA
S, LAKATTA EG, AND CAPOGROSSI MC. A functional ryanodine-sensitive intracellular Ca2⫹ store is present in vascular endothelial cells.
Circ Res 74: 151–156, 1994.
461. ZITT C, OBUKHOV AG, STRUBING C, ZOBEL A, KALKBRENNER F, LÜCKHOFF
A, AND SCHULTZ G. Expression of TRPC3 in Chinese hamster ovary
cells results in calcium-activated cation currents not related to
store depletion. J Cell Biol 138: 1333–1341, 1997.
462. ZÜNKLER BJ, HENNING B, GRAFE M, HILDEBRANDT AG, AND FLECK E.
Electrophysiological properties of human coronary endothelial
cells. Basic Res Cardiol 90: 435– 442, 1995.
81 • OCTOBER 2001 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 5, 2017
451. YAO X, KWAN HY, CHAN FL, CHAN NWK, AND HUANG Y. A protein
kinase G-sensitive channel mediates flow-induced Ca2⫹ entry into
vascular endothelial cells. FASEB J 14: 932–938, 2000.
452. YAO X, LEUNG PS, KWAN HY, WONG TP, AND FONG MW. Rod-type
cyclic nucleotide-gated cation channel is expressed in vascular
endothelium and vascular smooth muscle cells. Cardiovasc Res 41:
282–290, 1999.
453. YEH HI, ROTHERY S, DUPONT E, COPPEN SR, AND SEVERS NJ. Individual
gap junction plaques contain multiple connexins in arterial endothelium. Circ Res 83: 1248 –1263, 1998.
454. ZHANG A, CHENG TP, ALTURA BT, AND ALTURA BM. Mg2⫹ and caffeineinduced intracellular Ca2⫹ release in human vascular endothelial
cells. Br J Pharmacol 109: 291–292, 1993.
455. ZHANG H, INAZU M, WEIR B, BUCHANAN M, AND DANIEL E. Cyclopiazonic acid stimulates Ca2⫹ influx through non-specific cation channels in endothelial cells. Eur J Pharmacol 251: 119 –125, 1994.
456. ZHANG H, INAZU M, WEIR B, AND DANIEL E. Endothelin-1 inhibits
inward rectifier potassium channels and activates nonspecific cation channels in cultured endothelial cells. Pharmacology 49: 11–22,
1994.
1459