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(Circulation. 1997;96:3647-3654.)
© 1997 American Heart Association, Inc.

Articles

Ca²⁺ Release From Intracellular Stores Is an Initial Step in Hypoxic Pulmonary Vasoconstriction of Rat Pulmonary Artery Resistance Vessels

Craig H. Gelband, PhD; ; Henry Gelband, MD

From the Department of Physiology, University of Florida College of Medicine, Gainesville (C.H.G.), and the Division of Pediatric Cardiology, Department of Pediatrics, University of Miami (Fla) School of Medicine (H.G.).

Correspondence to Craig H. Gelband, Department of Physiology, University of Florida College of Medicine, PO Box 100274, Gainesville (C.H.G.), FL 32610. E-mail gelband{at}phys.med.ufl.edu

	Abstract

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Background A reduction in oxygen tension in the lungs is believed to inhibit a voltage-dependent K⁺ (Kv) current, which is thought to result in membrane depolarization leading to hypoxic pulmonary vasoconstriction (HPV). However, the direct mechanism by which hypoxia inhibits Kv current is not understood.

Methods and Results Experiments were performed on rat pulmonary artery resistance vessels and single smooth muscle cells isolated from these vessels to examine the role of Ca²⁺ release from intracellular stores in initiating HPV. In contractile experiments, hypoxic challenge of endothelium-denuded rat pulmonary artery resistance vessels caused either a sustained or transient contraction in Ca²⁺-containing or Ca²⁺-free solution, respectively (n=44 vessels from 11 animals). When the ring segments were treated with either thapsigargin (5 µmol/L), ryanodine (5 µmol/L), or cyclopiazonic acid (5 µmol/L) in Ca²⁺-containing or Ca²⁺-free solution, a significant increase in pulmonary arterial tone was observed (n=44 vessels from 11 animals). Subsequent hypoxic challenge in the presence of each agent produced no further increase in tone (n=44 vessels from 11 animals). In isolated pulmonary resistance artery cells loaded with fura 2, hypoxic challenge, thapsigargin, ryanodine, and cyclopiazonic acid resulted in a significant increase in [Ca²⁺]_i (n=18 cells from 6 animals) and depolarization of the resting membrane potential (n=22 cells from 6 animals). However, with prior application of thapsigargin, ryanodine, or cyclopiazonic acid, a hypoxic challenge produced no further change in [Ca²⁺]_i (n=18 from 6 animals) or membrane potential (n=22 from 6 animals). Finally, application of an anti-Kv1.5 antibody increased [Ca²⁺]_i and caused membrane depolarization. Subsequent hypoxic challenge resulted in a further increase in [Ca²⁺]_i with no effect on membrane potential (n=16 cells from 4 animals).

Conclusions In rat pulmonary artery resistance vessels, an initial event in HPV is a release of Ca²⁺ from intracellular stores. This rise in [Ca²⁺]_i causes inhibition of voltage-dependent K⁺ channels (possibly Kv1.5), membrane depolarization, and an increase in pulmonary artery tone.

Key Words: hypoxia • calcium • sarcoplasmic reticulum • potassium •

	Introduction

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In the lung, HPV is thought to serve as an adaptive mechanism by which pulmonary blood flow is diverted from poorly ventilated to well-ventilated lung parenchyma to optimize ventilation/perfusion matching.¹ However, reductions in PO₂ can lead to pulmonary arterial vasoconstriction, vascular remodeling, and pulmonary hypertension. The hypoxic pressor response is a unique physiological process that distinguishes the pulmonary vasculature from the systemic circulation, which usually dilates in response to hypoxia. The specific mechanism by which a decrease in PO₂ is sensed by the pulmonary vasculature and vasoconstriction is initiated is unknown.

HPV occurs in isolated pulmonary arteries denuded of endothelium, suggesting a direct effect of hypoxia on vascular smooth muscle.² Harder et al³ reported that hypoxia-induced vasoconstriction of small pulmonary arteries is accompanied by membrane depolarization. Additional studies have shown that hypoxia-induced increases in pulmonary vascular resistance can be potentiated by BAY K 8644 and inhibited by calcium channel blockers (verapamil, nifedipine, and nisoldipine) and the Kv channel blocker 4-AP, suggesting that HPV might involve membrane depolarization leading to Ca²⁺ entry through voltage-dependent Ca²⁺ channels.^4-7 Furthermore, the observation that a number of K⁺ channel inhibitors simulate HPV by increasing tension in intact pulmonary artery rings and elevate pulmonary artery pressure in isolated lungs^7,8 suggests that K⁺ channel inhibition may be an early critical event in the initiation of HPV. Recent electrophysiological studies have shown that an acute hypoxic challenge inhibits macroscopic whole-cell K⁺ currents, causing depolarization of the resting membrane potential in both freshly isolated and cultured pulmonary arterial smooth muscle cells.^8-11 These studies proposed that a hypoxic challenge inhibited a 4-AP–sensitive, voltage-dependent delayed rectifier K⁺ current that may be of the Kv1.5 class of K⁺ channels. These types of K⁺ channels have been shown in vascular smooth muscle, including pulmonary artery.¹²

Despite this recent progress, it is not clear by what cellular mechanism(s) hypoxia exerts its inhibitory effects on Kv channels. One hypothesis implies that hypoxia releases Ca²⁺ from intracellular stores and that it is Ca²⁺ that inhibits Kv channels.¹⁰ This is supported by recent evidence showing an early mobilization of intracellular free Ca²⁺ by hypoxia in cultured pulmonary arterial smooth muscle cells¹³ and the ability of intracellular free Ca²⁺ to directly inhibit Kv channels in a variety of smooth muscle cells,^14-16 including pulmonary arterial cells.¹⁰ Other studies have suggested a potential mechanism responsible for hypoxic inhibition of K⁺ channels that may involve hypoxia-induced changes in cytosolic redox status.^17-20

In light of this work, we examined the role of the sarcoplasmic reticulum in hypoxic modulation of vascular smooth muscle tone in resistance vessels and single cells of the rat pulmonary vasculature. Our objective was to identify and characterize which intracellular Ca²⁺ stores, either those activated by the second messenger IP₃ or those sensitive to the plant alkaloid ryanodine, are sensitive to hypoxia and to examine the role of hypoxia and sarcoplasmic reticulum in altering [Ca²⁺]_i and membrane potential in the rat pulmonary vasculature.

	Methods

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Tension Measurements
Male rats were euthanized, and the right lung was rapidly excised and placed in cold, oxygenated (95% O₂/5% CO₂) KHB containing (in mmol/L) NaCl 120, NaHCO₃ 20, KCl 4.2, KH₂PO₄ 1.2, MgCl₂ 0.5, CaCl₂ 1.8, and glucose 11; pH 7.38±0.3). A 0.5- to 1-cm segment of the fifth to seventh branch of the pulmonary artery was dissected free and cleaned of fat and adventitia. The internal diameter of these vessels was 200 µm. The vessels were denuded of endothelium by gentle rubbing of the internal surface of the vessel with filter paper or a tungsten wire. Ring segments (3 to 5 mm long) were mounted onto two triangular tungsten wires (65 µm in diameter) and placed into an isolated organ chamber. One triangle was mounted to a stable hook, while the top triangle was attached to a Gould strain gauge. The bath was maintained at 37°C in KHB solution (Po₂, 130±8 mm Hg). A resting force of 0.50 g was applied to the pulmonary artery segments. Vessel segments were initially equilibrated for 2 hours with alternating 5-minute exposures (with 15 minutes of washout) to KCl (80 mmol/L) and phenylephrine (1 µmol/L). All vessels were tested with acetylcholine (1 µmol/L) to assess our technique to remove the endothelium. Bath solutions were made hypoxic (PO₂, 26±3 mm Hg) by bubbling the KHB solution with 95% N₂/5% CO₂. The pH of this solution did not change (7.35±0.2). PO₂ values were obtained every 3 minutes from a blood gas analyzer for all hypoxic experiments. Ca²⁺-free KHB was made by replacing the CaCl₂ with EGTA (2 mmol/L).

Isolation of Single Smooth Muscle Cells
Single rat pulmonary artery smooth muscle cells were isolated by enzymatic digestion similar to that performed for the rat renal artery.²¹ In oxygenated KHB, a segment of the pulmonary artery was cleaned of fat and adventitia, cut into small pieces, and allowed to equilibrate for 45 to 60 minutes in oxygenated Ca²⁺-free KHB. The pieces of artery were resuspended in a Ca²⁺-free KHB digestion buffer for 30 to 45 minutes and gently stirred at 37°C. The Ca²⁺-free KHB digestion buffer contained (in mg/10 mL) collagenase 10 (Worthington 151 U/mg), protease 1.0 (Sigma type XXIV), trypsin inhibitor 20 (Sigma type II-S), ATP 1.1 (sodium salt), and BSA.²⁰ After digestion, the tissue was resuspended in oxygenated Ca²⁺-free KHB and gently triturated until a large number of elongated cells were observed. The isolated cells were then collected, resuspended in KHB, and stored at 4°C. Cells were used between 2 and 10 hours after isolation. All drugs used were purchased from Sigma Chemical Co unless otherwise specified.

Electrophysiological Methods
An aliquot of single pulmonary artery smooth muscle cells in suspension was added to the recording chamber (250 µL) mounted on a Nikon Diaphot microscope. The recording chamber was coated with cel-tak to ensure that the cells stuck to the bottom of the recording chamber and did not move during the experiments. Solutions were superfused through the chamber by gravity at a rate of 2.5 mL/min. When hypoxic solutions were superfused into the bath, O₂ reequilibration was avoided by having air jets of 100% N₂ pass over the experimental chamber. Experiments were performed at room temperature. Membrane voltage (under current clamp) was measured by the whole-cell configuration of the patch-clamp technique.²² Patch pipettes were made from borosilicate glass capillaries, pulled on a vertical puller (Narishige Scientific), and fire-polished with a microforge (Narishige Scientific). Pipettes had resistances of 1 to 3 M when filled with the appropriate solutions. Membrane potential was measured with an Axopatch 200A patch-clamp amplifier (Axon Instruments). Membrane potential was monitored on a digital oscilloscope (Hitachi Instrument), and data were stored on videotape with a digital VCR instrumentation recorder adaptor (Vetter) for later analysis. The bath solution contained (in mmol/L) NaCl 130, NaHCO₃ 10, KCl 4.2, H₂PO₄ 1.2, MgCl₂ 0.5, CaCl₂ 0 to 5, d-glucose 5.5, and HEPES 10 (pH 7.39±0.2 with NaOH). The pipette solution contained (in mmol/L) KCl 140, MgCl₂ 0.1, ATP (magnesium salt) 5, and HEPES 5 (pH 7.2±0.1 with NaOH). All drugs used were purchased from Sigma Chemical Co unless otherwise specified. Anti-Kv1.5 antibody was purchased from Alomone Laboratories (product No. APC-004).

Fluorescence Techniques
[Ca²⁺]_i in rat pulmonary artery smooth muscle cells was measured by fluorescence microscopy. To facilitate loading, the Ca²⁺ indicator fura 2 (pentapotassium salt, 50 µmol/L) was included in the patch pipette solution and dialyzed into the cell. This method of loading the dye will prevent some of the difficulties observed to occur when cells are loaded with the ester form of the dye (ie, leak from the cell, incomplete hydrolysis of the ester form, or sequestration into internal organelles). Background autofluorescence was measured before access was gained to the interior of the cell and subtracted from all fluorescence measurements.

The cells were illuminated alternately with ultraviolet light (10 Hz) of 340- and 380-nm wavelength with an IonOptix chopper-based, electronically controlled dual excitation imaging fluorescence system. Cell fluorescence (emitted light) was collected through a 510-nm barrier filter before acquisition by an ICCD camera (Phillips FTM800). Fluorescence signals were digitized on-line with an IBM-PC–compatible computer and IonOptix fluorescence imaging acquisition and analysis software. [Ca²⁺]_i can be calculated from the equation [Ca²⁺]_i =K_d(R-R_min)/(R_max-R)(S_f2/S_b2), where R is the F₃₄₀/F₃₈₀ fluorescence ratio, S_f2 is the 380-nm fluorescence signal in Ca²⁺-free solution, and S_b2 is the 380-nm fluorescence signal in Ca²⁺-containing solution. However, because of the uncertainties involved in the calculation of [Ca²⁺]_i by use of this equation,²³ the signals are reported as changes in F₃₄₀/F₃₈₀. This gives a relative indication of [Ca²⁺]_i. However, because [Ca²⁺]_i may need to be known at times, the basal level of [Ca²⁺]_i in these cells was 112±24 nmol/L (n=72 cells from 12 animals). Hypoxic challenge caused [Ca²⁺]_i to rise to 360±29 nmol/L (n=36 cells from 12 animals). Hypoxic challenge did not change the autofluorescence of cells that were not loaded with fura 2.

Data Analysis
Phenylephrine, an -adrenergic receptor agonist (10 µmol/L) that contracts blood vessels and increases [Ca²⁺]_i vascular smooth muscle, was used as a pharmacological mechanism to compare the data. Hypoxic contractions were normalized to a maximum phenylephrine contraction (10 µmol/L). Results are expressed as mean±SEM. Student's t test for unpaired observations and a one-way ANOVA were used to determine statistical significance. Differences were considered significant at P<.05. n corresponds to the number of tissue rings or cells examined.

	Results

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Ca²⁺ Dependence of Hypoxic Contractions
The first set of experiments was designed to investigate the Ca²⁺ dependence of HPV in pulmonary artery vessel rings. Fig 1 illustrates that resistance vessels of the pulmonary vasculature contract when a hypoxic challenge was applied to the vessel in Ca²⁺-containing and Ca²⁺-free solution. In Ca²⁺-containing solution (Fig 1A), hypoxic challenge caused a sustained contraction (0.48 g), whereas in Ca²⁺-free solution, only a rapid transient contraction was observed when the vessel was challenged by hypoxia (Fig 1B). These data are consistent with hypoxia causing Ca²⁺ release from intracellular stores and influx from the extracellular space in Ca²⁺-containing solution but only Ca²⁺ release from intracellular stores in Ca²⁺-free solution. Fig 1C illustrates that when the above data were normalized to the maximum phenylephrine contraction (10 µmol/L), hypoxia caused similar contractions in Ca²⁺-containing and Ca²⁺-free solution that were 75±4% and 68±3% of the peak phenylephrine contraction, respectively (n=32 vessels from 8 animals). A control phenylephrine contraction was used to normalize the data because it also causes Ca²⁺ release from internal stores and Ca²⁺ influx from the extracellular space.²⁴

View larger version (11K): [in this window] [in a new window]   Figure 1. Effect of extracellular Ca2+ on HPV. A, Hypoxic challenge resulted in sustained contraction of pulmonary artery rings in Ca2+-containing solution. B, Hypoxia causes rapid transient contraction of pulmonary artery rings in Ca2+-free solution. C, Mean effect of hypoxia on pulmonary artery tone in Ca2+-containing and Ca2+-free solutions (mean±SEM, n=32 cells from 8 animals). Hypoxic contractions were normalized to a 10 µmol/L phenylephrine contraction. No significant difference was observed for hypoxic challenge in Ca2+-containing or Ca2+-free solution.

Intracellular Ca²⁺ Stores Are Involved in the Hypoxic Response
At least two types of intracellular stores are involved in excitation-contraction coupling of vascular smooth muscle: IP₃-sensitive Ca²⁺ stores and CICR-sensitive Ca²⁺ stores.²⁴ To investigate the effect of hypoxia on these two Ca²⁺ stores, specific agents that alter the Ca²⁺ load of each of these storage sites were investigated. Thapsigargin and cyclopiazonic acid were used to deplete Ca²⁺ from IP₃-sensitive Ca²⁺ stores and ryanodine to deplete Ca²⁺ from CICR-sensitive Ca²⁺ stores.^24-26 Fig 2 illustrates the effect of hypoxia (Fig 2A), thapsigargin (5 µmol/L) plus hypoxia (Fig 2B), and ryanodine (5 µmol/L) plus hypoxia (Fig 2C) on pulmonary artery contraction in Ca²⁺-containing solution. Alone, hypoxia, thapsigargin, and ryanodine produced a sustained contraction that was 75% to 80% of a 10 µmol/L phenylephrine contraction (Figs 2A, 2B, 2C, and 3, n=44 vessels from 11 animals). However, after pretreatment of the tissue with either thapsigargin or ryanodine (Fig 2B and 2C), hypoxia, when challenged at the peak of contraction, resulted in no potentiation of the contraction (n=44 vessels from 11 animals). Results similar to that of thapsigargin were obtained with cyclopiazonic acid (5 µmol/L, Fig 3). Mean data for the above effects are summarized in Fig 3. In Ca²⁺-containing solution, hypoxia, thapsigargin, cyclopiazonic acid, and ryanodine caused a significant contraction of the pulmonary vessels compared with control (P<.01, n=44 vessels from 11 animals). The contractions were 77±5%, 79±4%, 81±3%, and 79±4% of the 10 µmol/L phenylephrine contraction, respectively. However, when the tissues were challenged by hypoxia after the thapsigargin, ryanodine, or cyclopiazonic acid, no significant potentiating effect was observed. The lack of a hypoxic effect on force when the vessels were pretreated with the thapsigargin, ryanodine, or cyclopiazonic acid was not due to a "ceiling" effect on tone, because KCl (80 mmol/L) could elicit a further increase in tone after pretreatment with the above pharmacological agents (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 2. Thapsigargin and ryanodine inhibit hypoxic contraction of pulmonary artery in Ca2+-containing solution. A, Control hypoxic contraction in Ca2+-containing solution. B, Thapsigargin (5 µmol/L) contracts vessel in Ca2+-containing solution, and subsequent hypoxic challenge in presence of drug produces no potentiation of contraction. Similar results were obtained in 44 vessels from 11 animals. C, Ryanodine (5 µmol/L) contracts vessel in Ca2+-containing solution, and subsequent hypoxic challenge in presence of drug produces no potentiation of contraction. Similar results were obtained in 44 vessels from 11 animals. Upper time bar applies to A and B; lower time bar to C.

View larger version (36K): [in this window] [in a new window]   Figure 3. Summary of effect of hypoxia (Hyp), thapsigargin (Tg), cyclopiazonic acid (Cpa), and ryanodine (Ryan) on pulmonary artery tone in Ca2+-containing and Ca2+-free solutions. All data are normalized to 10 µmol/L phenylephrine (Phe) contraction. Note that in Ca2+-free solution, hypoxia when applied to vessels pretreated with thapsigargin, cyclopiazonic acid, or ryanodine caused no significant contraction. Data are mean±SEM. *Significantly different from control phenylephrine contraction, P<.01.

Experiments were also performed in Ca²⁺-free solution (Fig 4). On their own, hypoxia, thapsigargin, and ryanodine produced a rapid transient contraction, suggesting that the contraction resulted only from Ca²⁺ release from intracellular stores. (Fig 4A, 4B, and 4C, n=44 vessels from 11 animals). With prior treatment of the tissue with either thapsigargin or ryanodine (Fig 4B and 4C), hypoxia was unable to elicit a contraction of the blood vessel when applied after the contraction was terminated (n=44 vessels from 11 animals). Results similar to that of thapsigargin were obtained with cyclopiazonic acid (Fig 3). Mean data for the above effects are summarized in Fig 3. In Ca²⁺-free solution, hypoxia, thapsigargin, cyclopiazonic acid, and ryanodine caused a significant contraction of the pulmonary vessels compared with the maximum phenylephrine contraction (P<.01, n=44 vessels from 11 animals). The contractions were 70±5%, 76±4%, 83±5%, and 76±3% of the 10 µmol/L phenylephrine contraction, respectively. Most importantly, when the tissues were challenged by hypoxia after treatment with thapsigargin, ryanodine, or cyclopiazonic acid, no significant contraction was elicited. The data observed in Ca²⁺-containing and Ca²⁺-free solution suggest that by depletion of intracellular Ca²⁺ stores, HPV can be prevented.

View larger version (10K): [in this window] [in a new window]   Figure 4. Thapsigargin and ryanodine inhibit hypoxic contraction of pulmonary artery in Ca2+-free solution. A, Control hypoxic contraction in Ca2+-free solution. Contraction is transient, suggesting intracellular Ca2+ release. B, Thapsigargin (5 µmol/L) transiently contracts vessel in Ca2+-free solution, and subsequent hypoxic challenge in presence of drug produces no effect. Similar results were obtained in 44 vessels from 11 animals. C, Ryanodine (5 µmol/L) contracts vessel in Ca2+-free solution, and subsequent hypoxic challenge in presence of drug produces no effect. Similar results were obtained in 44 vessels from 11 animals. Time bar applies to all three panels.

It was next necessary to illustrate that the above pharmacological effects were due to changes in [Ca²⁺]_i. Experiments were performed in Ca²⁺-containing solution with fura 2–loaded, freshly isolated pulmonary arterial cells. Fig 5 shows that hypoxia (Fig 5A), thapsigargin (5 µmol/L, Fig 5B), and ryanodine (5 µmol/L, Fig 5C) increased [Ca²⁺]_i over basal values. The increase in [Ca²⁺]_i by these agents was 2.2-, 2.15-, and 2.1-fold, respectively. The mean data on [Ca²⁺]_i are presented in Fig 6. Phenylephrine, hypoxia, thapsigargin, and ryanodine increased [Ca²⁺]_i 250±13%, 200±5%, 197±8%, and 175±16% over basal levels, respectively (P<.01, n=18 cells from 6 animals). However, when a hypoxic challenge was applied to the cells after treatment with thapsigargin or ryanodine, no potentiation of the change in [Ca²⁺]_i was observed (Fig 5B and 5C). Responses similar to those to thapsigargin were obtained with cyclopiazonic acid (5 µmol/L, 186±4%).

View larger version (18K): [in this window] [in a new window]   Figure 5. Thapsigargin and ryanodine inhibit hypoxia-induced changes in [Ca2+]i in pulmonary artery cells. A, Control hypoxic change in [Ca2+]i. Note that increase in [Ca2+]i is sustained and reversible. B, Thapsigargin (5 µmol/L) increases [Ca2+]i, and subsequent hypoxic challenge in presence of drug produces no potentiation. Similar results were obtained in 18 cells. C, Ryanodine (5 µmol/L) increases [Ca2+]i, and subsequent hypoxic challenge in presence of drug produces no potentiation of the signal. Similar results were obtained in 18 cells from 6 animals. Upper time bar applies to A and B; lower time bar to C.

View larger version (17K): [in this window] [in a new window]   Figure 6. Summary of effect of phenylephrine, hypoxia, thapsigargin, cyclopiazonic acid, and ryanodine on pulmonary artery [Ca2+]i. All data are normalized to basal levels of [Ca2+]i. Note that no change in percent increase in [Ca2+]i occurred when cells were challenged by hypoxia after treatment with thapsigargin, cyclopiazonic acid, or ryanodine. Data are mean±SEM. *Significantly different from resting [Ca2+]i, P<.01. Abbreviations as in Fig 3.

Like the contraction data, the lack of a hypoxic effect on [Ca²⁺]_i when the cells were pretreated with thapsigargin, ryanodine, or cyclopiazonic acid was not due to a "ceiling" effect on [Ca²⁺]_i, because KCl (80 mmol/L) could elicit a further increase in [Ca²⁺]_i after pretreatment with the above pharmacological agents (data not shown). These data suggest that the initiation of HPV is in part due to Ca²⁺ release from intracellular Ca²⁺ stores.

Effect of Ca²⁺ Release and Hypoxia on Membrane Potential
It is well established that hypoxia depolarizes vascular smooth muscle cells.^3,8-11 To date, however, no study has investigated the role of intracellular Ca²⁺ stores in hypoxia-induced membrane depolarization. Therefore, current-clamp experiments were performed to investigate the action of intracellular Ca²⁺ store depletion on membrane potential. Figs 7 and 8 illustrate the results of such experiments. Cells had a mean resting membrane potential of -57±4 mV (n=72 cells from 10 animals.) Hypoxia, thapsigargin (5 µmol/L), cyclopiazonic acid (5 µmol/L), and ryanodine (5 µmol/L) significantly depolarized pulmonary artery cells by 26±3, 28±4, 29±4, and 25±5 mV, respectively (Figs 7 and 8, P<.01, n=22 cells from 6 animals for each agent). However, hypoxia after application of thapsigargin, cyclopiazonic acid, or ryanodine resulted in no change in membrane potential (Figs 7 and 8, n=22 cells from 6 animals for each agent). This suggests that alteration of the Ca²⁺ load of the sarcoplasmic reticulum dramatically altered the electrical response observed during a hypoxic challenge.

View larger version (15K): [in this window] [in a new window]   Figure 7. Thapsigargin and ryanodine inhibit hypoxia-induced changes in membrane potential of pulmonary artery cells. A, Control hypoxic change in membrane potential. Note that hypoxia-induced depolarization is sustained and reversible. B, Thapsigargin (5 µmol/L) causes depolarization, and subsequent hypoxic challenge in presence of drug produces no potentiation. Similar results were obtained in 22 cells from 6 animals. C, Ryanodine (5 µmol/L) causes depolarization, and a subsequent hypoxic challenge in presence of drug produces no potentiation. Similar results were obtained in 22 cells from 6 animals. Upper time bar applies to A and B; lower time bar to C.

View larger version (17K): [in this window] [in a new window]   Figure 8. Summary of the effect of hypoxia, thapsigargin, cyclopiazonic acid, and ryanodine on pulmonary artery membrane potential. Note, no significant change in membrane potential occurred when cells were challenged by hypoxia after treatment with thapsigargin, cyclopiazonic acid, or ryanodine. Data are mean±SEM. *Significantly different from resting membrane potential, P<.01. Abbreviations as in Fig 3.

On the basis of our hypothesis that hypoxia initiates membrane depolarization by causing Ca²⁺ release from the sarcoplasmic reticulum, one would think that if a Kv channel were inhibited directly, hypoxia should still release Ca²⁺ but have no effect on membrane potential. Fig 9 illustrates cells in which [Ca²⁺]_i and membrane potential were simultaneously recorded. Application of a polyclonal antibody to the carboxyl terminus of Kv1.5 (1:200, predissolved in pipette solution) caused a change in [Ca²⁺]_i that preceded membrane depolarization. The t_1/2 for the change in [Ca²⁺]_i was 5±2 seconds, whereas the t_1/2 for the change in membrane potential was 9±3 seconds (n=16 cells from 4 animals). In this experiment, anti-Kv1.5 increased [Ca²⁺]_i approximately twofold and depolarized the membrane by 22 mV. Hypoxic challenge resulted in no change in resting membrane potential; however, a marked increase in [Ca²⁺]_i was observed (n=16 cells from 4 animals). It is evident that the change in [Ca²⁺]_i is reversed by reoxygenation but the change in membrane potential is not, because the antibody is placed inside the cell. The inset of Fig 9 shows a representative experiment in which the intracellular application of anti-Kv1.5 antibody inhibits Kv current. The inhibition of Kv current occurred in a time-dependent manner after the whole-cell configuration of the patch-clamp technique was acquired (Fig 9 inset). In this experiment, charybdotoxin (300 nmol/L) was present in the extracellular solution to inhibit Ca²⁺-activated K⁺ current. Boiled anti-Kv1.5 was without effect on the Kv current (data not shown). This suggests that hypoxia can still release [Ca²⁺]_i from intracellular stores but cannot potentiate the change in membrane potential.

View larger version (15K): [in this window] [in a new window]   Figure 9. An antibody to Kv1.5 inhibits hypoxia-induced changes in membrane potential but not [Ca2+]i. When measured in same cell, anti-Kv1.5 (1:200) resulted in approximately twofold increase in [Ca2+]i before membrane depolarization. Subsequent hypoxic challenge caused no potentiation of membrane potential effect, but more importantly, markedly increased [Ca2+]i. Similar results were obtained in 16 cells from 4 animals. Inset, Inhibition of Kv current by anti-Kv1.5. After whole-cell configuration was acquired, antibody perfused the cell and inhibited Kv current. Similar results were obtained in 7 cells from 3 animals.

	Discussion

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The present results and the findings of several investigators suggest that hypoxic mobilization of Ca²⁺ from intracellular stores^10,12 may represent an important early event in the initiation of HPV. We have extended these findings; our data suggest that hypoxic mobilization of [Ca²⁺]_i is from IP₃-sensitive and CICR-sensitive Ca²⁺ stores. These conclusions are based on our pharmacological data with thapsigargin, ryanodine, and cyclopiazonic acid, known agents that alter intracellular Ca²⁺ homeostasis in the pulmonary and systemic vasculature.^24-26 This release of intracellular Ca²⁺ would then inhibit K⁺ channels, leading to membrane depolarization^10,13-15 and thus promoting Ca²⁺ entry via voltage-dependent Ca²⁺ channels,^4,27-30 resulting in the initiation and maintenance of HPV. Our results are consistent with previous reports demonstrating (1) that depletion of intracellular Ca²⁺ stores with procaine attenuates HPV,³¹ (2) that hypoxic inhibition of pulmonary artery Kv current is mimicked by acute exposure of cells to caffeine,¹⁰ (3) that the hypoxic inhibition of Kv current is eliminated in cells in which intracellular Ca²⁺ stores have been depleted by caffeine or inclusion of BAPTA in the patch pipette,¹⁰ and (4) that the sustained component of the hypoxia-induced change in [Ca²⁺]_i is due to Ca²⁺ influx from the extracellular space.³⁰

The vasoconstriction of pulmonary arteries subjected to hypoxic challenge is unique to the pulmonary vasculature, because most other systemic arteries exhibit vasodilation in response to a hypoxia challenge.³² The ability of hypoxia to increase [Ca²⁺]_i via intracellular Ca²⁺ release and extracellular Ca²⁺ influx, inhibit Kv channels, and cause membrane depolarization seems to be a general biophysical characteristic of Kv channels. For example, Gelband et al,¹⁴ Ishikawa et al,¹⁵ and Gelband and Hume¹⁶ showed that agonists that increase [Ca²⁺]_i, such as angiotensin II and histamine, inhibit Kv in a number of different vascular and visceral smooth muscle cells, including pulmonary artery. Similarly, Lopatin and Nichols³³ and Komwatana et al³⁴ demonstrated that divalent cations inhibit Kv1.2 channels expressed in Xenopus oocytes and human parathyroid cells, respectively. Therefore, it seems that the pulmonary artery has evolved a specialized way of transducing the hypoxic signal.

Alternatives have been suggested for the hypoxia-induced inhibition of Kv current besides that involving intracellular Ca²⁺ release. Hypoxia may prevent the formation of oxygen radicals and hydroperoxides, which are produced during nominal oxidative metabolism.³⁵ Oxidants such as diamide and tert-butylhydroperoxide have been shown to attenuate HPV. This vasodilatory effect can also be produced by enzyme-substrate pairs (ie, xanthine/xanthine oxidase and glucose/glucose oxidase) that generate oxygen radicals or hydroperoxides.^36,37 The proposition that oxygen radicals and hydroperoxides are mediators of vascular tone is supported further by findings that superoxide dismutase and catalase (enzymes responsible for the degradation of oxygen radicals) inhibited xanthine/xanthine oxidase–induced reduction of HPV.³⁵ The mechanism(s) that underlie the redox model of HPV have yet to be identified but may involve a direct or indirect effect on Kv or Ca²⁺-activated K⁺ channel gating (ie, channel open probability) in pulmonary arterial cells.^17,19,20

Hypoxia may interfere with the function of the sarcoplasmic reticulum and increase [Ca²⁺]_i by eliminating Ca²⁺ sparks that serve to hyperpolarize and relax arterial smooth muscle.³⁸ If the fine balance between the actions of Ca²⁺ on Ca²⁺-activated K⁺ channels and Kv channels is disrupted, then a depolarization may take place. The fine balance in Ca²⁺-activated and Kv channel activity is primarily due to the magnitude of the change in [Ca²⁺]_i and the resting membrane potential of the cell. On the basis of our [Ca²⁺]_i measurements and the resting membrane potential of the cell, a small increase in Ca²⁺-activated K⁺ channel activity should be observed. However, even though the two single channels vary greatly in conductance, depolarization occurs. Similarly, 4-AP mimics the effect of hypoxia membrane potential [Ca²⁺]_i, and charybdotoxin does not (Reference 10¹⁰ and C.H. Gelband, unpublished observations). On the basis of these observations, it seems that Kv channels play a greater role in the regulation of hypoxic pulmonary vasoconstriction. Hypoxia may also alter [Ca²⁺]_i by disrupting the hypothesized superficial buffer barrier of vascular smooth muscle.³⁹ Finally, it is possible that the membrane depolarization observed may be due to the activation of Ca²⁺-activated Cl^- channels by intracellular Ca²⁺ release. However, the effects of hypoxia on membrane potential were not altered in the presence of either niflumic acid or 9-anthracene carboxylic acid, two Ca²⁺-activated Cl^- channels blockers (C.H. Gelband, unpublished observations).

Certainly, the idea that hypoxia may inhibit Kv current by a combination of a direct physical block (Ca²⁺) and by the metabolic state of the cell (redox status) is plausible. As previously stated, in isolated canine renal arterial cells, various agonists cause intracellular Ca²⁺ inhibition of Kv current¹⁶; however, hypoxia did not inhibit whole-cell K⁺ currents in these cells.⁸ These data may hold an answer to the puzzling question concerning the mechanism by which a reduction in PO₂ triggers Ca²⁺ release in pulmonary artery. Perhaps for hypoxia to exert its effects, not only does there have to be Ca²⁺ release from intracellular stores, but the redox status of the cell also has to respond to changes in Po₂ as well. Because redox status has been shown to influence pulmonary artery K⁺ channels^17,19,20 and the activity of sarcoplasmic reticulum Ca²⁺ release channels,⁴⁰ one may hypothesize that the pulmonary vasculature may have adapted itself for such a function. Further biochemical and electrophysiological experiments detailing the redox status of pulmonary artery Kv channels and Ca²⁺ release channels need to be performed to further elucidate this phenomenon.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine CICR = Ca2+-induced Ca2+ release HPV = hypoxic pulmonary vasoconstriction IP3 = inositol trisphosphate KHB = Krebs-Henseleit buffer Kv = voltage-dependent K+ current PO2 = alveolar oxygen tension

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health (HL-52189), the American Heart Association, Florida Affiliate, and the Council for Tobacco Research.

Received June 2, 1997; revision received August 8, 1997; accepted August 13, 1997.

	References

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