1 Justus Liebig University, 35392 Giessen, Germany; and 2 Veterans Affairs Medical Center, University of Minnesota, Minneapolis, Minnesota 55417
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ABSTRACT |
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Many studies indicate that hypoxic
inhibition of some K+ channels in the membrane of the
pulmonary arterial smooth muscle cells (PASMCs) plays a part in
initiating hypoxic pulmonary vasoconstriction. The sensitivity of the
K+ current (Ik), resting membrane
potential (Em), and intracellular Ca2+ concentration ([Ca2+]i) of
PASMCs to different levels of hypoxia in these cells has not been
explored fully. Reducing PO2 levels gradually
inhibited steady-state Ik of rat resistance
PASMCs and depolarized the cell membrane. The block of
Ik by hypoxia was voltage dependent in that low
O2 tensions (3 and 0% O2) inhibited
Ik more at 0 and 20 mV than at 50 mV. As
expected, the hypoxia-sensitive Ik was also
4-aminopyridine sensitive. Fura 2-loaded PASMCs showed a graded
increase in [Ca2+]i as
PO2 levels declined. This increase was reduced
markedly by nifedipine and removal of extracellular Ca2+.
We conclude that, as in the carotid body type I cells, PC-12 pheochromocytoma cells, and cortical neurons, increasing severity of
hypoxia causes a proportional decrease in Ik and
Em and an increase of
[Ca2+]i.
hypoxic pulmonary vasoconstriction; patch clamp; electrophysiology; ion channels; oxygen
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INTRODUCTION |
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THE REGIONAL DISTRIBUTION of pulmonary blood flow is determined in part by local variations in alveolar O2 tension. The pulmonary arterial vasoconstriction in response to hypoxia reduces the blood flow through poorly ventilated alveoli, matches lung perfusion to ventilation, and prevents systemic hypoxemia (19, 38). The small pulmonary arteries are unique in that they constrict in response to a fall in O2 tension, whereas systemic arteries tend to dilate. The "dose-response" relationship of hypoxia and pulmonary vasoconstriction has been demonstrated in the intact animal (33) and in the isolated perfused lung (32). In each case, the threshold for the onset of hypoxic pulmonary vasoconstriction (HPV) is ~60-70 mmHg. In human lungs, graded hypoxia significantly reduces the perfusion of the lung ventilated with a hypoxic gas mixture (10).
There is a large body of data from other O2-sensitive tissues, such as the carotid body (16-18), PC-12 cell line (45), pulmonary neuroepithelial body (39, 40), and neocortical neurons (12, 13), indicating that K+ channels play an essential role in their responsiveness to changes in O2. The PO2-channel activity dose-response curve is relevant to secretory behavior, neuronal excitability, and the physiology of O2 delivery and transport to the brain (14, 15). K+ current amplitude of the type I cells in the carotid body is inversely proportional to the degree of hypoxia (17). Lowering of PO2 also causes a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i; see Refs. 3-5 and 37). In the PC-12 cell line, the magnitude of hypoxia-induced inhibition of K+ channels shows a graded response to the severity of hypoxia (45). In cortical neurons, PO2 regulates the channel open probability, without changing the single-channel conductance. Exposure to progressively lower PO2 gradually reduces the open probability (12, 13). In contrast, how progressive changes in PO2 modify K+ channel activity, resting membrane potential (Em), and [Ca2+]i in pulmonary arterial smooth muscle cells is still uncertain. To address these questions, we have examined the direct effect of different O2 tensions on K+ channels, resting Em, and [Ca2+]i in freshly dispersed, rat pulmonary arterial smooth muscle cells.
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METHODS |
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The animal study was approved by the Institutional Animal Care and Use Committee of the Minneapolis Veterans Affairs Medical Center and conforms to current National Institutes of Health and American Physiological Society guidelines for the use and care of laboratory animals.
Cell isolation. Rat pulmonary artery smooth muscle cells (PASMC) were freshly dissociated for electrophysiological studies every day. Male Sprague-Dawley rats (body wt 300-400 g) were anesthetized with 50 mg/kg ketamine and 5 mg/kg xylazine, and the heart and lungs were removed en bloc. Resistance pulmonary arteries (4th-6th divisions) were dissected free and placed in Ca2+-free Hanks' solution (see Solutions). The arteries were then transferred to Hanks' solution containing 0.5 mg/ml papain, 1 mg/ml albumin, and 1 mg/ml dithiothreitol without EGTA and kept at 4°C for 30 min. After this time, the arteries were incubated at 37°C for 13 min. The arteries were washed thoroughly in enzyme-free Hanks' solution for at least 10 min and then maintained at 4°C. Several digestions were done each day to ensure cell viability. Gentle tituration produced a suspension of single cells, which was then separated into aliquots in a perfusion chamber on the stage of an inverted microscope (Diaphot 200; Nikon) for the studies.
Experimental protocol. After a brief period to allow a partial adherence to the bottom of the recording chamber, cells were perfused via gravity with a bath solution (see Solutions) at a rate of 2 ml/min for the recording of K+ current (Ik) and resting Em or [Ca2+]i. After equilibration under normoxic conditions (2 min), PASMCs were exposed to graded levels of hypoxia for 4 min followed by 4 min of reperfusion with normoxic extracellular solution to verify a recovery. All experiments were done using fresh cells isolated from at least three animals and were performed at 30°C. The electrophysiological studies were carried out in low light intensity because of the light sensitivity of amphotericin B.
Current recording.
Whole cell recordings were performed using the amphotericin-perforated
patch-clamp technique (26). Patch pipettes were pulled from glass tubes (PG 150T; Warner Instruments). The pipettes were fire-polished directly before the experiments and had a resistance of
2-3 M when filled with intracellular solution. The patch-clamp amplifiers were Axopatch 200A (Axon Instruments, Foster City, CA) in
all voltage- and current-clamp experiments. Offset potentials were
nulled directly before formation of a seal. Whole cell capacitance and
series resistance were corrected (usually 80%). Only cells with series
resistance <10 M
were kept. The effective corner frequency of the
low-pass filter was 1 kHz. The frequency of digitization was at least
two times that of the filter. For Em
experiments, cells were held in current clamp at their resting
Em (without current injection).
Em stability was always determined for at least
1 min before any recording. The data were stored and analyzed with
commercially available software (pCLAMP version 8.1; Axon Instruments).
Pulse protocols and analysis.
Smooth muscle cells from small pulmonary resistance vessels were
voltage-clamped at a holding potential of 70 mV. The standard protocol used to obtain current-voltage relationships consisted of
300-ms voltage-clamp pulses applied in 10-mV steps between
70 and +50
mV. Measuring the current at the end of the voltage-clamp pulse and
plotting this against the test potential obtained steady-state current-voltage relationships. Currents were normalized relative to
each cell's control (normoxic) current at +50 mV.
Measurement of [Ca2+]i. [Ca2+]i was measured by dual-excitation ratiometric imaging, using fura 2 (9). Freshly dispersed cells were transferred to the experimental chamber and incubated in Ca2+-free extracellular solution with 0.1 µM fura 2-AM and 0.8 µM pluronic acid for 20 min at room temperature. The plates were then washed with extracellular solution containing 2.0 mM Ca2+ and incubated at room temperature for a further 20 min. Plates were rewashed and placed on the stage of an inverted microscope and perfused with a warmed extracellular solution (30°C). This loading method allows low concentrations of fura 2 to be quickly introduced in the cells without the potential effects on cell morphology that may occur from long exposures to high concentrations. Changes in [Ca2+]i were measured using a cooled charge-coupled device camera (Hamamatsu) with MetaFluor image capture and analysis software (Universal Imaging, West Chester, PA). Measurements were made every 5 s. Background fluorescence was recorded from each dish of cells and subtracted before calculation of the 340- to 380-nm ratio. [Ca2+]i was calculated according to the method of Grynkiewicz et al. (9). A dissociation constant of 220 nM was calculated from in vitro calibration. Maximal and minimal ratio values were determined at the end of each experiment by first treating the cells with 1 µM ionomycin (maximal ratio) and then chelating all free Ca2+ with 10 mM EGTA (minimal ratio). Any cells not responding to ionomycin were disregarded, as were cells showing significant photobleaching. Peak increases in [Ca2+]i were measured during each intervention, and data are given as averaged peak values.
Solutions. The Hanks' solution contained (in mM) 145 NaCl, 4.2 KCl, 1 MgCl2, 1.2 KH2PO4, 10 HEPES, 10 glucose, and 0.1 EGTA (pH was adjusted to 7.4 by KOH). The extracellular or bath solution contained (in mM) 115 NaCl, 5.4 KCl, 2.0 CaCl2, 1 MgCl2, 10 glucose, 1 NaH2PO4, and 25 NaHCO3 (pH 7.4 when bubbled with 5% CO2). The standard intracellular pipette solution contained (in mM) 145 KCl, 1 MgCl2, 1 K2ATP, 0.1 EGTA, 10 HEPES 10, and 120 µg/ml amphotericin B (pH was adjusted to 7.2 by KOH).
The effect of hypoxia was studied by switching between normoxic and hypoxic perfusate reservoirs. Normoxic solutions were equilibrated with 21% O2, 5% CO2, and 74% N2. Hypoxic solutions were achieved by bubbling with 10% O2, 5% O2, 3% O2, and 0% O2 (plus 5% CO2-balance N2) for at least 20 min before cell perfusion and by blowing N2 over the surface of the experimental chamber using a modified dish (30). These procedures produced PO2 values in the experimental chamber of 140-160 mmHg (21% O2), 60-80 mmHg (10% O2), 35-44 mmHg (5% O2), 24-30 mmHg (3% O2), and 11-17 mmHg (0% O2), respectively. O2 levels were measured with a Rapidlab Chiron blood gas analyzer from samples taken directly from the experimental chamber containing the PASMC during perfusion, which allows an exact measurement of PO2. By the use of a small recording chamber (400 µl), high perfusion rate (2-3 ml/min), and short dead space, bath exchange could be achieved in <30 s. PCO2 was 36-42 mmHg, and pH was 7.37-7.42 under these conditions. Fura 2-AM and pluronic acid were obtained from Molecular Probes (Eugene, OR). All other compounds were purchased from Sigma Chemical (St. Louis, MO). All drugs were dissolved in Hanks' solution, with the exception of nifedipine, which was dissolved in ethanol as a stock solution. pH of solutions containing drugs was tested and corrected to eliminate potential pH-induced effects. Stock solutions in ethanol were diluted at least 1:10,000 in the bath solution. At this concentration, the vehicle alone had no effect on the baseline levels of Ca2+.Statistical analysis.
Numerical values are given as means ± SE of n cells.
Intergroup differences were assessed by a factorial ANOVA with post hoc analysis with Fisher's least significant difference test. P
values <0.05 were considered significant. In Figs. 1-8, the SE is
indicated when it exceeds the symbol size.
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RESULTS |
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Whole cell outward Ik in PASMCs under hypoxia. When the cells were superfused with a bath solution bubbled with different concentrations of O2 (10%, 5%, 3%, 0%), whole cell outward Ik in PASMC from resistance vessels was recorded after 4 min of exposure to the low O2 level (Fig. 1). Under these conditions, hypoxia rapidly inhibited Ik. This decrease in Ik was parallel to the decrease in PO2 levels. The averaged current-voltage relationships for Ik under normoxic and hypoxic conditions are shown in Fig. 2. The block of Ik under different hypoxic conditions was reversible to 90-97% (at +50 mV) after a 4-min recovery time, except after exposure to 0% O2 in the bath solution (77% recovery at +50 mV).
Effect of the K+ channel inhibitor 4-aminopyridine. To characterize the O2-sensitive outward Ik, their sensitivity to the classic K+ channel blocker 4-aminopyridine (4-AP) was examined. Representative Ik traces that were recorded during control and after exposure to 3% O2 or 5 mM 4-AP under hypoxic conditions (3% O2) are shown in Fig. 3A. Extracellular application of 5 mM 4-AP inhibited Ik to 17.6 ± 4.6% at 0 mV (n = 5), and no further decrease was induced by the additional application of bath solution containing 5 mM 4-AP and bubbled with 3% O2 (14.6 ± 4.0 at 0 mV; n = 5), suggesting that hypoxia may be acting on 4-AP-sensitive channels.
Effect of the Ca2+-activated K+ channel inhibitor iberiotoxin. To investigate the composition of the hypoxia-insensitive Ik in PASMC, their sensitivity to the Ca2+-activated K+ channel (KCa) blocker iberiotoxin (ITX) was examined. Representative Ik traces that were recorded during control and after exposure to 0% O2, or 100 nM ITX under hypoxic conditions (0% O2), are shown in Fig. 4A. When cells were dialyzed with extracellular solution bubbled with 0% O2, the current was inhibited to 38.6 ± 4.7% at +50 mV (n = 5; Fig. 4B). Application of ITX under hypoxia caused an additional inhibition of Ik to 11.6 ± 5.1% at +50 mV (n = 3).
Voltage dependence of the inhibition of steady-state outward
Ik by hypoxia.
The effect of hypoxia on Ik at several
potentials was also examined. Currents were normalized relative to each
cell's control (normoxic) current at +50, 0, and 20 mV and plotted
against the O2 level in the bath solution (Fig.
5). Low O2 tension at or
below 70 mmHg inhibited the whole cell K+ current. It was
remarkable that hypoxic inhibition of Ik was voltage-dependent, being greater at more negative
Em.
Effect of hypoxia on Em in PASMCs.
In rat PASMCs, the average resting Em value,
measured by the current-clamp technique, was 47.6 ± 2.0 mV
(Fig. 6). Superfusion with bath solution
containing 10% O2 had no effect on
Em (
47.2 ± 1.7, n = 5).
Decreasing the O2 concentration of the bath solution caused
further depolarization. Bath solution bubbled with 5% O2 depolarized to
42.0 ± 4.9 mV (n = 5), 3%
O2 to
35.8 ± 3.9 mV, and 0% O2 to
22.6 ± 6.4 mV. The depolarizing effect of 5 and 3%
O2 on resting Em was reversible. The
superfusion of the cell chamber with bath solution bubbled with 0%
O2 depolarized Em irreversibly during the recording period of 4 min.
Effects of hypoxia on [Ca2+]i in PASMCs. Average resting [Ca2+]i in rat PASMC under normoxic conditions was 91.3 ± 1.8 nM (n = 214). When the cells were superfused with the bath solution bubbled with different levels of O2, hypoxia caused an increase in [Ca2+]i in a dose-dependent manner (Fig. 7). Extracellular solution bubbled with 5 and 0% O2 increased [Ca2+]i by 184.5 ± 23.3 nM (n = 17) and by 512.1 ± 50.5 nM (n = 24), respectively. To determine whether Ca2+ influx from extracellular sources was required for the increase of [Ca2+]i, PASMCs were perfused with Ca2+-free extracellular solution (10 mM EGTA) during the 4 min of hypoxia. Removal of extracellular Ca2+ markedly reduced the response of [Ca2+]i to severe hypoxia, but there was still an increase in [Ca2+]i of 174.1 ± 38.8 nM (P < 0.001; n = 17), suggesting a role of intracellular stores in the increase of [Ca2+]i during hypoxia (Fig. 7). In another set of experiments, the hypoxic challenge was applied to PASMCs after treatment with 10 µM nifedipine (2 min), an L-type Ca2+ channel antagonist, or 30 µM La3+ (2 min), which is a nonselective blocker of Ca2+ entry. The addition of nifedipine or La3+ had no effect on resting [Ca2+]i (n = 28 and n = 26). The hypoxia-induced increase in [Ca2+]i was reduced markedly by La3+ (change in [Ca2+]i = 95.1 ± 17.9 nM; n = 17; P < 0.001) and by nifedipine (change in [Ca2+]i = 144.6 ± 33.4 nM; n = 17; P < 0.001; Fig. 7), suggesting that part of the increase is caused by Ca2+ influx via voltage-dependent Ca2+ channels.
Effects of KCl and 4-AP on [Ca2+]i in PASMCs. KCl (50 mM) caused an increase in [Ca2+]i of 743.7 ± 92.5 nM (n = 11; Fig. 8). Pretreatment of PASMCs with 10 µM nifedipine or 30 µM La3+ for 2 min before exposure to 50 mM KCl was begun almost completely abolished the KCl-induced increase in [Ca2+]i (change in [Ca2+]i = 69.7 ± 28.3 nM; n = 18; P < 0.001 for nifedipine and change in [Ca2+]i = 56.8 ± 19.6 nM; n = 11; P < 0.001 for La3+). The application of 5 mM 4-AP increased [Ca2+]i by 285.1 ± 41.9 nM (n = 21; Fig. 8). Nifedipine or La3+ significantly inhibited the 4-AP-induced increase in [Ca2+]i (change in [Ca2+]i = 60.6 ± 22.5 nM; n = 10; P < 0.001 for nifedipine and change in [Ca2+]i = 47.2 ± 14.8 nM; n = 9; P < 0.001 for La3+). These results suggest that the 4-AP-induced increase in [Ca2+]i is largely the result of Ca2+ influx through voltage-dependent Ca2+ channels, which are opened by the membrane depolarization resulting from decreased voltage-dependent K+ channel activity.
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DISCUSSION |
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Our observations demonstrate a graded response of Ik, Em, and [Ca2+]i to increasing hypoxia. The decrease in current started between 80 and 40 mmHg PO2 and was significant at 30 mmHg. The finding that electrophysiological and [Ca2+]i changes occur at these O2 tensions would be compatible with the levels of hypoxia reported to cause vasoconstriction in the isolated lung and intact animals. The hypoxia-induced inhibition of 4-AP-dependent voltage-gated K+ (Kv) channels in rat PASMCs causes membrane depolarization and an increase in [Ca2+]i, as previously reported (2, 43). More importantly, the effect of hypoxia in reducing Ik was maximal at more negative Em, close to the resting Em, reinforcing the conclusion that it may be physiologically relevant.
HPV is a physiological response whereby desaturated mixed venous blood is diverted away from hypoxic alveoli, thus optimizing the matching of perfusion and ventilation and preventing arterial hypoxemia. It seems likely that the smooth muscle cells are both sensors of hypoxia and effectors of vasoconstriction (31, 38). Several different mechanisms have been proposed to explain HPV. The first mechanism involves hypoxic inhibition of one or more K+ channels that set resting Em in the vascular smooth muscle cells of the small pulmonary resistance vessels, leading to cell depolarization, opening of voltage-gated Ca2+ channels, and myocyte contraction (1, 21, 25, 38, 42). The second proposal is that hypoxia induces release of Ca2+ from intracellular stores (28). This Ca2+ could contribute to contraction, either directly or through blockade of K+ channels (8, 24, 35). Finally, sensitization of actin/myosin might lead to more contraction at any given cytosolic Ca2+ level (44). HPV could in fact involve participation of two, or even all three, of these mechanisms.
It has been demonstrated that hypoxic vasoconstriction of pulmonary arterial smooth muscle cells is in part mediated by the inhibition of voltage-dependent K+ channels, leading to cell depolarization, Ca2+ influx, and myocyte contraction (1, 21, 25, 38, 42). The PO2-dependent K+ channel activity dose-response curve is relevant to the physiology of HPV. To determine how a gradual reduction in PO2 from normoxia to severe hypoxia inhibits K+ channels, we measured the voltage-current relationship in PASMCs from resistance arteries using the amphotericin-perforated patch-clamp technique. Hypoxia decreased the steady-state Ik and the decrease in current paralleled the decrease in PO2 levels. The hypoxia-inhibitory response was nearly completely reversed upon return to normoxia. These results are consistent with those of Zhu et al. (45), who showed that, in PC-12 cells, exposure to progressively lower PO2 gradually reduced the O2-sensitive Ik. Jiang and Haddad (12, 13) also showed, in excised patches from cortical neurons, a dose-dependent inhibition of open probability of single K+ channels by graded hypoxia, without alteration of single channel conductance.
In the present study, we observed that the blockade of
Ik by hypoxia is voltage dependent. This is
important in that it demonstrates that the hypoxic inhibition of
Ik is more marked at physiologically relevant
Em. This observation might be explained in two
ways. The first hypothesis involves the presence of different Kv
channels, with different sensitivities to hypoxia, in the PASMC. It
would suggest that the hypoxia-sensitive Kv channels are open during normoxia at more negative Em than the
non-hypoxia-sensitive Kv channels. After the Ik
was reduced by 5 mM 4-AP, no further decrease was caused by the
additional application of hypoxia, suggesting that hypoxia may be
acting on 4-AP-sensitive voltage-gated channels. The pharmacological
properties of this K+ channel are thus similar to those
described previously in rat PASMCs (1). Rabbit PASMCs are
also known to express O2-sensitive K+ channels
that are susceptible to block by 4-AP (7, 21). The
potential candidate Kv channel -subunits that could form O2-sensitive channels in PASMCs are Kv1.2 (11,
36), Kv1.5 (2, 36), Kv2.1 (2, 11, 23),
Kv3.1 (22), and Kv9.3 (11, 23). The second
explanation of the voltage dependence of hypoxic inhibition of
Ik relates to the fact that KCa
channels, which are not O2 sensitive in the PASMCs of the
adult rat, are only activated at more positive test potentials, as
shown in Fig. 4. Consequently, hypoxia might inhibit less of the total
Ik at more positive Em.
Membrane depolarization and hyperpolarization through inhibition and activation of K+ channels are important mechanisms regulating smooth muscle cell contraction and relaxation. Under current-clamp conditions, lowering PO2 from normoxia to different hypoxic levels resulted in progressive depolarization of resting Em. This observation is consistent with similar observations made in canine PASMCs (25) and cultured rat PASMCs (42). Although superfusion with bath solution containing 5 or 3% O2 depolarized Em of PASMCs reversibly, severe hypoxia caused depolarization that was not fully reversible in the few minutes permitted in our protocol.
In most proposed mechanisms of HPV, an increase of [Ca2+]i is necessary to elicit constriction of the vessels. In excitable cells, [Ca2+]i is increased by Ca2+ influx through Ca2+-permeable channels and/or by Ca2+ mobilization from intracellular Ca2+ stores (e.g., endoplasmic/sarcoplasmic reticulum). We have shown that [Ca2+]i begins to rise significantly in response to hypoxic challenge when PO2 is below 35-44 mmHg (5% O2) and is graded with the severity of hypoxia. This observation confirms work in rat carotid body type I cells (3-5, 37) and in porcine PASMCs (29).
The proposal that the hypoxic [Ca2+]i response results mostly from Ca2+ influx is further supported by the observation that it is considerably attenuated by 30 µM La3+ or 10 µM nifedipine. More specifically, the data with nifedipine imply the role of voltage-dependent L-type Ca2+ channels, which supports the hypothesis that membrane depolarization triggers the rise of [Ca2+]i. Our results are consistent with previous reports demonstrating that voltage-dependent Ca2+-channel blockers such as verapamil or SFK-525 significantly reduce HPV in isolated perfused lungs (20) and that nifedipine markedly inhibits the hypoxia-induced rise in [Ca2+]i in type I cells (4) and pulmonary rings (27). Omitting Ca2+ markedly reduced the hypoxia-induced rise of [Ca2+]i in our experiments. Similar observations have been reported in type I cells of the carotid body. The hypoxia-induced increase in [Ca2+]i is entirely inhibited in the absence of extracellular Ca2+ in these cells (4, 34). However, although the increase in [Ca2+]i is smaller in the absence of extracellular Ca2+ in PASMCs (8, 28), it is not absent, suggesting that both intracellular and extracellular Ca2+ are important. If the principal effect of hypoxia is the inhibition of particular voltage-dependent K+ channels, then 4-AP and high extracellular K+ would be expected to stimulate entry of Ca2+ through voltage-gated Ca2+ channels. In our study, 4-AP and KCl did in fact increase [Ca2+]i, primarily through entry of Ca2+ through voltage-gated Ca2+ channels. These results confirm previous findings in rat (41) and canine (6) PASMCs and provide additional insight that Kv are important regulators of [Ca2+]i in these cells.
Although the response of PASMCs to hypoxia almost certainly involves both influx of extracellular Ca2+ and release of Ca2+ from internal stores, this study focuses on the former. It provides evidence of a graded response of Ik, Em, and Ca2+ to hypoxia, as seen in other O2-sensitive tissues.
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ACKNOWLEDGEMENTS |
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A. Olschewski was supported by Deutsche Forschungsgemeinschaft Grant Ol 127/1-1. E. K. Weir was supported by Veterans Affairs Merit Review Funding and National Heart, Lung, and Blood Institute Grant RO1 HL-65322.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Kenneth Weir, VA Medical Center (111 C), One Veterans Dr., Minneapolis, MN 55417 (E-mail: weirx002{at}tc.umn.edu).
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.
August 9, 2002;10.1152/ajplung.00104.2002
Received 17 April 2002; accepted in final form 13 July 2002.
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