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 (E_m), 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 E_m, and [Ca²⁺]_i in freshly dispersed, rat pulmonary arterial smooth muscle cells.

	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 (I_k) and resting E_m 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 E_m experiments, cells were held in current clamp at their resting E_m (without current injection). E_m 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).

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.

View larger version (55K): [in this window] [in a new window]   Fig. 1.   Effect of hypoxia on whole cell outward K+ current of pulmonary artery smooth muscle cells (PASMCs). Representative 300-ms traces demonstrate K+ currents from PASMCs under normoxic conditions (left and right) and after a 4-min exposure to low O2 tension (middle). Currents were evoked from a holding potential of 70 to +50 mV in incremental depolarizing 10-mV steps with 5 cells in each group.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Averaged whole cell current-voltage (I-V) plots of steady-state outward K+ currents recorded from PASMCs. Currents were recorded during control and after a 4-min exposure to low O2 tension (measured at 290-300 ms) and normalized to maximum current under normoxia [measured current divided by maximum current (I/I0)] at +50 mV. Values are means ± SE.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Effect of 4-aminopyridine (4-AP) and low O2 tension on outward K+ current from PASMCs. A: representative K+ current traces were recorded during control and after exposure to 5 mM 4-AP or 5 mM 4-AP with 3% O2 by the voltage step from 70 to 0 mV. B: averaged whole cell I-V relationship of outward K+ currents normalized to maximum current under normoxia (I/I0) and after exposure to 5 mM 4-AP or 5 mM 4-AP with 3% O2. Values are means ± SE.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effect of low O2 tension and iberiotoxin (ITX) on outward K+ current from PASMCs. A: representative outward K+ current traces were recorded during control and after exposure to 0% O2 or 100 nM ITX under hypoxic conditions (0% O2) by a voltage step from 70 to +50 mV. B: averaged whole cell I-V relationship of outward K+ currents normalized to maximum current under normoxia (I/I0) and after exposure to 0% O2 or to 100 nM ITX in hypoxic conditions with 0% O2. Values are means ± SE.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Voltage dependence of the blockade of steady-state outward K+ currents recorded from PASMCs at low O2 tension. The currents, elicited by a test potential of +50 mV (), 0 mV (), or 20 mV (; holding potential 70 mV), were averaged from all of the cells tested and normalized to the maximal amplitude of each of the current records (n = 5). Connecting line was drawn by eye. Values are means ± SE. *P < 0.05 for difference between 20 and 0 and +50 mV; #P < 0.05 for difference between 0 and +50 mV.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effects of hypoxia on resting membrane potential (Em) measured with current-clamp (I = 0) in PASMCs. Em was measured after a 4-min exposure to 10% (n = 7), 5% (n = 7), 3% (n = 5), and 0% (n = 7) O2. Values are means ± SE. *P < 0.05 for difference from control; #P < 0.001 for difference from control.

View larger version (9K): [in this window] [in a new window]   Fig. 7.   Hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) measured in rat PASMCs. Superfusion of PASMCs with a bath solution bubbled with 5% (n = 17) and 0% (n = 24) O2 significantly increased [Ca2+]i. Preincubation with 30 µM La3+ (n = 17) or 10 µM nifedipine (2 min; n = 17) or omitting extracellular Ca2+ significantly decreased the hypoxia-induced increase in [Ca2+]i. Values are means ± SE. *P < 0.05 for difference from control; ***P < 0.001 for difference from control.

View larger version (10K): [in this window] [in a new window]   Fig. 8.   Effects of 50 mM KCl and 5 mM 4-AP on [Ca2+]i in PASMCs. Bath application of 50 mM KCl (n = 11) or 5 mM 4-AP (n = 21) increased [Ca2+]i. Pretreatment with 30 µM La3+ or 10 µM nifedipine (2 min) abolished the KCl- or 4-AP-induced increase in [Ca2+]i. Values are means ± SE. ***P < 0.001 for difference from control.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Whole cell outward I_k 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 I_k 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 I_k. This decrease in I_k was parallel to the decrease in PO₂ levels. The averaged current-voltage relationships for I_k under normoxic and hypoxic conditions are shown in Fig. 2. The block of I_k 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 I_k, their sensitivity to the classic K⁺ channel blocker 4-aminopyridine (4-AP) was examined. Representative I_k 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 I_k 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 I_k in PASMC, their sensitivity to the Ca²⁺-activated K⁺ channel (K_Ca) blocker iberiotoxin (ITX) was examined. Representative I_k 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 I_k to 11.6 ± 5.1% at +50 mV (n = 3).

Voltage dependence of the inhibition of steady-state outward I_k by hypoxia. The effect of hypoxia on I_k 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 I_k was voltage-dependent, being greater at more negative E_m.

Effect of hypoxia on E_m in PASMCs. In rat PASMCs, the average resting E_m 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 E_m (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 E_m was reversible. The superfusion of the cell chamber with bath solution bubbled with 0% O₂ depolarized E_m 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.

	DISCUSSION

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Our observations demonstrate a graded response of I_k, E_m, 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 I_k was maximal at more negative E_m, close to the resting E_m, 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 E_m 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 I_k 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 I_k. 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 I_k by hypoxia is voltage dependent. This is important in that it demonstrates that the hypoxic inhibition of I_k is more marked at physiologically relevant E_m. 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 E_m than the non-hypoxia-sensitive Kv channels. After the I_k 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 I_k relates to the fact that K_Ca 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 I_k at more positive E_m.

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 E_m. 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 E_m 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 I_k, E_m, and Ca²⁺ to hypoxia, as seen in other O₂-sensitive tissues.