Am J Physiol Lung Cell Mol Physiol
280: L1138–L1147, 2001.
Potassium channels regulate tone in rat pulmonary veins
EVANGELOS D. MICHELAKIS,1 E. KENNETH WEIR,2 XICHEN WU,1 ALI NSAIR,1
ROSS WAITE,1 KYOKO HASHIMOTO,1 LAKSHMI PUTTAGUNTA,3
HANS GUNTHER KNAUS,4 AND STEPHEN L. ARCHER1
Departments of 1Medicine (Cardiology) and 3Pathology, University of Alberta,
Edmonton, Alberta T6G 2B7, Canada; 2Department of Medicine (Cardiology),
Veterans Affairs Medical Center, Minneapolis, Minnesota 55455; and 4Department
of Biochemical Pharmacology, University of Innsbruck, Innsbruck 6020, Austria
Received 28 September 2000; accepted in final form 13 December 2000
return oxygenated blood from
the lungs to the left heart. Intrapulmonary vein (PV)
tone contributes significantly to the total pulmonary
vascular resistance (7, 22). PVs constrict in response
to a variety of agonists, including catecholamines
and serotonin (20), as well as to hypoxia (1, 45) and
dilate in response to alkalosis (17). Although PV
constriction has been implicated in the pathogenesis
of pulmonary edema due to both congestive heart
failure (13) and ascent to high altitude, (15, 23), this
has not been systematically studied (32). In addition,
although it is known that cells closely resembling
cardiomyocytes (CMs) are present in the media of
PVs in many mammals, a possible role of this “pulmonary myocardium” in the control of PV tone in
health and disease has not been explored (26, 29).
Very recently, it was reported that many patients
with atrial fibrillation have an ectopic electrical focus originating within the PVs (16). Therefore, PV
tone and electrophysiology may have important implications in human disease.
K⫹ channels play a major role in the control of
vascular tone in most vascular beds (28). In addition,
K⫹ channels in CMs, through their role in repolarization, determine action potential duration and
thus are important in the pathogenesis of arrhythmias. Studies with K⫹ channel blockers and openers
in perfused organs (5, 6) and vascular rings (10, 36)
have previously suggested a role of K⫹ channels in
the modulation of PV tone. However, the molecular
identity and the electrophysiology of K⫹ channels in
isolated muscle cells from the PVs have never been
studied. When K⫹ channels in vascular smooth muscle cells (SMCs) are inhibited, the basal efflux of K⫹
(down an intracellular/extracellular concentration
gradient of 140/4 mM) is decreased, and the cell
membrane depolarizes. This leads to the opening of
voltage-gated Ca2⫹ channels, inflow of Ca2⫹, and
contraction. In resistance pulmonary artery SMCs,
inhibition of voltage-gated K⫹ (KV) channels,
whether by 4-aminopyridine (4-AP) (2), hypoxia (4,
30, 42), endothelin (33, 34), or dexfenfluramine (38),
results in depolarization, the opening of voltagegated L-type Ca2⫹ channels, and vasoconstriction.
Thus we examined the hypothesis that K⫹ channels
are important in the control of resting PV tone. We
showed that K⫹ channels are functional and physiologically significant (blocking of K⫹ channels causes
PV constriction). To our knowledge, this is the first
description of the molecular identity and basic electrophysiology of K⫹ channels in the PVs.
Address for reprint requests and other correspondence: E. D.
Michelakis, Dept. of Medicine (Cardiology), Univ. of Alberta, WCM
Health Science Center, 8440 112 St., Edmonton, AB T6G 2B7,
Canada (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
inward rectifier potassium channels; voltage-gated potassium channels; venous tone; pulmonary circulation; pulmonary edema
THE PULMONARY VEINS
L1138
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.2 on June 14, 2017
Michelakis, Evangelos D., E. Kenneth Weir, Xichen
Wu, Ali Nsair, Ross Waite, Kyoko Hashimoto, Lakshmi
Puttagunta, Hans Gunther Knaus, and Stephen L. Archer. Potassium channels regulate tone in rat pulmonary
veins. Am J Physiol Lung Cell Mol Physiol 280: L1138–L1147,
2001.—Intrapulmonary veins (PVs) contribute to pulmonary
vascular resistance, but the mechanisms controlling PV tone
are poorly understood. Although smooth muscle cell (SMC) K⫹
channels regulate tone in most vascular beds, their role in PV
tone is unknown. We show that voltage-gated (KV) and inward
rectifier (Kir) K⫹ channels control resting PV tone in the rat.
PVs have a coaxial structure, with layers of cardiomyocytes
(CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have
both an inward current, inhibited by low-dose Ba2⫹, and an
outward current, inhibited by 4-aminopyridine. In contrast,
PVSMCs lack inward currents, and their outward current is
inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several KV, Kir, and large-conductance Ca2⫹-sensitive K⫹
channels are present in PVs. Immunohistochemistry showed
that Kir channels are present in PVCMs and PV endothelial
cells but not in PVSMCs. We conclude that K⫹ channels are
present and functionally important in rat PVs. PVCMs form
sphincters rich in Kir channels, which may modulate venous
return both physiologically and in disease states including pulmonary edema.
L1139
POTASSIUM CHANNELS IN PULMONARY VEINS
METHODS
Table 1. Design and cycling parameters
of K⫹ channel primers
Kv1.5
Kv2.1
Kv9.3
Kv4.3
Kir2.1
Kir3.1
Kir6.1
BKCa
␤-Actin
GenBank
Accession No.
Nucleotides
M27158
X16476
Y17607
U75448
AF021137
Y12259
D42145
AF135265
V01217
795–1242
319–736
1,565–2,132
1,502–1,859
119–402
699–1,451
430–811
866–1,499
2,160–2,445
Size, bp
Annealing
Temperature,
°C
Cycles
446
418
569
394
284
752
382
634
285
60
58
55
56
58
56
58
62
57
32
33
34
34
34
34
34
34
30
Kv, voltage-gated K⫹ channel; Kir, inward rectifier K⫹ channel;
BKCa, large-conductance Ca2⫹-sensitive K⫹ channels.
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Adult male Sprague-Dawley rats (250–350 g body wt) were
euthanized by a pentobarbital sodium overdose. PVs were
removed (fourth to fifth division from the left atrium) and
placed in cold Hanks’ balanced salt solution (HBSS). They
were then either used for the study of tone in isolated tissue
baths, fixed for immunohistochemistry and electron microscopy, enzymatically dispersed for patch clamping, or homogenized for immunoblotting and RT-PCR. All drugs were purchased from Sigma (St. Louis, MO) unless stated otherwise.
Tissue baths. Fourth to fifth division PV rings (internal
diameter 100 ⫾ 10 ␮m; n ⫽ 18) with intact endothelium
(preserved relaxation to 10⫺7, 10⫺6, and 10⫺5 M acetylcholine) were studied in Earle’s solution (37°C, pH 7.43 ⫾ 0.01,
PO2 110 ⫾ 5 mmHg), as previously described (2). Optimal
resting tension (at which maximal constriction to 80 mM KCl
occurred) was found to be 500 mg. The effects of 5 mM 4-AP
(pH 7.4), 10 ␮M glyburide (in ethanol), 5 mM tetraethylammonium (TEA), 100 nM iberiotoxin, 0.005–1 mM barium
chloride (Ba2⫹), and the vehicles were determined. In additional experiments, PV rings were denuded with a piece of
silk suture. The effects of 10⫺7, 10⫺6, and 10⫺5 M ACh and
Ba2⫹ were compared in intact versus endothelium-denuded
PV rings. Because ACh causes relaxation, in part by the
opening of K⫹ channels, these rings were preconstricted with
10⫺4 M PGF2␣ instead of KCl.
Cell dispersion and patch clamping. The adventitia was
removed, and the veins were opened longitudinally and
placed in Ca2⫹-free HBSS containing (in mM) 140 NaCl, 4.2
KCl, 1.2 KH2PO4, 1.5 MgCl2, 10 HEPES, and 0.1 EGTA, pH
7.4, for 20 min. They were then transferred to HBSS (4°C for
15 min) that contained (in mg/ml) 1.0 papain, 0.75 dithiothreitol, 0.8 collagenase, and 0.8 BSA. Next, the PVs were
heated to 37°C for 10 min and transferred to iced HBSS
supplemented with 1 mg/ml of glucose and triturated with a
Pasteur pipette. The cells were transferred to a perfusion
chamber on the stage of an inverted microscope for patchclamp studies and were left to attach for 10–15 min.
Whole cell patch-clamp recordings were performed as previously described (38). Electrodes (resistance 1–5 M⍀) were
filled with a solution that contained (in mM) 140 KCl, 1.0
MgCl2, 10 HEPES, 5 EGTA, and 10 glucose, pH 7.2. The
chamber containing the cells was perfused (2 ml/min) with a
solution containing (in mM) 145 NaCl, 5.4 KCl, 1.0 MgCl2,
1.5 CaCl2, 10 HEPES, and 10 glucose, pH 7.4 (extracellular
solution). The cells were voltage clamped at a holding potential of ⫺70 mV, and currents were evoked by steps from ⫺130
or ⫺110 to ⫹50 mV with 0.1-Hz test pulses of 200 ms
duration. Currents were filtered at 1 kHz and sampled at 2 or
4 kHz. Junction potentials were corrected, and series resistance was compensated ⬃80%. Data were recorded and analyzed with pCLAMP 6.02 software (Axon Instruments, Foster City, CA). Various K⫹ channel blockers were perfused in
random order. In some experiments, to study the “Ba2⫹sensitive” current, increasing doses of Ba2⫹ were superfused
onto cells in the presence of 5 mM 4-AP, 10 mM TEA, 10 ␮M
lanthanum (La; a nonspecific Ca2⫹ entry blocker), and 0.5
␮M TTX (a Na⫹ channel blocker). The doses were selected
based on preliminary experiments and previous work on
pulmonary artery (PA) SMCs. (2, 38)
Immunoblots. Immunoblots were performed as previously
described (4). We used a battery of K⫹ channel antibodies in
immunoblotting experiments on homogenized PVs and PAs
(fourth to fifth division) and brain (see Fig. 5A). Homogenates
of PVs and PAs were suspended in a buffer containing 10 mM
Tris, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 mM
EDTA, 0.1% Triton X-100, and 0.05 M dithiothreitol. Sam-
ples were then sonicated, and the proteins were isolated.
Equal amounts of PV and PA protein (25 ␮g) were loaded and
run on a 7.5% discontinuous SDS-polyacrylamide gel and
then transferred to a nitrocellulose membrane. Brain (7.5 ␮g
protein/lane) was used as a standard because it is abundant
in most types of K⫹ channels. The membrane was probed
with the primary anti-K⫹ channel antibody for 4 h (1:100–1:
500 dilution in Tris-buffered saline containing Tween 20 and
3% BSA) and subsequently incubated with horseradish peroxidase-linked secondary antibodies (1:3,000 dilution; Pierce,
Rockford, IL). Bands were visualized with enhanced chemiluminescence substrate (Amersham, Uppsala, Sweden). Antibody specificity was confirmed by competition experiments
in which antibodies were preincubated with the relevant
antigens at a ratio of 4:1 (except for Kv3.1, Kir2.1, and L-type
Ca2⫹ channel where the ratio was 1:1) for 1 h at room
temperature. All K⫹ channel antibodies were polyclonal and
obtained from Alomone Laboratories (Jerusalem, Israel) except the large-conductance Ca2⫹-sensitive K⫹ (BKCa) channel antibody, which was provided by Dr. Hans Gunther
Knaus.
RT-PCR. Total RNA was isolated from homogenized rat
PAs and PVs with a QIAGEN RNeasy Mini Kit (Missisauga,
ON). RNA (2 ␮g) was reverse transcribed with QIAGEN
Omniscript reverse transcriptase. Primers were designed
based on cloned rat sequences from GenBank. cDNA (1 ␮l)
was incubated with 150 ng of sense and 150 ng of antisense
primers and amplified in QIAGEN HotStarTaq Master Mix.
The cycling parameters were 95°C for 15 min, X°C for 1 min,
and 72°C for 1 min, where X is the annealing temperature
(Table 1) for the first cycle and 94°C for 30 s, X°C for 30 s, and
72°C for 1 min for the second to last cycle. The amplified PCR
products were run on ethidium bromide-stained 2% agarose
gels. All PCR products were sequenced, and a BLAST search
was run that confirmed the specificity of the product.
Immunohistochemistry. Immunohistochemistry was performed on paraffin-embedded, formaldehyde-fixed lungs
counterstained with hematoxylin as previously described (3).
We used primary antibodies against Kir channels (1:50 dilution for all three Kir antibodies used) as well as against
myosin heavy chain (MHC) or smooth muscle actin (SMA),
both diluted 1:200 (Chemicon International, Temecula, CA).
The tissue was exposed to primary antibody for 16 h at 4°C
and biotinylated secondary antibody for 20 min at 25°C (1:20
dilution; Link Biogenex Laboratories, San Ramon, CA). After
exposure to streptavidin peroxidase (20 min at 37°C), the
bands were revealed with diaminobenzidine.
Statistics. Values are expressed as means ⫾ SE. Intergroup comparisons were performed with Student’s t-test or a
L1140
POTASSIUM CHANNELS IN PULMONARY VEINS
factorial ANOVA as appropriate. Fisher’s probable least significant differences test was performed for post hoc comparisons.
RESULTS
Fig. 1. K⫹ channels regulate tone in
rat intrapulmonary veins (PVs). A:
mean data (left) and a representative
trace (right) of the effects of K⫹ channel blockers on PV tone. TEA, tetraethylammonium; IBTX, iberiotoxin; Glyb,
glyburide. Values are percent constriction to 80 mM KCl; n, no. of veins.
4-Aminopyridine (4-AP) and Ba2⫹ constricted PVs, suggesting that voltagedependent (KV) and inward rectifier
(Kir) K⫹ channels are important determinants of PV tone. Nifedipine significantly inhibited constriction to 4-AP,
suggesting that 4-AP causes constriction through depolarization and opening of the voltage-dependent L-type
Ca2⫹ channels. *P ⬍ 0.02 for dose-response effect of Ba2⫹. B: PV rings denuded of endothelium lost ACh-induced relaxation (left) and constricted
to low doses of Ba2⫹ (right). This suggests that Ba2⫹ constricts the PV muscle cells directly through Kir channel
inhibition.
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KV and Kir channels control PV tone. To determine
whether K⫹ channels play a role in the control of PV
tone, we studied the effects of various K⫹ channel
blockers on isolated PV rings with intact endothelium
in tissue baths (Fig. 1A). 4-AP caused significant constriction at 5 mM, a dose that preferentially blocks KV
channels (4). To determine whether KV channel inhibition causes constriction through depolarization and
opening of the voltage-gated L-type Ca2⫹ channels, we
studied the effects of 10 ␮M nifedipine on the 4-APinduced constriction (n ⫽ 3 rings). Nifedipine does not
itself alter tone, but it significantly inhibits 4-AP-induced constriction (Fig. 1A). Glyburide (10 ␮M) had no
effect on PV tone. TEA (5 mM) caused minimal constriction. Moreover, in four of the rings that constricted
in response to TEA, iberiotoxin, a more specific BKCa
channel blocker (10⫺7 M), had no effect on ring tension.
This suggested that the small amount of constriction to
TEA was not due to BKCa channel inhibition but rather
was the result of nonspecific inhibition of other classes
of K⫹ channels. Ba2⫹ causes a dose-dependent constriction in PVs at a threshold dose of 10 ␮M (Fig. 1A).
This dose is in the range that preferentially inhibits Kir
channels (28).
Endothelium-denuded PV rings lose dilatation to
ACh (Fig. 1B) but retain constriction to low doses of
Ba2⫹ (threshold dose, 5 ␮M). This suggests that Ba2⫹
acts directly on the PV media to cause constriction. The
endothelium-independent effects of Kir channel inhibition are important because it is known that the endothelium is abundant in Kir channels and that the
vasoconstrictive effects of Ba2⫹ might theoretically
have been mediated by the endothelium alone. We next
performed patch-clamping experiments to assess the
effects of these K⫹ channel blockers on K⫹ currents in
PV smooth muscle cells (Figs. 2 and 3).
Phenotypically and electrophysiologically distinct
SMCs in PVs. Enzymatic dispersion of fresh PVs
(fourth to fifth division) revealed two phenotypically
and electrophysiologically distinct cell types: 1) large
(length 32 ⫾ 7 ␮m) striated cells indistinguishable
from CMs on light microscopy (PVCMs; Fig. 2) that
beat spontaneously at ⬃60 Hz and 2) smaller (length
8 ⫾ 2 ␮m) nonstriated, spindle-shaped cells that resembled typical vascular SMCs seen in rat PAs (2)
(PVSMCs; Fig. 2). Whole cell patch-clamp recordings
showed that the two cell types have different electrophysiological phenotypes as well. PVCMs (n ⫽ 13) were
characterized by a rapidly inactivating, A-type current
and a significant inward current at hyperpolarizing
voltage steps (Fig. 2). In contrast, PVSMCs had a
noninactivating outward current and no inward current when isolated from the same veins and studied
with the same voltage-step protocol (Fig. 2). The appearance of the outward current of the PVSMCs, with
its low-amplitude spiky morphology, was consistent
with the idea of a large component of the current
resulting from BKCa channels. In PVCMs, most of the
outward current was inhibited by the KV channel
blocker 4-AP (5 mM; n ⫽ 7 experiments), whereas TEA
(5 mM; n ⫽ 5 experiments) and glyburide (10 ␮M; n ⫽
4 experiments) had no effect (data not shown). The
inward current was almost completely eliminated by
Ba2⫹ but was not affected by 4-AP (Fig. 2), TEA, or
glyburide (data not shown). In PVSMCs (n ⫽ 7 experiments), although the outward current was partially
inhibited by 4-AP (n ⫽ 6 experiments), it was significantly inhibited by 5 mM TEA (Fig. 2). Glyburide and
POTASSIUM CHANNELS IN PULMONARY VEINS
L1141
100 ␮M Ba2⫹ had no effects on PVSMC K⫹ current
(n ⫽ 4 experiments).
To determine the relative contribution of Kir channels to the inward current, we superfused PVCMs with
the Na2⫹ channel blocker TTX (0.5 ␮M) and an inhibitor of Ca2⫹ entry to block Ca2⫹ channels, La (10 ␮M).
To determine the contribution of Kir channels to outward current, we added the KV channel blockers 4-AP
(5 mM) and TEA (10 mM), which nonspecifically inhibit K⫹ channels but do not inhibit Kir channels (28).
Figure 3A shows that the outward current of PVCMs is
sensitive to 4-AP and that TEA⫹TTX⫹La does not
inhibit the current further. It also shows that the
majority of the inward current is insensitive to this
cocktail of blockers, confirming that it is due to Kir
channels, rather than to Na2⫹ or Ca2⫹ channels (Fig.
3A). Figure 3, B and C, shows that Ba2⫹ inhibits this
inward current in the presence of 4-AP⫹TEA⫹
TTX⫹La. Ba2⫹ (100 ␮M) not only significantly inhibited the inward current, but it also inhibited some of
the outward current occurring at negative membrane
potentials. The threshold dose of Ba2⫹ for current inhibition was 10 ␮M (data not shown). ACh (0.1 ␮M), an
activator of Kir3 channels, activated mostly the inward
current in PVCMs (Fig. 3D).
PV sphincters. The presence of both PVCMs and
PVSMCs in the media was confirmed by electron microscopy (Fig. 4). PVCMs have features of classic CMs,
i.e, striations, intercalated disks, and multiple large
mitochondria, as seen in Fig. 4. PVCMs and PVSMCs
are arrayed in a coaxial pattern, with PVSMCs closer
to the vein lumen, in contact with the endothelium.
The PVSMCs are surrounded by a sheath of PVCMs
(Fig. 4). The PVCM layer attenuates as the vein is
traced back from the left atrium toward the capillary
bed, but it is still evident in PVs up to the sixth
division.
KV and Kir channels were present in PVs. All tested
K⫹ channels were present in PVs: Kv1.1, -1.2, -1.3,
-1.4, -1.5, -1.6, -2.1, -3.1, and -4.2; BKCa; and Kir1.1,
-2.1, -3.1, and -3.2 (Fig. 5). L-type Ca2⫹ channels were
also present in PVs (Fig. 5). The immunoblots in Fig. 5
correspond to the major immunoreactivity bands in the
brain, and the channel proteins we identified were
found at similar molecular weights to those previously
published (4, 8, 21, 35, 40). The specificity of the antibodies is shown by the effective competition with the
relevant antigens in Fig. 5B. In some cases (e.g., Kv1.1,
Kv1.5, and Kv3.1), secondary bands were seen close to
the main band. These secondary bands were also effectively competed away by the relevant antigen, implying that they represent posttranslational modification
of the channels, probably glycosylation and/or phosphorylation. There are differences in the relative
amount of the K⫹ channel protein expressed between
the PVs and PAs. For example, certain KV channels
(Kv1.1–1.3 and -3.1) and Kir channels (Kir1.1 and -3.1)
are more abundant in PVs than in PAs. In contrast,
Kv1.4, Kv1.5, Kv2.1, and BKCa are more abundant in
PAs (Fig. 4). Care was taken to load the same amount
of protein from PAs and PVs on the gels, and this was
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Fig. 2. Morphologically and electrophysiologically distinct smooth muscle
cells (SMCs) in the media of rat PVs.
Mean (⫾SE) data [current-voltage
(I-V) curves; left] and representative
traces (right) from whole cell patch
clamping of freshly isolated PV cardiomyocytes (CMs) (n ⫽ 13) and PVSMCs
(n ⫽ 7) from the 4th to 5th division of
rat PVs are shown. Insets: morphological differences between the PVCMs
(A) and PVSMCs (B). Doses were 5 mM
4-AP, 1 mM Ba2⫹, and 5 mM TEA.
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POTASSIUM CHANNELS IN PULMONARY VEINS
confirmed by similar Ponceau staining (data not
shown).
RT-PCR showed that mRNA for several K⫹ channels
(Kv1.5, Kv2.1, Kv4.3, Kv9.3, BKCa, Kir2.1, Kir3.1, and
Kir6.1) was present in the PVs (Fig. 6). Despite similar
amounts of ␤-actin mRNA, there were differences in
the amount of mRNA for some K⫹ channels in PAs
versus PVs. Whereas mRNA from Kv1.5 and BKCa was
more abundant in the PAs, mRNA from Kir3.1 was
more abundant in the PVs (Fig. 6). The differences in
the mRNA levels correlated with the differences in the
expressed proteins between the two types of vessels.
Kir channels are known to be abundant in the endothelium. Because homogenized vessels are used in immunoblotting and RT-PCR, to study the presence of Kir
channels directly in PV muscle, we performed immunohistochemistry.
The PV sphincters consist of CMs enriched in Kir
channels. Electron microscopy demonstrated that the
PVCMs closely resembled atrial CMs, whereas the
PVSMCs had the classic vascular SMC appearance
(Fig. 4). To better elucidate the phenotype of these two
cell types, we first performed immunohistochemistry
with antibodies against the CM-specific MHC and
SMA. Indeed, PVCMs were strongly positive for MHC
and negative for SMA (Fig. 7). In contrast, PVSMCs
were positive for SMA and negative for MHC (Fig. 7).
Kir1.1, Kir2.1, and Kir3.2 were abundant in the
PVCMs (Fig. 7), and these cells displayed inward current in patch clamping experiments (Figs. 2 and 3). In
contrast, these Kir channels were absent in PVSMCs
(Fig. 7), which did not display inward currents (Fig. 2).
Furthermore, Kir channels were also expressed in the
endothelial cells of PVs (Fig. 7). In summary, histology
revealed an intriguing structure in the intrapulmonary
PVs, composed of a spinchterlike arrangement of
PVCMs. The PVCMs formed circular arrays that surround medium-sized PVs. These spinchterlike structures stained intensely for several Kir channels.
DISCUSSION
To our knowledge, this is the first study of the molecular identity and basic electrophysiology of K⫹ channels in PVs. We used a multifaceted approach and
report two important findings. 1) KV and Kir channels
are important in the control of resting PV tone because
their blockade results in PV constriction, and 2)
PVCMs form spinchterlike structures that surround
the large- and medium-sized PVs with unique electro-
Downloaded from http://ajplung.physiology.org/ by 10.220.33.2 on June 14, 2017
Fig. 3. Kir currents in PVCMs. A: effects of 4-AP and 4-AP⫹TEA⫹TTX⫹ La on PVCM current density. The
outward current was 4-AP sensitive (as shown in Fig. 2) but not TEA, TTX, or La sensitive. The inward current was
essentially insensitive to all the above blockers. B and C: effects of low-dose Ba2⫹ on current density in the presence
of 4-AP⫹TEA⫹TTX⫹La over the more physiological membrane potentials ⫺110 to ⫺30 mV. C: representative
trace from B also showing the electrophysiology protocol. Note that 100 ␮M Ba2⫹ inhibited both the inward and the
outward current. D: ACh mostly increased the inward current in the presence of 4-AP⫹TEA⫹TTX⫹La. n, No. of
experiments. P values compared with control.
POTASSIUM CHANNELS IN PULMONARY VEINS
L1143
physiology characterized by the presence of functional
Kir channels.
K⫹ channels are important in the control of vascular
tone in most vascular beds (28). Our tissue bath experiments showed that this is also true for rat PVs. It
appears that KV channels control PV tone because
4-AP (but not TEA, iberiotoxin, or glyburide) significantly constricted PV rings (Fig. 1). The mechanism of
the 4-AP-induced constriction in PVs, as in the PAs (4,
41) or the rabbit (37) and human ductus arteriosus
(27), is thought to be depolarization, which, in turn,
enhances the opening of voltage-gated L-type Ca2⫹
channels. Nifedipine eliminated the 4-AP-induced vasoconstriction in all three tissues, confirming this hypothesis (Fig. 1).
In contrast to our findings, Halla et al. (17) showed
that 4-AP does not constrict piglet PV rings (500-␮M
diameter). Our PVs were significantly smaller (110-␮M
diameter). Our laboratory (2) has previously shown
that, at least in the rat, there is diversity in the expression and/or function of KV channels in proximal
versus distal PAs. KV current is predominant in distal
resistance arteries, whereas Ca2⫹-sensitive K⫹ (KCa)
current is predominant in proximal conduit arteries.
Consistent with this electrophysiological diversity,
4-AP constricts the distal much more than the proximal PAs (2). A similar diversity of KV channels in the
PVs could explain the different 4-AP effects observed
between the two studies. In addition, possible species
differences in the expression and function of PV KV
channels need to be considered.
Our pharmacology studies excluded a role of ATPsensitive K⫹ (KATP) or BKCa channels in regulating
resting tone in normal rat PVs. However, we did not
examine the role of these channels in conditions of
raised PV tone because it might occur in disease states.
It has been shown that KATP channel openers dilate
preconstricted porcine (10) and lamb (36) PV rings and
inhibit the PV constrictor response to serotonin in
perfused lungs in the dog (5). The effects of KATP
channel openers on PV tone were shown to be partially
(10) or entirely (36) endothelium dependent. In one of
these studies (36), glyburide (10 ␮M) was shown to
have no effect on basal PV tone. These data are in
agreement with our finding that glyburide had no
effect on isolated PVCM and PVSMC K⫹ current. Inhibition of KCa channels with TEA has been shown (6)
to increase PV tone and potentiate the effects of serotonin on PVs in perfused dog lungs. Although the dose
that was used is relatively small (1 mM), TEA is a
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Fig. 4. The “cardiac muscle” in PVs.
Electron microscopy from rat lung sections at the level of 5th division PVs is
shown. A: cross-section of striated cells
closely resembling CMs were arranged
in a circular manner external to a thin
layer of nonstriated SMCs. B: longitudinal section at the same magnification. C: box from A at higher magnification revealing characteristics of
typical CMs. E, endothelial cell; CL,
collagen fibers; ID, intercalated disks;
M, mitochondria.
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POTASSIUM CHANNELS IN PULMONARY VEINS
nonspecific KCa blocker. An indication of this nonspecificity is seen in Fig. 1. Although some of our PV rings
constricted slightly to TEA, the same rings did not
constrict to iberiotoxin, a specific BKCa blocker. It
Fig. 6. RT-PCR showed the presence of mRNA for several K⫹ channels in PVs and PAs (4th to 5th division). There were differences in
mRNA levels for certain channels between the PVs and PAs that
were concordant with the differences in protein expression in the
immunoblot experiments (see text). The lack of DNA contamination
is shown by the absence of any signal in the “no-RT” band. All
products were sequenced and found to be identical to the predicted
product (see also Table 1). Nos. at left, bp.
needs to be emphasized that diversity in expression
and function of K⫹ channels among species may explain the differences between the current study and
the work of others.
Ba2⫹ caused a dose-dependent constriction of PV
rings (Fig. 1). This is likely due to inhibition of the
outward component of Kir currents at the physiological
membrane potentials observed in PVCMs (Fig. 3, B
and C). The majority of the inward current in PVCMs
is sensitive to low doses of Ba2⫹ (⬍100 ␮M) but is
resistant to TTX and La. This provides strong evidence
that the 4-AP-TTX-TEA-La-resistant current (both inward and outward) is indeed a result of Kir channels. A
contribution of nonspecific cation channels, which have
been identified in human atria (14), to the inward
current of PVCMs cannot be excluded. The molecular
identity of the Kir current in PVCMs cannot be determined without single-channel patch clamping or molecular targeting studies and is complicated by the lack
of additional specific inhibitors of Kir channels. Very
recently, the important role of Kir2.1 in the control of
cerebral arterial tone was shown with the use of gene
targeting (44).
Although the molecular identity of the inward current remains uncertain, there are clues provided by
current morphology. PVCMs have a strongly inward
rectifying current, consistent with a role for Kir2.1, a
known strong inward rectifier found in vascular SMCs
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Fig. 5. K⫹ channels are expressed in rat PVs. A: immunoblots showed the presence of many types of K⫹ channels
in rat PVs and PAs (4th to 5th division) and brain. There were qualitative differences in the number of K⫹ channels
expressed in PVs vs. PAs (see text). BKCa, large-conductance Ca2⫹-sensitive K⫹ channel. B: antibody specificity
was confirmed by competition experiments in which antibodies were preincubated with their relevant antigens,
effectively diminishing immunoblot intensity as expected. Nos. at left, molecular mass in kDa.
POTASSIUM CHANNELS IN PULMONARY VEINS
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(12). In contrast, Kir1.1 is a weak inward rectifier (11),
suggesting that perhaps most of the Kir current in
PVCMs is due to Kir2.1. Members of the Kir3 family
are G protein-dependent channels and conduct current
only after stimulation with agonists like ACh. (31) The
fact that ACh increases the PVCM inward K⫹ current
suggests that the Kir3.1/3.4 heteromultimer, known to
be the ACh-sensitive channels in atrial myocytes (24,
39, 43), is also physiologically important in PVCMs.
The fact that ACh does not dilate the denuded PV rings
(Fig. 1B) might be partially explained by the lack of
significant effects of ACh on the PVCM outward current (Fig. 3D). We have shown that mRNA and expressed protein for both Kir2.1 and Kir3.1 are present
in PVCMs (Figs. 5–7; antibodies against Kir3.4 are not
commercially available).
Although Kir channels are abundant in the nervous system and in the myocardium, their presence
and function in the vasculature has only been reported in the coronary and cerebral microcirculation
(28). We are not aware of any reports showing the
presence of Kir1.1 (previously known as ROMK1) in
the vasculature. Here, we show that Kir1.1 is
strongly expressed in PVCMs and PV endothelial
cells with the use of both immunoblotting (Fig. 5)
and immunohistochemistry (Fig. 7). We remain uncertain as to its function.
Because Kir currents and Kir proteins are only
present in PVCMs and not in PVSMCs (Fig. 7), we
speculated that the observed PV constriction to Ba2⫹
was due to contraction of PVCMs in the outer layer of
the PV media. In other words, the presence of CMs in
the media conferred Ba2⫹ responsiveness to the PVs.
Paes de Almeida et al. (29) first systematically studied
the “cardiac muscle” in the PVs in 1975. They showed
that the PVCMs have action potentials and responses
to ACh similar to those in CMs from the left atrium.
Having demonstrated electrical continuity between the
left atrium and the PVs with propagation of the action
potentials toward the lung, they hypothesized that a
rhythmic, valvelike action of the “striated musculature” of the PV during atrial systole optimized forward
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Fig. 7. Sphincters enriched in Kir channels in PVs. PVs were immunostained (brown) with myosin heavy chain
(MHC) and smooth muscle actin (SMA) antibodies (A) or Kir antibodies (B and C). Background counterstaining was
with hematoxylin. PVCMs were MHC positive and SMA negative, whereas the PASMCs were SMA positive and
MHC negative. Note that the alveoli in A, middle, show that these PV segments were intrapulmonary. Kir channels
were present in the PVCM and PV endothelial cells but not in PVSMCs. PVCMs were arranged in a coaxial and
spinchterlike manner, clearly shown in the PV transverse sections. E, endothelium.
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POTASSIUM CHANNELS IN PULMONARY VEINS
E. D. Michelakis and S. L. Archer were supported by the Alberta
Heritage Foundation for Medical Research, the Heart and Stroke
Foundation of Canada, and the Canadian Institutes for Health
Research. E. D. Michelakis was also supported by the Alberta Lung
Association. E. K. Weir was supported by the Minnesota Heart
Association and a Veterans Affairs Merit Review grant.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
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