Departments of 1 Medicine (Cardiology) and 3 Pathology, University of Alberta, Edmonton, Alberta T6G 2B7, Canada; 2 Department of Medicine (Cardiology), Veterans Affairs Medical Center, Minneapolis, Minnesota 55455; and 4 Department of Biochemical Pharmacology, University of Innsbruck, Innsbruck 6020, Austria
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ABSTRACT |
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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.
inward rectifier potassium channels; voltage-gated potassium channels; venous tone; pulmonary circulation; pulmonary edema
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INTRODUCTION |
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THE PULMONARY VEINS 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 voltage-gated 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.
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METHODS |
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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 µm; n = 18) with intact endothelium (preserved
relaxation to 107, 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 µM 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 patch-clamp 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 MImmunoblots. 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. Samples were then sonicated, and the proteins were isolated. Equal amounts of PV and PA protein (25 µg) were loaded and run on a 7.5% discontinuous SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane. Brain (7.5 µg 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 µg) was reverse transcribed
with QIAGEN Omniscript reverse transcriptase. Primers were designed
based on cloned rat sequences from GenBank. cDNA (1 µl) 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.
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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 factorial ANOVA as appropriate. Fisher's probable least significant differences test was performed for post hoc comparisons.
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RESULTS |
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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 µM nifedipine on the
4-AP-induced constriction (n = 3 rings).
Nifedipine does not itself alter tone, but it significantly inhibits
4-AP-induced constriction (Fig. 1A). Glyburide (10 µM) 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 (107 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 µM (Fig.
1A). This dose is in the range that preferentially inhibits
Kir channels (28).
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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 µm) striated cells indistinguishable from CMs on light microscopy (PVCMs; Fig. 2) that beat spontaneously at ~60 Hz and 2) smaller (length 8 ± 2 µm) 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 µM; 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 100 µM 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 µM) and an inhibitor of Ca2+ entry to block Ca2+ channels, La (10 µM). 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 µM) 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 µM (data not shown). ACh (0.1 µM), 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.
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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 confirmed by similar Ponceau
staining (data not shown).
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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).
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DISCUSSION |
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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 electrophysiology 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-µM diameter). Our PVs were significantly smaller (110-µM 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 ATP-sensitive 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 µM) 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 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 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 µM) 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 (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 atrial output. Our finding of spontaneous beating of the PVCMs as well as the formation of sphincters further supports their hypothesis. It is very interesting that cells closely resembling the pacemaking sinus node cells have also been described in the media of the PVs (26). The effects of ACh on the PVs that Paes de Almeida et al. (29) reported in 1975 could be explained by our finding of Kir3.1 channels in PVCMs (Figs. 5-7) and the effects of ACh on PVCM K+ current (Fig. 3D).
In this study, PVCMs penetrated deep into the PV segments, whereas in normal humans, the PV cardiac muscle is thought to be restricted to the extrapulmonary veins. In rats, the PV cardiac muscle that normally exists in the PVs has previously been reported to extend even further away from the atrium on exposure to chronic hypoxia (19) or after experimental myocardial infarction (25). If this pathological PV remodeling also occurs in humans, the PVCMs, particularly the cells in sphincters, might be critically involved in the pathogenesis of pulmonary edema or arrhythmias in patients with chronic hypoxia or heart failure after myocardial infarction. The localization of PVCMs has not been adequately studied in human disease states. In one study (9), an increased amount of "myocardial fibers" was described in the PV media of patients with dilated cardiomyopathy. The presence of functional sphincters in the PVs might provide new insight into the pathogenesis of pulmonary edema because it could result from PV constriction regardless of the left ventricular performance or the left atrial filling pressures.
Several of the KV channels that have been shown to be oxygen sensitive (Kv1.2, Kv1.5, Kv2.1, and Kv9.3) (18) or that have been directly implicated in the initiation of hypoxic pulmonary vasoconstriction (Kv1.5 and Kv2.1) (4) are also present in the rat PVs (Figs. 5 and 6). Although our findings in the rat cannot necessarily be extrapolated to other species, they raise the intriguing possibility that these channels might also mediate acute hypoxic PV constriction and contribute to high-altitude pulmonary edema in humans (1, 45).
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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: emichela{at}cha.ab.ca).
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.
Received 28 September 2000; accepted in final form 13 December 2000.
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