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(Circulation Research. 1995;77:131-139.)
© 1995 American Heart Association, Inc.

Articles

[Ca²⁺]_i Inhibition of K⁺ Channels in Canine Pulmonary Artery

Novel Mechanism for Hypoxia-Induced Membrane Depolarization

Joseph M. Post, Craig H. Gelband, Joseph R. Hume

From the Department of Physiology, University of Nevada School of Medicine, Reno.

Correspondence to Dr Joseph R. Hume, Department of Physiology, University of Nevada School of Medicine, Reno, NV 89557-0046.

	Abstract

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Abstract Experiments were performed on smooth muscle cells isolated from canine pulmonary artery to identify the type of K⁺ channel modulated by hypoxia and examine the possible role of [Ca²⁺]_i in hypoxic K⁺ channel inhibition. Whole-cell patch-clamp experiments revealed that hypoxia (induced by the O₂ scavenger, sodium dithionite) reduced macroscopic K⁺ currents, an effect that could be prevented by strong intracellular buffering of [Ca²⁺]_i. The inhibitory effects of hypoxia were mimicked by acute exposure of cells to caffeine and could be prevented by caffeine pretreatment, suggesting an important obligatory role of [Ca²⁺]_i in hypoxic inhibition of K⁺ currents. Exposure of cells to low concentrations of 4-aminopyridine (4-AP, 1 mmol/L) prevented hypoxic inhibition of macroscopic K⁺ currents, whereas low concentrations of tetraethylammonium were without effect, suggesting that the target K⁺ channel inhibited by hypoxia is a voltage-dependent delayed rectifier K⁺ channel, which is inhibited by [Ca²⁺]_i. Hypoxia failed to consistently modify the activity of large-conductance (118 picosiemens [pS] in physiological K⁺) Ca²⁺-activated K⁺ channels in inside-out membrane patches but reduced open probability of smaller-conductance (25-pS) delayed rectifier K⁺ channels in cell-attached membrane patches. In inside-out membrane patches, 1 µmol/L Ca²⁺ added to the cytoplasmic surface significantly reduced open probability of small-conductance (25-pS) 4-AP–sensitive delayed rectifier K⁺ channels. Whole-cell current measurements using symmetrical K⁺ to increase driving force for small currents active near the cell's resting membrane potential revealed the presence of a 4-AP–sensitive K⁺ current that activated near -65 mV and was inhibited by hypoxia. Simultaneous measurements of changes in [Ca²⁺]_i, using the Ca²⁺ indicator indo 1, and membrane potential revealed that hypoxia causes an initial rise of [Ca²⁺]_i, which precedes hypoxia-induced membrane depolarization. It is concluded that in canine pulmonary arterial cells an early key event in hypoxic pulmonary vasoconstriction is release of Ca²⁺ from caffeine-sensitive intracellular Ca²⁺ stores, which causes inhibition of delayed rectifier K⁺ channels and membrane depolarization, possibly leading to subsequent activation of Ca²⁺ entry through voltage-dependent Ca²⁺ channels.

Key Words: pulmonary artery • hypoxia • K⁺ channels

	Introduction

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In the normal lung, hypoxic pulmonary vasoconstriction (HPV) is thought to serve as an adaptive mechanism by which blood flow is diverted from poorly ventilated to better ventilated regions to optimize ventilation/perfusion matching.¹ However, severe reductions in alveolar oxygen tension, as in obstructive lung disease, can lead to pulmonary vasoconstriction, vascular remodeling, and pulmonary hypertension. The exact mechanism responsible for HPV is presently unknown, and the contribution of the endothelium to HPV is controversial. Removal of the endothelium has been reported to abolish HPV^{2 3} or to have little effect on HPV.^{4 5} The recent demonstration that HPV occurs in isolated single pulmonary arterial smooth muscle cells⁶ suggests that the underlying mechanism may be intrinsic to pulmonary smooth muscle cells and that factors released from the endothelium may modulate HPV.

With regard to intrinsic cellular mechanisms, it is known that a reduction in PO₂ from normoxic to hypoxic levels causes depolarization of the resting membrane potential⁷ and that HPV is attenuated by organic Ca²⁺ channel antagonists,^{7 8 9} suggesting an important role for depolarization-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels. Furthermore, the observation that a number of K⁺ channel inhibitors simulate HPV by increasing tension in intact pulmonary arterial rings and elevate pulmonary arterial pressure in isolated lungs^{10 11} suggests that K⁺ channel inhibition may be a critical early event in the initiation of HPV. This hypothesis has received support from recent studies demonstrating that hypoxia inhibits macroscopic whole-cell K⁺ currents, causing depolarization of the resting membrane potential in both acutely isolated and cultured pulmonary arterial smooth muscle cells.^{11 12} In spite of this recent progress, it is not clear at this time exactly which type of K⁺ channel is inhibited by hypoxia or by what mechanism hypoxia exerts its inhibitory effects. Although the exact type of K⁺ channel involved is uncertain, other studies have suggested a potential mechanism responsible for hypoxic inhibition of K⁺ channels that may involve hypoxia-induced changes in cytosolic redox status.^{13 14 15 16}

In our earlier study of hypoxic inhibition of whole-cell K⁺ currents in acutely isolated canine pulmonary arterial smooth muscle cells,¹¹ it was concluded that a Ca²⁺-sensitive K⁺ channel was involved, since the inhibitory effects of hypoxia could be prevented by chelation of intracellular Ca²⁺. In contrast, the study by Yuan et al¹² concluded that hypoxia inhibited a Ca²⁺-insensitive K⁺ channel in cultured rat pulmonary arterial cells. In light of recent new data showing an early mobilization of intracellular free Ca²⁺ by hypoxia in cultured pulmonary arterial smooth muscle cells¹⁷ and the ability of intracellular free Ca²⁺ to directly inhibit voltage-dependent delayed rectifier K⁺ channels in a variety of smooth muscle cells,^{18 19 20} we have reexamined hypoxic modulation of K⁺ channels in acutely isolated canine pulmonary arterial smooth muscle cells with the objective of identifying the type of K⁺ channel modulated by hypoxia as well as examining the potential role of [Ca²⁺]_i in hypoxic K⁺ channel inhibition.

	Materials and Methods

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Cell Preparation and Experimental Solutions
Mongrel dogs were euthanatized with an overdose of pentobarbital sodium (45 mg/kg IV). Second, third, and fourth branches of the pulmonary artery were removed and cleaned. The solutions and isolation technique used to disperse canine pulmonary arterial smooth muscle cells have been described in detail previously.¹¹ Macroscopic membrane currents were measured by using the whole-cell configuration, and unitary currents were measured by using the inside-out configuration of the patch-clamp technique,²¹ as described in the accompanying article in this issue of Circulation Research.²⁰ For whole-cell experiments, repetitive voltage ramps were applied at a frequency of 0.07 Hz during control conditions and continued during the various experimental interventions.

The standard external solution used for whole-cell recordings contained (mmol/L) NaCl 130, NaHCO₃ 10, KCl 4.2, KH₂PO₄ 1.2, MgCl₂ 0.5, CaCl₂ 1.5, glucose 5.5, and HEPES 10 (pH 7.4 with NaOH). In some experiments, the NaCl was replaced with 135 mmol/L KCl. The patch electrode for whole-cell recordings contained (mmol/L) potassium gluconate 110, KCl 20, ATP (dipotassium) 2.0, phosphocreatine 2.0, HEPES 5.0, and EGTA 1.0 (pH 7.2 with NaOH). Whole-cell experiments were performed at 36±1°C. Unitary K⁺ currents in cell-attached and inside-out patches were measured by using a bath solution containing (mmol/L) KCl 140, HEPES 10, EGTA 0.1, CaCl₂ 0.05, and glucose 5.5 (pH 7.2 with Tris). The pipette solution contained (mmol/L) NaCl 140, HEPES 10, glucose 5.5, and KCl 5.5 (pH 7.2 with Tris). In selected inside-out patch experiments, symmetrical K⁺ solutions were used by replacing NaCl with KCl in the pipette solution. Measurements of single-channel K⁺ currents were performed at room temperature.

The O₂ scavenger sodium dithionite (1 mmol/L) was used to establish hypoxic conditions in most experiments, since it provides a simple and consistent method of lowering PO₂ and maintaining low PO₂ in an open recording chamber. Sodium dithionite was added to the standard external solution, and pH was then adjusted to 7.4 with NaOH. PO₂ of these solutions was 5 mm Hg, as determined with a blood/gas analyzer. In some experiments, oxygen tension was changed by bubbling the solution, whereas normoxic solutions (PO₂, 130 mm Hg) were obtained by aeration with a 20% O₂/5% CO₂/balance N₂ gas mixture, and hypoxic solutions (PO₂, 30 mm Hg) were obtained by aeration with a 5% CO₂/balance N₂ gas mixture. To avoid O₂ reequilibration, air jets of hypoxic gas were passed over the experimental chamber during protocols examining the effects of hypoxia. The experimental chamber had a volume of 1 mL, and complete exchange of solutions required 1 minute.

Measurement of [Ca²⁺]_i
Indo 1 (pentapotassium salt, 50 µmol/L) was included in the patch pipette solution and dialyzed into the cell. [Ca²⁺]_i was measured as described in our accompanying article.²⁰

Statistics
Results are expressed as mean±SEM. Statistical significance was evaluated by using a Student's t test for unpaired observations. Differences were considered significant at P<.05; n corresponds to the number of cells examined. Membrane currents were measured from the zero current level.

	Results

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Hypoxic Inhibition of K⁺ Current Is Linked to [Ca²⁺]_i
To examine the effects of hypoxia on delayed rectifier K⁺ currents, the contribution of Ca²⁺-activated K⁺ channels to macroscopic membrane currents was minimized by including 1 mmol/L EGTA in the pipette intracellular dialysis solution and confining current measurements to potentials more negative than +40 mV. In our previous study of isolated canine pulmonary arterial cells,¹¹ hypoxic inhibition of K⁺ currents (obtained by bubbling solutions with a hypoxic gas mixture) was shown to be insensitive to intracellular concentrations of EGTA up to 5 mmol/L. Sodium dithionite–induced hypoxia also inhibited macroscopic K⁺ currents (Fig 1A) and depolarized the resting membrane potential from -58.2±4.2 to -38.6±7.0 mV (n=5; see also Fig 9) in freshly dispersed canine pulmonary arterial smooth muscle cells. Fig 1A (right panel) also illustrates the time course for hypoxic inhibition of K⁺ current. The onset for inhibition of K⁺ current occurred within 1 minute after exposure to sodium dithionite and continued to increase during the subsequent time period. Hypoxia inhibited K⁺ currents an average of 21.0±4.8% at 10 mV during ramp depolarizations from -70 to +40 mV (holding potential, -70 mV; n=7).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of hypoxia on macroscopic K+ currents and its dependence on [Ca2+]i. A, Effects of hypoxia on K+ currents elicited by repetitive ramp depolarizations from -70 to 40 mV in a cell dialyzed with 1 mmol/L EGTA. B, Effects of hypoxia on K+ currents elicited by ramp depolarizations from -70 to 40 mV in a cell dialyzed with 10 mmol/L BAPTA. Membrane currents measured at times a, b, and c are illustrated on the left; time courses of peak outward currents (+10 mV) are shown on the right. Rate of voltage depolarization was 0.034 mV/ms.

View larger version (26K): [in this window] [in a new window]   Figure 9. Recordings showing the effects of hypoxia on [Ca2+]i and the membrane potential of a canine pulmonary arterial cell. Transient exposure to hypoxic solution increased [Ca2+]i (indo 1 fluorescence ratio) and depolarized the pulmonary arterial smooth muscle cell. The bath solutions contained charybdotoxin (100 nmol/L) and niflumic acid (100 µmol/L) to reduce the activity of large-conductance Ca2+-activated K+ channels and Ca2+-activated Cl- channels, respectively. Note that upon making the bath solution hypoxic, the rise of [Ca2+]i occurred before the depolarization of the membrane potential. The effects of hypoxia on [Ca2+]i and membrane potential were reversible upon reexposure of the cell to normoxic solutions. F indicates change in fluorescence ratio. Similar results were obtained in two other cells.

We next examined whether sodium dithionite–induced hypoxia inhibits a Ca⁺-sensitive component of K⁺ current. Initial experiments investigated whether changes in [Ca²⁺]_i altered the effects of hypoxia on K⁺ current. Buffering [Ca²⁺]_i by dialyzing cells with 10 mmol/L BAPTA prevented hypoxic inhibition of K⁺ current over the voltage range from -70 to +40 mV (Fig 1B). The percentage of K⁺ current present after exposure to hypoxia was 101.0±5.9% of the normoxic control at 10 mV (n=5). These results are consistent with our previous study¹¹ and suggest that the effects of hypoxia on K⁺ current are linked to the level of intracellular free Ca²⁺. It is noteworthy that the ability of BAPTA to prevent hypoxic inhibition of K⁺ currents also argues against possible nonspecific effects of sodium dithionite on membrane currents (see also Fig 2B).

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of caffeine on macroscopic K+ currents and on hypoxic inhibition of K+ currents. A, Effects of acute exposure to caffeine (10 mmol/L) on macroscopic K+ currents elicited by repetitive ramp depolarizations from -70 to 40 mV. B, Effects of prolonged incubation in caffeine (10 mmol/L, >10 minutes) on hypoxic inhibition of K+ currents. Membrane currents measured at times a and b are illustrated on the left; time courses of peak outward currents (+10 mV) are shown on the right. Rate of voltage depolarization was 0.034 mV/ms.

The link between intracellular Ca²⁺ and its regulation of K⁺ current was also tested by examining the effects of caffeine. Caffeine acutely causes a transient elevation in intracellular Ca²⁺ by triggering release of Ca²⁺ from the sarcoplasmic reticulum (SR), eventually causing a reduction in intracellular Ca²⁺ by preventing reuptake of Ca²⁺ into the SR in vascular smooth muscle cells.^{22 23} Acute exposure to 10 mmol/L caffeine produced a reduction of K⁺ current, most prominent at negative membrane potentials (by an average 34.9±2.9% of control at 10 mV, n=6; Fig 2A). This reduction of K⁺ current may be attributed to release of SR Ca²⁺, which inhibits the delayed rectifier component of K⁺ current,^{19 20} since it could be prevented by dialyzing cells with 10 mmol/L BAPTA (data not shown). The effects of acute caffeine exposure inhibited K⁺ current with a time course that resembles the inhibition produced by hypoxia (Fig 1A). In some cells, after prolonged exposure to caffeine (>10 minutes) sufficient to deplete intracellular Ca²⁺ stores, the effects of hypoxia on macroscopic K⁺ currents were examined. In these cells, prolonged exposure to caffeine eliminated any subsequent hypoxic inhibition of K⁺ current (Fig 2B). Mean K⁺ current amplitude after exposure to hypoxia was 108.3±1.8% of control at 10 mV (n=3). These results suggest that SR Ca²⁺ release may play an obligatory role in hypoxic inhibition of K⁺ currents.

Hypoxia Inhibits a 4-Aminopyridine–Sensitive K⁺ Current
Previous whole-cell current studies suggest the presence of at least two distinct K⁺ channels on the basis of different slope conductances, noise characteristics, and pharmacology of macroscopic currents¹⁹ : low concentrations of 4-aminopyridine (4-AP) preferentially inhibit low-noise delayed rectifier K⁺ current at more negative potentials, whereas low concentrations of tetraethylammonium (TEA) inhibit higher-noise Ca²⁺-activated K⁺ currents at more positive potentials. The effects of hypoxia on K⁺ current were tested in the presence of either 4-AP (1 mmol/L) or TEA (1 mmol/L) to investigate whether hypoxia preferentially inhibits the TEA-sensitive or 4-AP–sensitive components of macroscopic K⁺ current. Hypoxia failed to inhibit K⁺ current when cells were preexposed to 4-AP (1 mmol/L) (Fig 3A). Preincubation with 4-AP (1 mmol/L) completely eliminated hypoxic inhibition of K⁺ current at 10 mV (n=5). The effect of hypoxia on K⁺ current in the presence of TEA (1 mmol/L) was also investigated to determine if hypoxic inhibition of K⁺ current resulted in part from inhibition of large-conductance Ca²⁺-activated K⁺ channels. In contrast to 4-AP, preincubation with TEA (1 mmol/L) did not prevent hypoxic inhibition of K⁺ current (Fig 3B). Hypoxia inhibited K⁺ current at 10 mV by 35.3±2.9% (n=4). These results suggest that the major effect of hypoxia involves inhibition of 4-AP–sensitive delayed rectifier K⁺ current.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of hypoxia on 4-aminopyridine (4-AP, 1 mmol/L)–sensitive and tetraethylammonium (TEA, 1 mmol/L)–sensitive macroscopic K+ currents. A, Effects of hypoxia in cell pretreated with 4-AP (1 mmol/L). B, Effects of hypoxia measured in cell pretreated with TEA (1 mmol/L). Membrane currents were elicited by repetitive ramp depolarizations (0.034 mV/ms) from -70 to 40 mV. Membrane currents measured at times a and b are illustrated on the left; time courses of peak outward currents (+10 mV) are shown on the right.

Effects of Hypoxia and Ca²⁺ on Unitary K⁺ Channels
Measurement of unitary K⁺ currents revealed the presence of small and large K⁺ channel conductances during step or ramp depolarizations using inside-out membrane patches (Fig 4). These channels had conductances of 240±12 and 69±10 picosiemens (pS), respectively, in symmetrical K⁺ solution (Fig 4). Previously, we have demonstrated with inside-out patches that increasing free [Ca²⁺] on the cytoplasmic side from 4 to 100 nmol/L reversibly increases NxP(open) [where N is the number of functional channels in a patch and P(open) is the opening probability] of the large-conductance K⁺ channel.¹⁴ The conductance and Ca²⁺ sensitivity of this K⁺ channel suggest that it is similar to other TEA-sensitive large-conductance Ca²⁺-activated K⁺ channels observed in several vascular preparations.^{24 25}

View larger version (17K): [in this window] [in a new window]   Figure 4. Unitary K+ channels in inside-out membrane patches. A, Recording showing example of single K+ channel currents elicited by ramp depolarization in inside-out patches in symmetrical K+ solution. Arrows denote smaller-conductance channels. B, Graph showing mean current-voltage relations for the small-conductance () and large-conductance () channels accumulated from 10 and 17 patches, respectively. Mean slope conductances () were 69 and 240 picosiemens, respectively; SEM for some points is not shown since it was smaller than the symbol. Data points were fit by linear regression.

Although the TEA whole-cell data (Fig 3) suggest that large-conductance Ca²⁺-activated K⁺ channels are not the target site for hypoxia-induced K⁺ channel inhibition, our previous study,¹¹ which first implicated a Ca²⁺-sensitive K⁺ current, has sometimes been construed to mean a Ca²⁺-activated K⁺ current.¹² Thus, it seemed important to directly test the effects of hypoxia on the large-conductance Ca²⁺-activated K⁺ channel by using inside-out patches. Open probability of the large-conductance Ca²⁺-activated K⁺ channel did not significantly change when switching from normoxia [NxP(open), 0.285±0.19; PO₂, 130 mm Hg] to hypoxia [NxP(open), 0.439±0.20; PO₂, 33 mm Hg; n=8] in symmetrical K⁺ solution with inside-out patches (Fig 5; holding potential, 40 mV). These data are consistent with the previous whole-cell experiments suggesting that hypoxia does not inhibit TEA-sensitive large-conductance Ca²⁺-activated K⁺ channels.

View larger version (25K): [in this window] [in a new window]   Figure 5. Effect of hypoxia on large-conductance Ca2+-activated K+ channels. A, Large-conductance Ca2+-activated K+ channels during normoxia. B, Large-conductance Ca2+-activated K+ channels during hypoxia. Top panels are recordings. Bottom panels are amplitude histograms. NxP(open) [where N is the number of functional channels in a patch and P(open) is the opening probability] of the large-conductance Ca2+-activated K+ channel did not significantly change when switching from normoxia to hypoxia in symmetrical K+ solutions with inside-out patches (holding potential, 40 mV). In these experiments, hypoxic solutions were obtained by bubbling with 100% N2, which reduced PO2 from 130 mm Hg (normoxic) to 33 mm Hg. Histograms were constructed from 1-minute recordings made before and 4 minutes after exposure to hypoxic solutions.

The effects of hypoxia on the smaller-conductance delayed rectifier K⁺ channels were tested in cell-attached membrane patches. Most patches examined contained large-conductance Ca²⁺-activated K⁺ channels, which were dramatically stimulated by exposure to hypoxic solutions, making evaluation of the effects of hypoxia on the smaller-conductance delayed rectifier K⁺ channels difficult. The effects reported here were measured in the few membrane patches that contained no evidence of large-conductance Ca²⁺-activated channels before or after exposure to hypoxia. In these experiments, hypoxia was induced by incubating the cell in extracellular hypoxic solutions achieved by bubbling with 100% nitrogen (see "Materials and Methods"). When a physiological K⁺ gradient was used, the conductance of the small channel was 25.5±1.2 pS (n=6). As shown in Fig 6, hypoxia significantly reduced NxP(open) of the 25-pS channel at membrane holding potentials of -20, 0, and +20 mV.

View larger version (23K): [in this window] [in a new window]   Figure 6. Effect of hypoxia on delayed rectifier K+ channels in cell-attached membrane patches. A, Recordings showing small-conductance K+ channels measured at +20 mV by using a physiological K+ gradient during normoxia (left) and then during hypoxia (right). In this experiment, hypoxic solutions were obtained by bubbling with 100% N2, which reduced PO2 from 130 mm Hg (normoxic) to 33 mm Hg. B, Bar graph summarizing the effects of hypoxia on NxP(open) [where N is the number of functional channels in a patch and P(open) is the opening probability] at -20, 0, and +20 mV (data accumulated from three patches). When a physiological K+ gradient was used, the conductance of the small channel was 25 picosiemens and the large channel was 152 picosiemens. Cell membrane potential was zeroed by bathing cells in high K+ solution.

Because the whole-cell results suggested that hypoxic inhibition of K⁺ current may be due to [Ca²⁺]_i inhibition of a 4-AP–sensitive K⁺ channel, we directly tested the effects of 4-AP and changes in cytoplasmic Ca²⁺ on the activity of small-conductance K⁺ channels with inside-out membrane patches. In these experiments, charybdotoxin (100 nmol/L) was included in the pipette solution (of inside-out patches) to reduce the activity of large-conductance Ca²⁺-activated K⁺ channels. 4-AP (1 mmol/L) added directly to the intracellular surface of inside-out patches reduced NxP(open) of the 25-pS channels from 0.399±0.2 to 0.157±0.08 at 0 mV (n=3; data not shown). In many experiments, the presence of residual activity of large-conductance Ca²⁺-activated channels precluded the ability to assess the effects of Ca²⁺ on the smaller-conductance channels, but in two patches in which the large-conductance channel was not present, the application of 1 µmol/L Ca²⁺ to the cytoplasmic surface of the membrane reduced NxP(open) of the 25-pS delayed rectifier K⁺ channel from 1.81±0.18 to 0.69±0.50 (Fig 7). In four other patches, it was possible to show an inhibitory effect of Mg²⁺ on the small-conductance K⁺ channels, similar to that previously shown for the effects of Mg²⁺ on delayed rectifier K⁺ channels in patches from canine renal arterial cells.¹⁹ The inhibitory effects of Mg²⁺ were much less potent than those of Ca²⁺, with 1 mmol/L Mg²⁺ reducing mean NxP(open) of the 4-AP–sensitive 25-pS channel from 1.87 to 0.87 (data not shown). This suggests that under normal physiological conditions, although some competition between intracellular Mg²⁺ and Ca²⁺ is expected, Ca²⁺ is a much more potent ligand for the inhibitory binding site on delayed rectifier K⁺ channels in pulmonary arterial cells.

View larger version (29K): [in this window] [in a new window]   Figure 7. Effect of Ca2+ on delayed rectifier K+ channels in inside-out membrane patches. A, Delayed rectifier K+ channels (25 picosiemens) in Ca2+-free bath solution (estimated free [Ca2+]i, 4x10-9 mol/L). B, Delayed rectifier K+ channels (25 picosiemens) in same patch after changing to a bath solution containing 1 µmol/L Ca2+. Top panels are recordings. Bottom panels are amplitude histograms. Ca2+ at 1 µmol/L reduced NxP(open) [where N is the number of functional channels in a patch and P(open) is the opening probability] of the delayed rectifier K+ channels from 1.81±0.18 to 0.69±0.50 (holding potential, 20 mV; n=2). In these experiments, the pipette solution contained 100 nmol/L charybdotoxin to reduce the activity of large-conductance Ca2+-activated K+ channels.

[Ca²⁺]_i Inhibition of 4-AP–Sensitive K⁺ Channels Causes Hypoxic Membrane Depolarization
Although previous studies have suggested some contribution of 4-AP–sensitive K⁺ channels to the resting membrane potential of vascular smooth muscle cells,^{11 19 26 27} direct demonstration of 4-AP–sensitive K⁺ channels at the resting membrane potential is difficult, because of the small conductance and low driving force of these channels when a physiological K⁺ gradient is used. Earlier experiments using ramp depolarizations provided evidence for a 4-AP–sensitive component of membrane current, with an activation threshold near -30 mV, significantly more positive than the mean resting membrane potential of acutely isolated pulmonary arterial cells (Fig 3). It seems particularly important to demonstrate a contribution of 4-AP–sensitive K⁺ current at more physiological potentials given that ATP-sensitive K⁺ currents can modulate membrane potential of pulmonary arterial cells when intracellular ATP levels are low²⁸ and since an inwardly rectifying K⁺ current has recently been identified as an important determinant of resting membrane potential in certain types of vascular smooth muscle cells.²⁹

To facilitate the identification of membrane currents activated at negative membrane potentials, voltage ramps were applied from -120 to 20 mV with symmetrical K⁺ solutions. With these solutions, the driving force for inward K⁺ currents at negative potentials was increased since the K⁺ equilibrium potential (E_K) was shifted to near 0 mV. Fig 8A shows the effects of changing [K⁺]_o from 5.4 to 140 mmol/L on membrane currents activated by ramp depolarizations from -120 to 20 mV. With [K⁺]_o of 5.4 mmol/L, only small outward currents could be detected over the voltage range from -45 to 20 mV. After changing to symmetrical 140 mmol/L K⁺, a large inward current was activated by the voltage ramp with a threshold near -65 mV. The current reversed close to the predicted value of E_K and became outward at more positive membrane potentials. Fig 8B shows results obtained in another cell, which indicates that most of the inward K⁺ current activated at negative membrane potentials was blocked by 4-AP (1 mmol/L).

View larger version (12K): [in this window] [in a new window]   Figure 8. Recordings showing the effect of hypoxia on 4-aminopyridine (4-AP)–sensitive K+ currents active near the resting membrane potential. A, Effect of changing [K+]o on K+ current activated by ramp depolarization from -120 to 20 mV is shown. Changing [K+]o from 5.4 to 140 mmol/L increased the driving force for K+ at more negative potentials. [K+]o at 140 mmol/L changed K+ current from outward to inward at potentials more negative than the K+ equilibrium potential and enhanced K+ current amplitude. B, Inward whole-cell K+ current was blocked by 4-AP (1 mmol/L) in high-K+ solution. C, Inward K+ current at negative potentials was inhibited after changing to hypoxic high-K+ bath solution. In this experiment, initial holding potential was -70 mV, and the rate of ramp depolarization was 0.028 mV/ms (nisoldipine [1 µmol/L] was present throughout experiment). All current measurements were made 2 minutes after each experimental intervention.

It is noteworthy that no evidence for the presence of an inwardly rectifying K⁺ current was observed in pulmonary arterial smooth muscle cells as has been demonstrated in cerebral arterial cells.²⁹ Furthermore, in three cells 0.5 mmol/L Ca²⁺ was found to have little effect on the amplitude of inward K⁺ currents. In these experiments, the possible contribution of ATP-sensitive K⁺ currents was minimized by dialyzing cells with an intracellular solution containing 2 mmol/L ATP. In three cells, sodium dithionite–induced hypoxia reduced the peak amplitude of the 4-AP–sensitive inward K⁺ current by an average of 45.3±2.1% (Fig 8C). These data leave little doubt that hypoxia inhibits a 4-AP–sensitive K⁺ channel, which normally contributes to maintenance of the resting membrane potential of pulmonary arterial smooth muscle cells. It should be noted that these cells in the presence of nisoldipine still exhibited hypoxic inhibition of K⁺ currents, in contrast to earlier results¹¹ in which nisoldipine seemed to attenuate the hypoxic response. The difference might be related to the time of nisoldipine exposure: in the earlier experiments, cells were bathed in nisoldipine-containing solutions for long times (up to an hour), which might have resulted in depletion of [Ca²⁺]_i stores.

Previous experiments using caffeine and strong intracellular Ca²⁺ buffers suggest that hypoxic inhibition of 4-AP–sensitive K⁺ channels may be mediated by release of Ca²⁺ (Figs 1 through 3), and single-channel studies provided direct evidence that physiological concentrations of Ca²⁺ applied to the cytoplasmic surface of inside-out membrane patches directly inhibit 4-AP–sensitive 25-pS K⁺ channels (Fig 7). This hypothesis predicts that exposure of pulmonary arterial smooth muscle cells to hypoxia should elicit an early increase in [Ca²⁺]_i, which should precede the observed depolarization of the resting membrane potential. To test this prediction, cells were dialyzed with the Ca²⁺ indicator indo 1 to simultaneously monitor changes in Ca²⁺_i and membrane potential during hypoxia. As shown in Fig 9, sodium dithionite–induced hypoxia reversibly increased [Ca²⁺]_i and depolarized the resting membrane potential in isolated pulmonary arterial smooth muscle cells. In three cells, hypoxia produced a change in mean fluorescence from 0.91±0.2 to 1.4±0.02 and a change in mean resting membrane potential from -54.0±1.5 to -31.7±1.5 mV. The time course of the response demonstrates that the depolarization is indeed preceded by an increase in [Ca²⁺]_i. In these experiments, charybdotoxin and niflumic acid were included in the bath solution to prevent possible activation of Ca²⁺-activated K⁺ or Cl^- channels, respectively.

	Discussion

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The present results and the recent findings of several investigators suggest that hypoxic mobilization of Ca²⁺ from intracellular stores^{17 30} may represent an important initial event in the induction of HPV. Our data suggest that hypoxic mobilization of [Ca²⁺]_i may directly cause inhibition of 25-pS 4-AP–sensitive delayed rectifier K⁺ channels, leading to membrane depolarization, which (although not directly shown in the present study) may promote Ca²⁺ entry via voltage-dependent Ca²⁺ channels.^{7 8 9 31} These results are consistent with previous reports demonstrating that (1) 4-AP is an effective pulmonary vasoconstrictor,³² (2) depletion of intracellular Ca²⁺ stores with procaine attenuates HPV,³³ and (3) chronic pulmonary hypoxia is associated with decreased activity of delayed rectifier K⁺ channels.³⁴

Other studies have demonstrated that pulmonary arterial smooth muscle cells contain several types of K⁺ channels on the basis of different pharmacological and biophysical characteristics. ATP-modulated K⁺ channels, which assist in the regulation of resting membrane potential in pulmonary arterial cells,²⁸ do not appear to be inhibited by hypoxia, since dialysis of the cells with ATP did not prevent hypoxic inhibition of K⁺ current.¹¹ In addition, the observation that the ATP-sensitive K⁺ channel blocker glibenclamide does not cause a pressor response or potentiate hypoxic constriction in perfused rat lungs³² argues against a role for ATP-modulated K⁺ channels during the hypoxic response.

Ca²⁺-activated K⁺ channels do not appear to be inhibited by hypoxia, since the Ca²⁺-activated K⁺ channel antagonist TEA (1 mmol/L) does not prevent hypoxic inhibition of K⁺ current. In addition, hypoxia does not significantly alter open probability of the large-conductance K⁺ channels in inside-out membrane patches. Intuitively, this would seem logical, since the direct relation between hypoxia-induced contraction and increase in [Ca²⁺]_i^{17 30} would be expected to augment rather than inhibit the activity of Ca²⁺-activated K⁺ channels, which we did observe in some cell-attached membrane patches. Thus, the effects of [Ca²⁺]_i on these channels would tend to oppose the membrane depolarization induced by hypoxia, which might contribute to the transient nature of the hypoxic constrictor response.

We previously suggested that hypoxia may inhibit a Ca²⁺-sensitive K⁺ channel, since high buffering of [Ca²⁺]_i with BAPTA prevented hypoxia from inhibiting macroscopic K⁺ currents in pulmonary arterial smooth muscle cells.¹¹ In contrast to our results, it has been suggested that hypoxia modulates the activity of Ca²⁺-insensitive K⁺ channels,¹² since buffering [Ca²⁺]_i with EGTA did not prevent hypoxic inhibition of K⁺ current. These conflicting results can be explained by the fact that BAPTA is a much faster Ca²⁺ buffer than EGTA³⁵ and would be expected to be more effective in buffering a fast intracellular Ca²⁺ release process than EGTA. Similar analogies exist in chromaffin cells³⁶ and in presynaptic terminals,³⁷ where BAPTA is much more effective than EGTA in suppressing Ca²⁺-activated K⁺ channels or evoked transmitter release, respectively. It is also noteworthy that the dependence of hypoxic inhibition of pulmonary arterial K⁺ currents on [Ca²⁺]_i described here is also supported by the observation that the hypoxic response is mimicked by acute exposure of cells to caffeine, which initially liberates Ca²⁺ from internal stores, and that the hypoxic inhibition of 4-AP–sensitive K⁺ channels is eliminated in cells in which intracellular Ca²⁺ stores have been depleted by caffeine. It was also shown that 4-AP–sensitive 25-pS K⁺ channels are directly inhibited by Ca²⁺ applied to the cytoplasmic membrane surface. This conductance for delayed rectifier K⁺ channels is slightly higher than that reported in some previous studies of vascular smooth muscle³⁸ and airway smooth muscle³⁹ but is well within the range recently reported for visceral smooth muscle.⁴⁰

In summary, all of these data are consistent with our earlier conclusions regarding the involvement of Ca²⁺-sensitive K⁺ channels in the hypoxic pulmonary response.¹¹ In addition, our data confirm and extend the earlier results of Salvaterra and Goldman,¹⁷ who showed that the hypoxia-induced elevation of [Ca²⁺]_i in cultured pulmonary arterial cells consisted of an early release of Ca²⁺ from the SR and a later influx of extracellular Ca²⁺. Although membrane depolarization and subsequent extracellular Ca²⁺ entry has long been suspected to represent an important cellular event in hypoxic pulmonary constriction,^{7 8 9 11} our data suggest a novel mechanism that links intracellular Ca²⁺ release to subsequent membrane depolarization. In contrast to the results of Salvaterra and Goldman, who showed that the early Ca²⁺ release phase occurred within 50 to 70 seconds of the onset of hypoxia, our data showed a slower onset for the inhibition of K⁺ current in response to hypoxia (Figs 1 through 3). This slower rate might be due to some [Ca²⁺]_i buffering by the inclusion of 1 mmol/L EGTA in our dialysis pipettes or due to a slower rate at which PO₂ was reduced in our experiments (see "Materials and Methods").

The hypoxic vasoconstrictor response of pulmonary arteries is a rather unique response, since many other systemic arteries usually exhibit vasodilation in response to hypoxia.⁴¹ Yet the ability of [Ca²⁺]_i to inhibit sarcolemmal delayed rectifier K⁺ channels and cause membrane depolarization seems to be a rather general phenomenon that has been previously observed in a number of different vascular and visceral smooth muscle cells.^{18 19} The phenomenon may be even more general, since divalent cations have recently been shown to block K⁺ channels in human parathyroid cells⁴² and to block K_v1.2 K⁺ channels expressed in Xenopus oocytes.⁴³ This raises the important question of how such a general phenomenon might be reconciled with the uniqueness of the pulmonary response to hypoxia. Indeed, it has been shown (eg, in isolated canine renal arterial cells) that various agonists cause [Ca²⁺]_i inhibition of delayed rectifier K⁺ channels and membrane depolarization by liberation of Ca²⁺ from both ryanodine- and caffeine-sensitive and -insensitive Ca²⁺ stores,²⁰ yet hypoxia does not inhibit whole-cell K⁺ currents in these cells.¹¹ Although the mechanism by which low PO₂ triggers Ca²⁺ release in the pulmonary artery is unknown, it may be that hypoxic modulation of this site confers the uniqueness of the pulmonary response to hypoxia, and there is precedence for the ability of cellular redox status to influence the activity of SR Ca²⁺-release channels.⁴⁴ Thus, in contrast to earlier suggestions^{15 16} that hypoxic inhibition of pulmonary K⁺ channels may involve direct redox regulation of a sarcolemmal K⁺ channel, our results would suggest that such an effect is indirect and due to cellular redox regulation of the activity of an SR channel or transporter. Consistent with earlier observations that HPV does not cause elevation of inositol 1,4,5-tris-phosphate levels,⁴⁵ our data would suggest that hypoxic release of Ca²⁺ involves a caffeine-sensitive Ca²⁺ store in acutely isolated pulmonary arterial cells.

	Acknowledgments

 
This study was supported by National Institutes of Health (NIH) grant HL-49254 (Dr Hume) and a grant from the American Heart Association, Nevada Affiliate, Inc (Dr Post). Drs Post and Gelband were supported by NIH postdoctoral fellowships. Canine pulmonary arteries were graciously made available from a program project grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-41315). Expert technical assistance was provided by Lan Xue, Michael Sokoloff, and Sarena Keane.

Received August 15, 1994; accepted March 20, 1995.

	References

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