112
Tutorial in Molecular and Cellular Biology
Intracellular Calcium, Currents, and
Stimulus-Response Coupling
in Endothelial Cells
Herbert M. Himmel, A. Richard Whorton, and Harold C. Strauss
Vascular endothelium appears to be a unique organ. It not only responds to numerous hormonal and
chemical signals but also senses changes in physical parameters such as shear stress, producing
mediators that modulate the responses of numerous cells, including vascular smooth muscle, platelets,
and leukocytes. In many cases, the initial response of endothelial cells to these diverse signals involves
elevation of cytosolic Ca2+ and activation of Ca2+-dependent enzymes, including nitric oxide synthase and
phospholipase A2. Both the release of Ca2+ from intracellular stores, most likely the endoplasmic
reticulum, and the influx of Ca2+ from the extracellular space contribute to the [Ca 2+ ]| increase. The most
important trigger for Ca2+ release is inositol 1,4,5 -trisphosphate, which is generated by the action of
phospholipase C, a plasmalemmal enzyme activated in many cases by the receptor-G protein cascade.
Ca2+ influx appears to be related to the activity of receptor-G protein-enzyme complex and to the degree
of fullness of the endoplasmic reticulum but does not involve voltage-gated Ca2+ channels. The magnitude
of the Ca2+ influx depends on the electrochemical gradient, which is modulated by the membrane
potential, Vm. Under basal conditions, Vm is dominated by a large inward rectifier K+ current. Some
stimuli, e.g., acetylcholine, have been shown to hyperpolarize Vm, thus increasing the electrochemical
gradient for Ca 2+ , which appears to be modulated by activation of Ca2+-dependent K+ and CI" currents.
However, the lack of potent and specific blockers for many of the described or postulated channels (e.g.,
nonselective cation channel, Ca2+-activated Cl~ channel) makes an estimation of their effect on
endothelial cell function rather difficult. Possible future directions of research and clinical implications
are discussed. (Hypertension 1993;21:^12-127)
KEY WORDS • calcium • endothelium • ion channels
D
uring the last decade, it has become increasingly clear that the endothelial cells lining
blood vessels are more than a barrier between
blood and surrounding tissues. In fact, the endothelium
is a highly sophisticated, regionally differentiated organ
that regulates vascular tone in both conduit and smaller
resistance vessels1"3 and plays a role in hemostasis,
cellular proliferation, inflammation, and immunity.4"11
Endothelial cells communicate Immorally with their
neighboring smooth muscle cells by synthesis and release of vasoactive compounds such as endothelin,12
prostaglandins,13 and endothelium-derived relaxing factor (EDRF), 14 which is composed of nitric oxide15 and
related nitrosothiols.16 In addition, direct electrotonic
interaction between endothelial and vascular smooth
muscle cells (VSMCs) has also been proposed.17-18 The
intracellular signal that links systemic or local external
stimuli to the synthesis and release of EDRF and
prostaglandins appears to be the level of free cytosolic
calcium, [Ca2+]j. Furthermore, [Ca 2+ ]| also seems to be
From the Departments of Pharmacology and Medicine, Duke
University Medical Center, Durham, N.C.
Supported in part by grant Hi475/1-1 from the Deutsche Forschungsgemeinschaft and grants HL-45132, HL-19216, HL-42444,
and HL-44740 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.
Address for correspondence: Harold C. Strauss, MD, Box 3845,
Duke University Medical Center, Durham, NC 27710.
involved in the contraction of endothelial cells and the
increased permeability of microvessels in response to
inflammatory agents (for review, see Reference 19).
Thus, a detailed knowledge of intracellular Ca2+ homeostasis is essential for our understanding of the
physiology, pathophysiology, and pharmacology of endothelial cells. The following sections review the importance of the endothelium in various disease states and
the mechanisms underlying cytoplasmic Ca2+ regulation
under basal and stimulated conditions.
Importance of the Endothelium in Various
Disease States
Increased systemic vascular tone, which is thought to
result from enhanced circulating hormones (e.g., norepinephrine, angiotensin II), is among the most common hemodynamic findings in patients with heart failure.20 However, heightened vasoconstriction correlates
poorly with the plasma levels of these substances.20 The
poor correlation could possibly be explained by the
involvement of local, endothelium-dependent factors,
such as an imbalance of endothelium-derived relaxing
and contracting factors.21"23 This view is supported by
numerous studies conducted in isolated vascular segments as well as in intact laboratory animals and human
subjects in a variety of disease states, including systemic
and coronary atherosclerosis, pulmonary and systemic
hypertension, and heart failure (Table 1).
Himmel
et al
113
Calcium and C u r r e n t s in Endothelial Cells
TABLE 1. Vasodilator Responses Under Various Pathological Conditions
Endothelium-dependent
vasorelaxation
Animal/tissue
Isolated vascular segments
Pig coronary artery
Rabbit thoracic aorta
Rat mesenteric artery
Rat aorta
Monkey coronary
microvessel
Human epicardial
coronary artery
Intact laboratory animals
Dog femoral artery
Rat thoracic aorta and
pulmonary artery
Rat
Human subjects
Coronary artery
Coronary artery
Pathological condition
Agents tested
Impairment
Agents tested
Atherosclerosis and
hypercholesterolemia
BK, 5-HT, ADP
Y
Nitroprusside
N
24,25
Atherosclerosis
Hypertension
ACh, A23187
Y
Y
Y
Y
Nitric oxide
Nitric oxide
N
N
Adenosine, nitroprusside
N
26
27
28
29
Y
Isoprenaline, nitroglycerin
N
30
Nitrogrycerin
Nitroglycerin
N
N
31
32
Hypertension
ACh
ACh
Atherosclerosis
BK, ACh, A23187
Atherosclerosis
Substance P, BK,
A23187
ACh
Y
Heart failure
ACh, ADP (A23187)
Y(N)
Hypertension
/V-Monomethyl
arginine
Y
Cor. atherosclerosis
ACh
ACh
Y
Y
Nitroglycerin, papaverin
N
N
34,35
Dipyridamole
Substance P
Y at stenosis,
N distal
Isosorbide dinitrate
N
37
Nitroprusside
Adenosine
3839
Nitroglycerin
N
N
N
Nitroprusside
N
42
Forearm blood flow
Coronary blood flow •»
Hypertension
ACh
ACh
Forearm blood flow 1
Cardiomyopathy and
heart failure
a-ANP
Y
Y
Y
Methacholine
Y
f
Forearm blood flow J
Impairment Reference
Heart failure
Angina pectoris;
normal angiogram
Cor. atherosclerosis
Coronary artery
Endothelium-independent
vasorelaxation
33
36
40
41
BK, bradykinin; 5-HT, serotonin; ADP, adenosine diphosphate; Y, yes; N, no; ACh, acetylcholine; Cor., coronary.
Isolated Vascular Segments
In ring preparations of coronary arteries of pigs with
hypercholesterolemia and atherosclerosis, endothelium-dependent relaxation in response to bradykinin,
serotonin, and ADP was impaired24-25 (Table 1). Similar
observations have been made in small coronary resistance arteries (diameter <200 fim) of atherosclerotic
monkeys, in which endothelium-dependent vasodilation
was also found to be depressed.29 The impairment of
EDRF-mediated relaxation (in response to substance P,
bradykinin, and calcium ionophore A23187) has been
confirmed in isolated human epicardial coronary arteries as well.30
Intact Laboratory Animals and Human Subjects
In a canine model of pacing-induced heart failure, the
acetylcholine-mediated relaxation of the femoral artery
was depressed, whereas vasomotor responses to nitroglycerin and norepinephrine were unchanged.31 As predicted from the experiments on isolated vascular segments, patients with both mild and severe coronary
atherosclerosis reacted to acetylcholine, an endothelium-dependent vasodilator, with vasoconstriction or decreased vasodilation, whereas their response to nitroglycerin was normal.34-35 Endothelial dysfunction was
evident in coronary resistance arteries in patients suffering from angina pectoris who had normal coronary
angiograms.36 The forearm vasculature of patients with
essential hypertension also showed reduced response to
acetylcholine, whereas the response to nitroprusside
was indistinguishable from that of healthy control subjects.38-39 Several studies in patients with heart failure
caused by ischemic or idiopathic cardiomyopathy report
attenuated endothelium-dependent vasodilation for
both forearm and coronary blood flow.40-42
The endothelium may also contribute to the increased vasomotor tone associated with certain forms of
hypertension. For example, the plasma concentrations
of immunoreactive endothelin, the most potent endothelium-derived vasoconstrictor peptide,12-43 were elevated in patients with severe systemic essential hypertension,44-45 secondary pulmonary hypertension,46 and
chronic heart failure.47 These studies point to a putative
role for endothelium-derived contracting factors in the
pathophysiology of hypertension.
Regardless of whether impaired local vasodilation,
increased vasotonus, or both are involved in cardiovascular disorders such as heart failure and hypertension,48-49 a better understanding of the role of endothelial cells and the mechanisms underlying signal
transduction and [Ca2+], regulation is likely to result in
new therapeutic approaches.
Signal Transduction Mechanisms in
Endothelial Cells
The endothelium-dependent relaxation of vascular
tone is mediated by many compounds such as vasoactive
114
Hypertension
Vol 21, No 1 January 1993
FIGURE 1. Schematic shows endothelial
cell model of mechanisms involved in
signal transduction and ion transport R,
receptor; G, guanine nucleotide binding
regulatory protein; PLC, phospholipase C;
PLA2, phospholipase A2; PG, prostaglandins; IP3, inositol 1,4,5-trisphosphate;
IP4, inositol
1,3,4,5-tetrakisphosphate;
DG, diacylgtycerol; PKC, protein kinase
C; ER, endoplasmic reticulum; IKl, K+
inward rectifier; lr^A), transient A-type K+
current; IK<C*)> calcium-activated K+ currznt; IK(AOI)I acetylcholine-activated K+
current; INS, nonselective cation current;
iNS(m), nonselective stretch-activated cation current; IK(ltr), stretch-activated K+
current
peptides (bradykinin, substance P) and autacoids (acetylcholine, histamine, ATP, serotonin), which depend
on receptors on the endothelial cell surface. However,
other stimuli, such as shear stress, hypoxia, and calcium
ionophores, are receptor independent. The effects of
the vasoactive constrictor peptide endothelin have been
reviewed recently.43
Some of the compounds that produce vasodilation are
also neurotransmitters or cotransmitters. These substances cause vasoconstriction when acting directly on
VSMCs, as would be expected after release from
perivascular nerves. For example, ATP released from
perivascular nerves leads to P^-purinoceptor-mediated
contraction of vascular smooth muscle, whereas luminal
ATP acts on P^-purinoceptors located on endothelial
cells, resulting in EDRF-mediated vasodilation.50-52 It
is therefore essential to identify the sources of vasoactive substances that induce endothelium-dependent responses by binding to endothelial cell receptors. One
readily available and well-known source is cellular blood
constituents such as platelets and erythrocytes, which
release serotonin, ATP, and ADP.52-53 Another potential source is the endothelium itself, as choline acetyltransferase, substance P, serotonin, vasopressin, angiotensinogen, angiotensin II, histamine, ATP, and
endothelin have been localized in endothelial cells from
various blood vessels with the use of biochemical techniques and immunocytochemical staining in combination with electron microscopy.54-59 In fact, human umbilical vein and rabbit aortic endothelial cells have been
shown to release ATP, substance P, acetylcholine, and
bradykinin into the culture medium.60-63 However,
there is no conclusive evidence as to the physiological
significance of this release mechanism in vivo.
One ubiquitous transduction pathway for receptorgenerated signals is through GTP-binding regulatory
proteins (G proteins),64-66 although growth factors67"69
and protein kinase C agonists70-71 may act independently
of G proteins (Figure 1). The subsequent activation of
effectors (e.g., phospholipase C, phospholipase A2, adenyryl cyclase) produces intracellular second messengers such as inositol 1,4,5-trisphosphate (IP3), diacylglycerol, arachidonic acid, and cyclic AMP72-74 and
activation of ion channels,75 resulting in Ca2+ influx into
the cytoplasm (Figure 1). This chain of events results in
an increase in [Ca2+]i (see below), which activates
Ca2+-dependent enzymes such as nitric oxide synthase76-77 or phospholipase A2. Finally, vasoactive compounds such as EDRF, putative endothelium-derived
hyperpolarizing factor (EDHF), and prostaglandin I2
are formed and released.7879 However, documentation
of a particular signal transduction pathway under defined conditions (species, regional vascular origin, conditions for cell isolation, culture, and experiment) is
sometimes incomplete. Although a reasonable amount
of information is available with respect to muscarinic,
histaminergic, and purinergic stimuli, bradykinin, and
thrombin (see Table 2), little is known about shear
stress, an important physiological stimulus.
Although most of the stimuli listed in Table 2 need G
proteins for signal transduction, their role in muscarinic
and purinergic transmission has yet to be confirmed.
Although a few of these agonists transduce their signal
via pertussis toxin-sensitive G proteins, as shown in
functional studies for a2-adrenergic, serotoninergic, and
histaminergic transduction,80'114 functional as well as
biochemical experiments have failed to demonstrate an
effect of pertussis toxin for the majority of agonists
(Table 2). Experiments using A1F4"123 or nonhydroh/zable GTP analogues point to the presence of pertussis
toxin-insensitive G proteins in the signal transduction
cascade for bradykinin, thrombin, and purinergic compounds.81-82-101 However, recent evidence suggests that
the transduction mechanism for bradykinin receptors
probably involves pertussis toxin-sensitive as well as
pertussis toxin-insensitive G proteins.83-84
Despite the diversity in receptors and G proteins,
receptor activation appears to be followed by the increase in [Ca2+], and by the release of EDRF. The Ca2+
mobilization step can be modulated by both diacylgrycerol-activated protein kinase C and cyclic AMP-dependent protein kinase. For example, phorbol myristate
acetate has been shown to inhibit bradykinin- and
ATP-induced Ca2+ release in bovine aortic endothelial
cells, whereas cyclic AMP had the opposite effect and
cyclic GMP had no effect.85 Although endothelial cells
have been shown to release arachidonic acid metabolites (e.g., prostaglandins I2 and E2 and thromboxane
Himmel et al Calcium and Currents in Endothelial Cells
TABLE 2.
Signal Transdnction Pathways for Some Endothelium-Dependent Modulators of Vascular Tone
Compound
PTX
Bradykinin
Thrombin
ACh,
carbachol
115
T?
M,,M3
M2
M,
Effector
enzyme
Vasoactive
compound
Endothelial
cell type
PLC
EDRF/NO
PLA2
PLC
PLA2
PLC
PLA2
PGI2
EDRF/NO
PGI2
EDRF/NO
BPA,BA,
PA.PCA,
HUV, BBMV
IP3t
EDHF?
M?
Histamine
H,
PLC
EDRF/NO
ATP/ADP
H2
p
AC
PLC
PLA2
EDRF/NO
PGI2
Reference
80-82
< 83-89
90-100
•
HUV, PA
Rat aorta
BA,MBM
Rat FA+MA
DogHA+PV
BA, RA,
GPCA
[MBM
HUV, GPPA
BA
fBA,HUV
| PA, DBA
80, 101-105
106-111
17,112,113
100, 105,
.114-117
118
80, 119-122
R, receptor, G, guanine nucleotide binding protein; PTX, pertussis toxin sensitivity, IP3, inositol 1,4,5-trisphosphate; +, yes; - , no; PLC,
phospholipase C; EDRF/NO, endothelium-derived relaxing factor/nitric oxide; BPA, bovine pulmonary artery, BA, bovine aorta; PA,
porcine aorta; PCA, porcine coronary artery HUV, human umbilical vein; BBMV, bovine brain microvessels; PLA2, phospholipase A2;
PGI2, prostacyclin; ACh, acetylcholine; ?, not known; MBM, mouse brain microvessels; FA+MA, femoral artery and mesenteric artery
HA+PV, hepatic artery and portal vein; EDHF, endothelium-derived hyperpolarizing factor; I^AQ), acetjicholine-activated K+ current;
RA, rabbit aorta; GPCA, guinea pig coronary artery; GPPA, guinea pig pulmonary artery AC, adenylate cyclase; DBA, dog basilar artery.
A2), which requires the activation of phospholipase A 2 ,
it is often not clear whether this release of metabolites
is solely due to an increase in [Ca 24 ],. 8687
Although muscarinic receptor-induced, EDRF-mediated vasodilation and the concomitant hyperpolarization
of VSMCs are well known, there has been some confusion until recently with respect to the receptor subtypes
and the signal transduction pathways in endothelial
cells.io6-ioe.u2 Binding and functional data109-124-126 but
not autoradiographic findings127 suggest that endothelial
cells possess muscarinic receptors, which are heterogeneous with respect to subtype, vascular region, and
species. However, interpretation of these data requires
some caution, because muscarinic receptors can also be
found on smooth muscle cells and neurons. A recent
study comparing cultured aortic endothelial cells and
VSMCs showed that cholinergic stimuli enhanced prostacyclin and cyclic GMP production in endothelial cells
but not in VSMCs.110 These effects were mediated by the
activation of M2- and M3-receptors but not the M r
receptor subtype,110 which is in apparent conflict with a
radioligand binding study that reported the presence of
M,-receptors onry in isolated bovine aortic endothelial
cells.128 These apparent discrepancies could be explained
by altered expression of muscarinic receptors in cultured
as opposed to freshly isolated endothelial cells. Indeed,
Northern blot analysis identified mRNA transcripts for
the M r , M2-, and M3-receptor subtypes in freshly isolated endothelial cells, whereas cultured endothelial cells
contained messenger RNA (mRNA) for only the M2receptor subtype.111 Because muscarinic receptor subtypes are differentially coupled to phospholipase C and
adenylate cyclase,129 the gradual loss of the M r and
M3-receptor subtypes during cell culture may account for
different functional responses to muscarinic stimulation
in vivo or in freshly isolated cells versus cultured endothelial cells.
Functional studies in intact vessels suggest that M2receptors mediate EDRF release and that endotheliumdependent hyperpolarization of VSMCs is caused by the
M,-dependent synthesis and release of an EDHF.130-132
However, the chemical nature of EDHF and its mechanism of action are a matter of speculation.133 Furthermore, since it has not yet been determined whether
nitric oxide hyperpolarizes the plasma membrane of
VSMCs, this compound may also be an EDHF. 134135
The acetylcholine-induced hyperpolarization in endothelial cells is primarily mediated by an inwardly rectifying K+ current, I^AQ)) and is independent of G
proteins17'112; however, the receptor subtype and the
relation to EDRF and EDHF remain to be established.
Blood flow-induced forces (e.g., shear stress, stretch)
are among the most important physiological stimuli and
also among the least understood. Vasodilation due to
shear stress is associated with increased release of
EDRF, prostaglandin I2, or both.136 The rise in prostacyclin production in endothelial cells exposed to increases in flow or shear stress137-139 is probably due to
phospholipase A2 activation in response to enhanced
[Ca2+],,140 although shear stress-induced increases in
[Ca2+], are not observed in a consistent manner.141 The
increase in [Ca2+]f is probably caused by IP3-mediated
(shear stress,142 stretch143) Ca2+ release or influx of Ca2+
via a nonselective cation channel (see below). The
observed hydrodynamic hyperpolarization of endothelial cells,144 which may be due to a flow-activated
inwardly rectifying K+ channel (see below), increases
the electrochemical gradient for and thus the influx of
Ca2+. Over time, shear stress also affects the cytoskeleton.7 However, because the studies cited above were
performed with endothelial cells from various species
and various sites, the steps linking shear stress to
cellular responses have to be further elucidated.
116
Hypertension
Vol 21, No 1 January 1993
1000 -i
1000 -\
800 -
800 600 -
600 -
O 400 -
(M
V
200 -
400 -
0.1
200 -
1
10
100
1000
BK Concentration (nM)
10
20
30
40
Time (min)
FIGURE 2. Graph shows elevation of [Ca2+], in bovine endothelial cells treated with bradykinin (BK). Confluent monolayers of
endothelial cells grown on glass coverslips were loaded with 20 (iMfura 2-AMfor 30 minutes and placed in a cuvette across the
diagonaL The cuvette was placed in a spectrofluorometer maintained at 37°C and perfused at 3 ml/min with Hanks' balanced salt
solution. Cells were stimulated with bradykinin by stopping buffer flow and injecting 20 ml solution containing thepeptide at doses
indicated over a 2-minute period; thereafter, buffer was immediately restarted. Fluorescence was continuously monitored
throughout the experiment. Data represent Ca2+ from a slide of cells exposed to a single dose of bradykinin. Each experiment was
repeated at least three times. Inset: Line graph shows dose-response curve for bradykinin. Each point represents mean±SEM,
n=4.
Agonist-Evoked Ca 2+ Transient
Our understanding of the basis of Ca2+ mobilization
in endothelial cells after exposure to extracellular ligands has evolved through the use of different techniques, including intracellular fluorescent Ca2+ indicator dyes. Many groups have shown that several ligands,
including bradykinin, ATP, histamine, and thrombin,
can elevate [Ca2+]i in endothelial cells in monolayer
culture from a basal level of 50-100 to 400-800
2+
spike is reached within
nM.86.88,89,iouo2,ii9,i2o The Ca
20-30 seconds, followed by a plateau phase that persists
for 5-8 minutes (Figure 2). A variety of experiments
have shown that the initial component of the Ca2+
transient results from release from intracellular stores,
whereas the delayed component contributing to the
plateau phase is dependent on Ca2+ influx from the
extracellular space.8914514* In general, agonists that
increase [Ca2+], in endothelial cells are coupled by
receptors and G proteins to phospholipase C. Generation of IP3 is thought to activate IP3 receptors in internal
stores, resulting in Ca2+ mobilization. The plasmalemmal Ca2+ influx pathway is incompletely characterized;
however, both Ca2+ depletion of intracellular stores and
inositol 1,3,4,5-tetrakisphosphate (IP4) have been
shown to activate Ca2+ influx through the plasmalemma
(see below).
Other mediators such as diacylglycerol and Ca2+ may
act as second messengers in regulating both the Ca2+
and subsequent cellular response. In addition, arachidonic acid and sphingosine derivatives, which have been
shown recently to mediate Ca2+ release and the inhibition of several key enzymes, such as protein kinase C
and Ca2+/calmodulin-dependent protein kinase,147 may
also be involved; although, in the case of the latter, the
mechanism of action is not understood.
Studies in brain cells, hepatocytes, pancreatic acinar
cells, and VSMCs provide evidence, with limited confirmation from endothelial cells, that IP3 binds to a
receptor on the endoplasmic reticulum (ER) (recently
reviewed; see References 148-153). On average, IP3
releases 50% of the Ca2+ stored in the ER pool. The
remaining Ca2+ can be released by calcium ionophores
or by agents such as thapsigargin145154-157 or 2,5di(ter/-butyl)-l,4-benzohydroquinone (BHQ), 145158 - 160
which inhibit the Ca2+-ATPase in the ER. Thus, it
seems that these cells contain several distinct Ca2+
storage compartments, which may be involved in agonist-induced Ca2+ release. In fact, it has been suggested
that different Ca2+ pools may exist in a number of cells
and participate in a coordinated fashion in IP3-mediated
Ca2+ release and subsequent Ca2+-activated Ca2+ release, leading to such phenomena as Ca2+ waves and
oscillations.161"167 Although these pools may be con-
Himmel et al Calcium and Currents in Endothelial Cells
tained within the ER, it has been proposed that the
IP3-sensitive Ca2+ pool is localized in a unique membrane compartment termed calciosome, which contains
the Ca2+ binding protein calsequestrin.168 The IP3 binding site, which can be blocked by heparin and other
compounds,169-171 and the Ca2+ release channel that it
controls are probably separate domains within the same
protein.148'153 ^-sensitive Ca2+ release channels isolated from aortic smooth muscle (not yet confirmed in
endothelial cells) have a single-channel conductance of
approximately 10 pS and are blocked by cinnarizine and
flunarizine but not by the classic calcium channel blockers.148-153 Furthermore, the function of the channel
protein can be modulated by phosphorylation (see
Reference 153). However, the molecular mechanisms
involved in Ca release from the ER are still unclear.
On the one hand, there is evidence to support the
presence of a Ca2+ leak channel, an IP3-gated Ca2+
channel, and an ATP-driven Ca2+ pump in the ER
membrane (Figure 1), which are responsible for basal as
well as stimulus-induced Ca2+ release and Ca2+ reuptake. On the other hand, a Ca2+ leak channel is not
required to account for the continuous cycling of Ca2+
across the ER membrane, because the passive efflux
("leak") of Ca2+ from the ER could occur through the
same channel that mediates the IP3-stimulated release.
The influx of extracellular Ca 2+ , which constitutes the
delayed component of the agonist-stimulated increase
in [Ca2+]i, is still poorly understood with respect to both
its pathway and control mechanisms. The influx pathway
is probably a receptor-mediated or second messengeroperated, calcium-permeable, nonselective cation channel.172-173 A recent study has implicated both intracellular Ca2+ and IP* in controlling the slow influx of
extracellular calcium,75 which is in contrast to an earlier
report174 in which IP3 and IP4 did not regulate Ca2+
influx. Other studies, however, indicate that the Ca2+
content of the ER controls the Ca2+ permeability of the
plasmalemma.145146-175 The data on which this model is
based were gathered using at least two different experimental approaches that use the measurement of 45Ca2+
fluxes or [Ca2+], by means of fluorescent dyes. One
approach in endothelial cells uses repeated agonist
stimulation of IP3-sensitive Ca2+ release under conditions of normal extracellular Ca 2+ , nominally Ca2+-free
solutions, or solutions in which Mn 2+ replaces
Ca2+.146-176 The other approach uses compounds such as
thapsigargin or BHQ for IP3-independent emptying of
the intracellular Ca2+ pool and activation of the Ca2+
entry pathway in endothelial cells.145 As has been
pointed out recently,177178 Ca2+ and Mn2+ influx after
depletion of intracellular Ca2+ stores could also occur
through two different pathways that are controlled by
different mechanisms. The inability to discern whether
cellular responses to external agonists reflect a variety
of pathways thus may reflect limitations of the current
experimental techniques, including the degree of expression of a particular pathway in a given cell.
Mn2+ has been a useful tool in addressing this question, because Mn2+ influx is not detectable under basal
conditions, and Mn2+ influx after exposure to an agonist
should therefore reflect influx through the plasmalemma alone. Using histamine as an agonist, Jacob146
demonstrated that when internal Ca
stores were
discharged by transiently exposing cells in a Ca2+-free
117
solution to histamine and then to extracellular Mn 2+ ,
Mn2+ entry was seen. By exposing the cells to extracellular Ca 2+ , Jacob was able to show that Mn2+ influx was
dependent on the duration of exposure to extracellular
Ca 2+ . This was interpreted to mean that 1) the rate of
Mn2+ influx depends on the degree of repletion of
internal stores by Ca 2+ , and 2) the mechanism for
activation of the Ca2+ influx in the plasmalemma is IP3
independent.146 Similar observations have been made
using BHQ or thapsigargin to deplete the intracellular
Ca2+ stores.145 This mechanism has been postulated by
Putney179 to be the capacitivery coupled mechanism for
Ca2+ influx into the cytosol. According to these data,
extracellular Ca2+ could enter a restricted cytosolic
compartment and be taken up into the ER by a Ca2+ATPase. The internal Ca2+ store would thereby control
the plasmalemmal channel, which would then be progressively closed as the [Ca2+] in the internal stores
increased. A leak pathway between the restricted compartment and the major part of the cytosol would
account for the refilling transient seen in many cells. A
corollary of this capacitative model is the close apposition of ER and plasmalemma. Although the link between these two structures is not known, one could
speculate about a gap junction-like pore for Ca2+
transport or other connections involving parts of the
cytoskeleton.7180
Electrophysiological Properties
Exposure to a variety of stimuli has been shown to
transiently increase [Ca2+]( in endothelial cells. Within a
few seconds of exposure to various ligands, [Ca2+]i
increases from basal levels to a peak, which is followed
by a maintained plateau phase. The initial peak is
unaffected by removal of extracellular Ca2+; however,
the plateau phase depends on the presence of extracellular Ca2+ and, in fact, has been shown to depend on the
calcium electrochemical gradient.181-183 Although these
cells have been demonstrated to be nonexcitable,184-193
i.e., they do not have voltage-activated Ca2+ channels
(for the only exception, see References 194 and 195),
the dependence of the delayed component of the Ca
transient on the calcium electrochemical gradient indicates that knowledge of the electrical pathways is essential to our understanding of both the regulation of
membrane potential in endothelial cells and calcium
homeostasis within these cells (also compare earlier
publications: References 178 and 196-199). The different current pathways are summarized in Table 3.
Basal Conditions
Membrane potential. Numerous investigators have
measured resting potential in endothelial cells from
various preparations using the "tight seal" technique,
and a wide range of values has been reported, from
approximately - 8 0 up to nearly 0 mV (4.0-5.4 mM
[K+]o). i7.iB2-i86,i88-i9o,2O2-2O4.2i 1-213 when resting potential was measured after a high seal resistance had been
obtained, the relatively hyperpolarized values and the
response of the resting potential to changes in [K+]o
suggest (Mullins-Noda modified constant field equation) that the membrane is selectively permeable to K+
over Na + , with a PN./PK of 0.027 (assuming a Na-K
pump coupling ratio of 3:2).183 Accurate measurement
of the resting potential of endothelial cells is a prereq-
118
TABLE 3.
Current
Hypertension
Vol 21, No 1 January 1993
Properties of Various Ionk Currents in Vascular Endothellal Cells
Activation,
agonist*
Blockade
Ba 2 + ,Cs +
Ba 2 + ,Cs +
Ba2+
U,
a
+
Whole-cell current
slope conductance
-266 pS/pF§
~230pS/pF§
-104 pS/pF§
-160 pS/pF
-150-160 pS/pF§
-130-220 pS/pF§
Single-channel
conductancet
25 pS (150 K+)
36 pS (140 K + )
Permeability
ratioj
26 pS (145 K+)
23 pS (140 K+)
35 pS (200 K + )
27 pS (140 K+)
Ba 2+
-250-360 pS/pF
VD
4-AP
-121 pS/pF§
BA
186
ACh
ACh
Atropine
Atropine
—70 pS/pF
- 8 6 pS/pF§
BA
RA
17
202
BA
BA
BPA
BA
BA
RA
203
188
192
197
200,204
205
360pS
PA
206
V
382 pS (140 CT)
BA
207
VH
Thrombin, BK
[Ca2+], t , BK
[Ca2+], T , IP* (ATP,
BK)
[Ca2+]i t .histamine,
A23187
VH, histamine,
thrombin
VH?
Histamine
12 pS (140 K+)
X 2+ :Na + >2:l#
BA
BPA
BPA
BA
203
184
192
75
. . . ||
HUV
190
28pS(140K + )
K:Na:Ca=l:0.9:0.2
HUV
208
-50pS(140K + )
26.4 pS (140 K+)
K:Na:Ca-l:l:100
K:Na:Ca=l:l:15.7
HUV
RPA
193
209
BA
187
PA
210
•K<ACS)
VD, [Ca2+], t
[Ca ],T (BK)
[Ca 2+ ],t (BK)
[Ca2+],T (ATP)
[Ca 2+ ],t (BK)
[Ca2+],T (ACh)
-150 pS (140 K+)
2+
ICKO
la
INS
Endothelial
cell type Reference
BA
186
BPA
184
BA
188
BA
17
BA, BPA
191
BPA
192
BA
200
HUV
193
BPA
201
Heparin
- 8 0 pS/pF§
Increase
- 7 1 pS/pF§
Increase
Increase
V,[Ca 2+ ],t
(A23187)
Ba 2 + ,Cs +
Shear stress
40 pS (140 K+)
9pS
...J
- 5 0 pS/pF§
Increase
3.2 pA/pF
2.5 pSH
Increase
20-25 pS (140 K+)
91 pS/pF
Shear stress
39 pS
2+
K + -Na +
2+
+
+
Ca 2 + :Na + -6:l
Iu, inward rectifier, Ba , extracellular Ba ; Cs , extracellular Cs ; BA, bovine aorta; BPA, bovine pulmonary artery; HUV, human
umbilical vein; IK(A), transient outward current; 4-AP, 4-aminopyridine; IK(AO.)» acetylcholine-activated K+ current; ACh, acetylcholine; RA,
rabbit aorta; I*<ci), Ca2+-activated K+ current; BK, bradykinin; ATP, adenosine trisphosphate; Io<c.), Ca2+-activated Cl~ current; PA, pig
aorta; IQ, Q " current; I ^ , agonist-activated nonselective cation current; IP 4 , inositol 1,3,4,5-tetrakisphosphate; RPA, rat pulmonary artery,
IK(HI). stretch-activated K+ current; l^^m), stretch-activated nonselective cation current.
"Voltage-activated (V) by hyperpolarization (H) or depolarization (D); [Ca2+]i t (BK), activated by increased cytosolic Ca 2+
concentration due to effect of bradykinin.
tl50 K + , conductance determined in symmetrical K+ solutions with concentrations of 150 mM.
tlici, IK(A). IK<ACII)» IK(C), lK<nr) are carried mainly by K + , as inferred from 1) ion substitution experiments, 2) reversal potential
measurements, 3) change in reversal potential per 10-fold change in [K+] ratio.
{Estimated from linear parts of current-voltage relation, assuming cell capacitance of 30 pF.
IINonselectivity based on ion substitution experiments and shifts of reversal potential.
^Determined in excised patches with Mn2+ as major charge carrier.
#X 2+ stands for Mn 2+ , Ca 2+ , Ba 2+ .
Himmel et al
00
en
4
Calcium and Currents in Endothelial Cells
5
1
119
h
o
••
m
UJ
o
o
</>
UJ
a.
O
400-
_
2 -
2000 -,
B
400-
1000 -
0-
%
0-
-400-
-400-
-1000J
-BOO-
-120
-60
60
-800
-120
0
-60
60
J
-120
-60
Vm (mV)
FIGURE 3. Tracings show Ca2+ transient and whole-cell currents in a single, voltage-clamped, bovine aortic endothelial cell
(H2218R08). Panel A: Time course offura-2 fluorescence ratio. Fura-2 (pentapotassium salt, 50 fiM) was loaded via a patch
electrode; intracellular fura-2 was alternately excited at 340 and 380 nm; intensity of emitted light was measured at 510 nnu
Horizontal bar denotes exposure of the cell to bradykinin (5 fiM). Numbers (1-7) at various points along the Ca2+ transient
correspond to times that respective current traces were recorded (depicted in panels B-D). Panels B-D: The cell was voltage
clamped at a holding potential of —40 mV and stimulated with ramp pulses (—120 to +60 mV; duration, 160 msec) at 2-second
intervals. Before the beginning of each ramp, the cell was clamped to -120 mV for 5 msec to allow for settlement of the capacitance
transient (blanked in panels B-D). Holding current is displayed at left of panels B-D. Note the different ordinate in panel C.
During initial seconds of [Ca2+/, increase, there is no change in inward current (2) and only a slight increase in outward current
(2). This is consistent with Ca 2+ released from endoplasmic reticulum. While [Ca 2+]t reaches its peak value (3^*4^*5), outward
current increases dramatically, which could be interpreted as activation of a Ca2+-dependent K+ current. The concomitant slight
increase in inward current could be caused by the inward rectifier or the activation of a Cl~ current. The small shift of reversal
potential to a more positive value, however, suggests that a nonselective cation current is probably activated as well Note the
considerable linearization of the I-V relation. Traces 6 and 7 (panel D) show return of the I-V relation to its normal N-shape, and
[Ca2+], approaches control levels again.
uisite to understanding the basic electrophysiology and
roles of various ion channels in the plasmalemma. The
wide variability in observed resting potentials may
reflect differences among species, level of the vascular
tree, and tissue culture conditions. However, accurate
measurement of the resting potential can be problematic in small single cells with high input impedance, in
which the magnitude of the leakage conductance due to
seal resistance becomes critical. The size of the leakage
conductance determines the degree of variance between
the true and measured resting potential. On the other
hand, measurement of resting potential in a confluent
monolayer is less dependent on seal resistance. Mehrke
et al213 have demonstrated that the histogram of the
resting potentials of bovine aortic endothelial cell
monolayers is bimodal, with one peak around —25 mV
and another peak around - 8 5 mV. The two peak values
and the transition between them can be explained by an
N-shaped current-voltage relation of the endothelia]
cell membrane. In contrast, resting potential measure-
ments in cultured guinea pig coronary endothelial cell
monolayers showed a reasonable Gaussian distribution
around -35 mV. One interpretation of these differences in resting potential values is that the regional
differences in electrophysiological properties exist between endothelial cells at different levels of the vascular
tree.
Ionic currents. Numerous studies of voltage-clamped
bovine aortic and pulmonary artery endothelial cells
have shown that the basal current-voltage relation of
these cells inwardly rectifies near the resting potential
(Figure 3 and Table 3). In the range of potentials from
- 4 0 to +50 mV, endothelial cells display very small,
time-independent outward currents, whereas hyperpolarization to voltages negative to the reversal potential
results in a large inward current (Figure 3). Unitary
current recordings obtained using the patch-clamp technique demonstrate a channel with permeation and
gating properties comparable to those reported for the
inward rectifier in other cell types. Experiments per-
120
Hypertension
Vol 21, No 1 January 1993
formed by other investigators have shown that the
single-channel conductance estimated from inward current was 25-27 pS, and no evidence of current reversal
was observed (Table 3). As Campbell et al183 have
pointed out, this time-independent inward rectifying
background current is the primary determinant of resting potential in these cells. It has been postulated that
endothelial cells are electrically coupled not only to
each other but also to VSMCs.1718 In fact, in terminal
arterioles, numerous gap junctions between endothelial
cells and the underlying one or two layers of VSMCs
have been found in a recent electron microscopic study
on primate hearts. Myoendothelial gap junctions were
found to be more abundant in coronary resistance
vessels than in large coronary arteries.214-218 Thus, the
maintenance of a relatively hyperpolarized resting potential has been postulated to be of importance to both
endothelial cell function and VSMC relaxation.1718^219
Repeated studies have failed to demonstrate the
presence of a delayed rectifier current in endothelial
cells. However, one investigator183 noted the presence
of a fast-rising, rapidly inactivating outward current
analogous to the transient outward or A current in
approximately one third of the cells analyzed.
Agonist-Evoked Conductance Changes
Transmembrane potential measurements after exposure to a variety of agonists (e.g., bradykinin and ATP)
have revealed a transient hyperpolarization that parallels the agonist-induced transient rise in cytosolic Ca2+
levels. The magnitude of the hyperpolarization depends
on the resting potential recorded in the cell. In cells
with low resting potential (-10 to - 3 0 mV), agonists
induced a large hyperpolarization, whereas in those
with membrane potential more negative than - 8 0 mV,
the hyperpolarization was much smaller in amplitude .18^21^213 Although Ca2+ influx occurs during the
delayed phase of the Ca2+ transient, the anticipated
depolarization does not occur, because the magnitude
of the inward current is too small to offset the outward
current generated by the Ca2+-activated K+ channel or
the Ca2+-actfvated Cl~ channel.
The absence of voltage-gated Ca2+ channels in endothelial cells obtained from conduit vessels suggests that
exposure to an agonist such as bradykinin activates a
receptor-mediated Ca2+ influx pathway,184 which contributes to the Ca2+ transient in these cells (a receptoroperated Ca2+ channel has also been described in other
cells; see References 220-222). Johns et al184 have
reported that thrombin and bradykinin rapidly activate
a large inward current in bovine pulmonary artery
endothelial cells. Ionic substitution experiments suggest
that the influx pathway does not discriminate between
Na+ and Ca2+; i.e., the pathway is nonselective. On the
other hand, other investigators have failed to demonstrate an inward current during the early stages (<30
seconds) after exposure to bradykinin192 (Figure 3). One
can dismiss the explanation that those cells failing to
demonstrate this inward current during the early stages
were unresponsive to bradykinin, because an increase in
outward current resulting from activation of calciumactivated K+ channels was reported in one such study
(see below). One explanation for this apparent discrepancy is that in the experiments reported by Johns et
al,184 shear stress was generated by the flow of agonist
solution over the cell surface. However, it should also be
noted that, although a variety of nonselective cation
channels have been described in endothelial cells (Table 3), it is not known if endothelial cells from different
species or different levels of the vascular tree have
different types (isoforms) of the nonselective channels
expressed in their plasmalemma.
In contrast to the controversy over the inward current
changes early during exposure to the agonist, we and
others75'190'193'20*223 (see Figure 3) have observed a
delayed (>30 seconds) increase in inward current that
developed after exposure to agonist. Bregestovski et
al190 and, more recently, Nilius and Riemann193 have
described an inward, nonselective cation current evoked
by histamine in human umbilical vein endothelial cells,
which showed a latency of >60 seconds and was mimicked by application of A23187 (also compare Reference 209). The unitary conductance of 20-26 pS and
the reversal potential measurements suggest that the
channel is nonselective, although when internal K+ was
replaced with Na + , only inward currents were observed.
Histamine in the pipette solution was unable to elicit
single-channel activity but clearly induced single-channel activity in cell-attached membrane patches if present in the bath solution. Although both extracellular
and cytosolic Ca2+ were necessary for histamine to
activate the channel, Bregestovski's experiments190 allow no unequivocal conclusion with respect to the
activation mechanism. These unitary currents have also
been observed in the absence of agonist, as has a higher
conductance, nonselective cation channel.208 A more
recent study has demonstrated a lower conductance,
Ca2+-permeable, IP4- and Ca2+-sensitive channel in
endothelial cells.75 With the use of Mn2+ as the charge
carrier and patch-clamp techniques (cell-attached, inside-out, and outside-out patches), single-channel activity was recorded with a slope conductance of 2.5 pS and
a mean open time of 230 msec. IP3 was ineffective, and
inositol 1,3,4,5,6-pentakisphosphate (IP5) was much less
effective than IP4 in enhancing channel activity. Finally,
n \ did not activate the channel at a Ca2+ concentration
of 0.1 fiM, suggesting some cooperatrvity between Ca2+
and rP4 in activating the channel. Although this channel
shares many of the attributes of the receptor-operated
channel predicted from whole-cell recordings, the identification of more than one type of channel that is
permeable to Ca2+ in the plasmalemma suggests that
additional experiments will be needed to clarify the
relative contributions of these pathways to Ca 2+ influx
in endothelial cells.
Recent studies have provided some insight into the
ionic mechanism underlying the transient hyperpolarization of membrane potential that has been observed
after exposure to a variety of agonists, such as bradykinin. Voltage-clamp studies have demonstrated that
when intracellular Ca2+ concentration is not buffered,
exposure to an agonist results in a transient increase in
outward current197 (Figure 3).
Single-channel studies in cell-attached and inside-out
patches using standard patch-clamp technique have
demonstrated a large, calcium-activated K+ channel
(150±10 pS) that is also voltage dependent. The physiological relevance of this channel is uncertain, as it was
observed infrequently.203 Another calcium-activated K+
channel was observed when ATP was applied outside
Himmel et al Calcium and Currents in Endothelial Cells
the pipette. Since the same channel was activated when
the cell surface around the pipette was exposed to
A23187, the authors concluded that exposure to ATP
activated a Ca2+-dependent channel.204 The unitary
conductance at negative potentials was 40±2 pS ([K+],
212 mM), and the conductance became increasingly
nonlinear near the zero current potential value. Open
probability, which was analyzed in inside-out patches,
was found to be negligible (Po<0.05) at [Ca2+] of 0.4 /AM
and increased substantially at [Ca2+] >1.0 jtM. This Po
versus [Ca2+] relation implies that, during a typical Ca2+
transient, the open probability does not attain its maximal value. As can be seen in Figure 3, the increase in
outward currents follows the increase in [Ca2+]1; although the exact relation between I^cj or open probability of the calcium-activated K+ channel and [Ca2+]i in
single endothelial cells remains to be established. Thus,
our understanding of the role of the different calciumactivated K+ channels during agonist-evoked responses
is still incomplete. The combination of fluorescent calcium indicator and electrophysiological techniques has
been used to identify which channels were activated
after the increase in intracellular calcium concentration
induced by exposure to agonists.192 The experiments
showed that variable outward currents could be elicited
during successive voltage ramps, apparently because of
the opening and closing of one or more large channels.
Because channel openings were sufficiently long at only
one voltage (+40 mV), conclusions regarding the conductance of one of the channels were limited; therefore,
direct validation of the channel by type that is activated
during the calcium transient is necessary. Additional
experiments using simultaneous measurement of [Ca2+]i
and single-channel currents are still needed to resolve
the unanswered questions concerning regulation of
channel gating, relative contribution of different channel types to the calcium-activated potassium current,
and identification of relative apparent Kj values for
different pharmacological agents in blocking these currents. Although a variety of Ca2+-activated K+ channels
in VSMCs and in fibroblasts have been reported to be
sensitive to charybdotoxin, iberiotoxin, or tetraethylammonium ions,224-227 this has yet to be confirmed in
endothelial cells.
It is of interest that a muscarinic-activated K+ current
has also been identified in bovine and rabbit aortic endothelial cells. The cholinergjc K+ current is also inwardly
rectifying, with a slope conductance of approximately 80
pS/pF at -110 mV (see Table 3; References 17 and 202).
As a result, activation of this current also contributes to
the hyperpolarization induced by acetylcholine.
Mechanically Evoked [Ca2+] and
Conductance Changes
Vascular endothelium is subjected to flow-related
forces (shear stress, pressure, stretch), the principal one
of which is shear stress. It is sensed by the endothelial
surface and varies considerably (20-50-fold) throughout
the vascular system (for review, see Reference 228).
Shear stress evokes changes in morphology and release
of vasodilators,228^29 which may be mediated in part by
changes in membrane conductance properties and cytosolic Ca2+ levels. Cytosolic Ca2+ measurements have
revealed that endothelial cells may respond to shear
stress with an initial transient rise in [Ca ], followed by
121
a small, elevated plateau.140141 This response is uniformly
seen but can be quite variable. Perfusion of rabbit iliac
arteries at varying flow rates induced proportional increases in vessel diameter, which were antagonized by
N°-methyl-L-arginine.230 BaCl2 and antagonists of the
calcium-activated K+ channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation.230 When
cultured endothelial cells were added to a flow chamber
containing a vascular ring without endothelium and were
stimulated subsequently by flow, they relaxed the vascular ring, and this relaxation is inhibited by Ba 2+ , tetraethylammonium ion, or charybdotoxin.230 These data
suggest that flow activation of a potassium channel
(possibly the calcium-activated K+ channel) is necessary
for release of nitric oxide. However, it should be pointed
out that these data still do not exclude the possibility that
a hyperpolarization of the endothelial cells is propagated
electrotonically to VSMCs, contributing to their relaxation. Lansman et al187 have described single, stretchactivated channels in porcine aortic endothelial cells.
These channels were activated by applying suction to the
patch-pipette in the cell-attached configuration and were
shown to be cation selective and Ca2+ permeable. As a
result, Ca2+ influx through these channels might be
sufficient to account for the elevation in cytosolic Ca2+
and release of vasodilators during exposure to shear
stress.
A different type of shear stress-activated current has
been described in bovine aortic endothelial cells.210
Using flows that resulted in wall shear stress comparable to those occurring in the arterial system in vivo,
Olesen et al210 demonstrated the transient activation of
an inwardly rectifying, highly selective K+ current. This
flow-induced current caused a hyperpolarization of endothelial cells, which could be propagated to electrotonicalry coupled VSMCs, resulting in their relaxation.
Pumps, Exchangers, and Cotransporters
In addition to the ion channel-mediated permeation
pathways, a Na+.K^-ATPase,18*189-231 a Na+-Ca2+ exchanger,232"234 a Na + -H + exchanger,200'231 •235 and a Ca 2+ ATPase236-237 have been demonstrated in the plasmalemma of endothelial cells (see Figure 1). Several
groups238-241 provided evidence that a Na+-K+-Cl"
cotransport system is a major pathway for Na+ and K+
transport in bovine aortic endothelial cells. The ion flux
by this transporter can be modulated by a variety of
compounds: It is increased by Ca 2+ , bradykinin, vasopressin, and angiotensin II and decreased by acetylcholine, histamine, norepinephrine, and the cyclic nucleotides cyclic AMP and cyclic GMP.
Although the maintenance of resting [Ca2+] in endothelial ceDs appears to be little affected by the voltagedependent Na+-Ca2+ exchanger,233 its activity could
be modulated indirectly by compounds that affect the
Na+,K+-ATPase or the Na+-K+-CT cotransporter, thus
changing electrochemical gradients. Yet another modulatory role in [Ca 2+ ]| homeostasis could be played by
the Na + -H + exchanger, because, among other processes,
intracellular Ca2+ release, Ca2+-ATPases, and Ca2+
binding depend on the pH. 235 Thus, pumps, exchangers,
and cotransporters could influence the milieu in which
aforementioned mechanisms of [Ca2+]i homeostasis are
active.
122
Hypertension
Vol 21, No 1 January 1993
Future Directions and Conclusions
As knowledge of endothelial cell function has
evolved, our understanding of Ca2+ homeostasis has
markedly increased. Ca2+ influx into the cytosol from
both the ER and the extracellular space through the
plasma membrane contribute to the Ca2+ transient.
Although endothelial cells are inexcitable, Ca ^-permeable channels (Table 3) are responsible for Ca2+ influx,
whereas Ca2+-activated K+ channels and inward rectifier K+ channels are responsible for maintaining the cell
at a relatively hyperpolarized potential during the plateau phase of the Ca2+ transient to maximize the
calcium electrochemical gradient. The redundancy in
ion channel types points to the importance of these
pathways in the function of endothelial cells as well as
the need for identifying their relative contribution to
Ca2+ and K+ fluxes. In addition, there is little information about the blocking action of a variety of compounds
on the individual currents. This limits the extrapolation
of the contribution of a particular current to the function of the endothelial cell.
Many clinical and laboratory studies suggest the
involvement of the endothelium in disease states such as
atherosclerosis, hypertension, and heart failure. However, the pathophysiological basis of endothelial cell
dysfunction remains to be elucidated. More information
is also needed with respect to signal transduction pathways and receptor subtypes mediating the actions of
endothelium-dependent vasoactive compounds.
One major problem that has not yet been mentioned
is the source or sources of the available evidence. A
number of studies have dealt with the remarkable
regional heterogeneity of the regulation of vascular tone
and have shown differences not only between arterial
and venous vasculature but also between large arteries,
arterioles, and capillaries. Small arteries, arterioles, and
capillaries are generally considered the most important
with respect to systemic vascular resistance. However,
the majority of endothelial cell data available and
presented in this paper has been collected from large
arteries of multiple animal species, as well as from
human umbilical vein endothelial cells, which are probably not representative of the endothelial cell from the
resistance vessels. Therefore, our present ideas should
be reevaluated in microvascular endothelial cells.
Finally, the application of molecular biological techniques to study ion channels in endothelial cells will be
of interest not only to investigators studying these cells
but also to investigators studying ion channels in general, given the plethora of stretch-activated, receptoroperated, and nonselective channels in endothelial
cells.
Acknowledgment
We thank Steffani Webb for invaluable editorial assistance
during the preparation of this manuscript.
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