Effects of hypoxia in porcine pulmonary arterial myocytes: roles of KV channel and endothelin-1

James S. K. Sham, Benjamin R. Crenshaw Jr., Li-Hua Deng, Larissa A. Shimoda, and J. T. Sylvester

Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21224


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of acute hypoxia on intracellular Ca2+ concentration ([Ca2+]i) and cell length were recorded simultaneously in proximal and distal pulmonary (PASMCs) and femoral (FASMCs) arterial smooth muscle cells. Reducing PO2 from normoxia to severe hypoxia (PO2 < 10 mmHg) caused small but significant decreases in length and a reversible increase in [Ca2+]i in distal PASMCs and a small decrease in length in proximal PASMCs but had no effect in FASMCs, even though all three cell types contracted significantly to vasoactive agonists. Inhibition of voltage-dependent K+ (KV) channel with 4-aminopyridine produced a greater increase in [Ca2+]i in distal than in proximal PASMCs. In distal PASMCs, severe hypoxia caused a slight inhibition of KV currents; however, it elicited further contraction in the presence of 4-aminopyridine. Endothelin-1 (10-10 M), which itself did not alter cell length or [Ca2+]i, significantly potentiated the hypoxic contraction. These results suggest that hypoxia only has small direct effects on porcine PASMCs. These effects cannot be fully explained by inhibition of KV channels and were greatly enhanced via synergistic interactions with the endothelium-derived factor endothelin-1.

vasoconstriction; calcium; voltage-dependent potassium channel; pulmonary arterial smooth muscle cell


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE REDUCTION in alveolar PO2 causes reversible hypoxic pulmonary vasoconstriction (HPV) in intact animals and isolated perfused lungs (13). Despite extensive research, the exact mechanism of this response remains unclear. Previous work indicated that HPV required extracellular Ca2+ and L-type Ca2+ channels (36, 37, 53) but was not mediated by vasoactive substances such as angiotensin, histamine, serotonin [5-hydroxytryptamine (5-HT)], or leukotriene (34, 37, 48). HPV has been demonstrated in isolated pulmonary arteries (4, 6, 27, 30, 32, 33, 45, 46), indicating that HPV may be mediated through a direct effect on these vessels. Hypoxic responses may depend on size and location, with small distal "resistant" arteries thought to be more responsive than large proximal "conduit" vessels (3, 32, 33).

In recent years, new information has been obtained about the direct effects of hypoxia on pulmonary arterial smooth muscle. Harder et al. (19) were the first to report that hypoxia caused membrane depolarization of smooth muscle in small pulmonary arteries. This result was confirmed subsequently in pulmonary arterial smooth muscle cells (PASMCs) (15, 43, 44, 64). Exposure of PASMCs to hypoxia also increased intracellular Ca2+ concentration ([Ca2+]i) and myosin light chain phosphorylation and caused contraction (10, 11, 15, 33, 38, 43, 47, 56, 66). Moreover, patch-clamp studies demonstrated that hypoxia inhibited delayed rectifier voltage-dependent K+ (KV) currents in PASMCs (2, 44, 64). These hypoxic effects might be specific for PASMCs because they were not observed in myocytes of systemic arteries (33, 56, 64). Because KV channels are important regulators of resting membrane potential in PASMCs (2, 43, 49, 64), it was proposed that the initiation of HPV involves inhibition of KV channels, which leads to membrane depolarization, activation of L-type Ca2+ channels, Ca2+ influx, increase in [Ca2+]i, and cell contraction (2, 59, 64). Other studies, however, showed that hypoxia released Ca2+ from intracellular stores independent of Ca2+ influx (15, 47), membrane depolarization (17, 43, 45), or inhibition of KV channels (15). Thus the role of KV channels in initiating HPV requires further elucidation.

Besides its direct effects on PASMCs, hypoxia may act indirectly through the release of factors derived from the endothelium; however, the role of the endothelium in HPV remains controversial. Some reports (22, 29, 45, 46) showed that removal of the endothelium completely or partially abolished HPV, whereas others (6, 40, 65) found little or no effect of endothelium removal. Recent evidence suggests that endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor, may mediate HPV. Plasma levels of ET-1 were elevated during acute hypoxia (25), and infusion of ET-1 antagonists prevented acute HPV in the rat, dog, pig, and fetal lamb (7, 23, 42, 57, 61) and blocked or reversed pulmonary hypertension associated with prolonged exposure to hypoxia (8). Moreover, hypoxic contractions of isolated pulmonary arteries from rats and pigs were endothelium dependent (30, 31, 46, 58) and in the pig could be blocked by ET-1 antagonists (31, 50).

In this study, we examined the direct effects of hypoxia on [Ca2+]i and length in PASMCs from adult pigs to determine whether hypoxia could directly activate myocytes from vessels exhibiting predominantly endothelium-dependent hypoxic response (14, 22, 29). Specific experiments were designed to characterize the effects of hypoxia on proximal and distal PASMCs and femoral arterial smooth muscle cells (FASMCs) and to examine the involvement of KV channels in and the modulatory effect of ET-1 on the hypoxic responses. Our results demonstrate that hypoxia causes small contractions and increases in [Ca2+]i in distal PASMCs under severe hypoxia but has little or no effect in proximal PASMCs or FASMCs. Hypoxic responses of distal PASMCs could not be explained completely by the inhibition of 4-aminopyridine (4-AP)-sensitive K+ channels but was enhanced significantly by a low concentration of ET-1, which by itself did not alter cell length or [Ca2+]i.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Cell Preparation

Porcine PASMCs were isolated as described by Clapp and Gurney (9). In brief, male and female pigs (~35 kg) were anesthetized with ketamine (20 mg/kg im) followed by pentobarbital sodium (12.5 mg/kg iv) and exsanguinated through the femoral arteries. Proximal (5- to 10-mm-ID) and small (0.2- to 0.7-mm-ID) intralobar pulmonary arteries were excised and placed in modified Krebs-Ringer bicarbonate solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2 CaCl2, 25 NaHCO3, and 11.1 glucose, 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin bubbled with 21% O2-5% CO2 to achieve a pH of 7.4. The adventitia was carefully removed with forceps under a dissecting microscope. The endothelium was removed by opening the vessel longitudinally and rubbing the intimal surface with a cotton swab. The cleaned vessels were washed and cut transversely into small pieces, transferred to a vial with 10 ml of dissociation medium containing (in mM) 0.16 CaCl2, 110 NaCl, 5 KCl, 2 MgCl2, 10 HEPES, 10 NaHCO4, 0.5 KH2PO4, 0.5 NaH2PO4, 10 glucose, 0.04 phenol red, 0.49 EGTA, and 10 taurine, 3.2 U/ml of papain (type IV), 0.02% bovine serum albumin (BSA; type V), 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin, pH titrated to 7.4 with NaOH, and stored overnight at 4°C to allow complete penetration by the enzyme. The next morning, papain was activated by adding 0.1 mM dithiothreitol and raising the temperature to 37°C. After 30 min of digestion, the tissue was transferred to fresh dissociation medium. Gentle trituration with a wide-bore pipette yielded long relaxed single smooth muscle cells, which were stored at 5-7°C for use within 8 h.

FASMCs were isolated with the same method with a minor modification. After overnight incubation and 30 min of papain digestion, the tissues were transferred to dissociation medium containing collagenase (400 U/ml, type I; Sigma) for an additional 10 min of digestion. The myocytes were dispersed in fresh medium by trituration.

Measurement of [Ca2+]i and Cell Shortening

Freshly isolated myocytes were placed in a laminar flow cell chamber on the stage of a Nikon Diaphot inverted microscope and incubated with 2.5 µM indo 1-AM, a membrane-permeant Ca2+ fluorescent dye, for 30 min at room temperature (~22°C) under an atmosphere of 21% CO2-5% CO2. The cells were then washed by superfusion with Krebs bicarbonate solution for 30 min at 35°C to remove extracellular indo 1 and allow complete deesterification of cytosolic indo 1-AM.

[Ca2+]i of isolated single myocytes was measured by exciting indo 1 at 365 nm via a ×40 fluorescence oil-immersion objective (Fluor ×40, Nikon, Japan). Emission fluorescence at 405 (F405) and 495 (F495) nm were detected by two photomultiplier tubes, and the signals were amplified with a dual-emission fluorometer (Biomedical Instrumentation Group, University of Pennsylvania, Philadelphia, PA). Photobleaching of indo 1 was minimized by using a neutral density filter (ND-3, Omega Optics, Brattleboro, VT) and an electronic shutter (Vincent Associates, Rochester, NY). The shutter was opened for 35 ms every 2 s, and the fluorescence signals during the open period were integrated with a sample-and-hold circuit. Hence, in a recording period of 1 h, the cells were actually exposed to 0.1% of excitation light for a total of 63 s. The protocols were executed, and data were collected online with a Labmaster analog-to-digital interface (Axon Instruments, Foster City, CA) and the pClamp software package (Axon Instruments). [Ca2+]i was calculated with the equation of Grynkiewicz et al. (18): [Ca2+]i = KdB(R - Rmin)/(Rmax - R), where R is the ratio of (F405 - F405,bg) to (F495 - F495,bg), where F405,bg and F495,bg are the background fluorescence values at 405 and 495 nm, respectively. Rmin and Rmax are the fluorescence ratios measured in situ in myocytes permeabilized with the Ca2+ ionophore 4-bromo A-23187 (10 µM) with external solutions containing 10 mM EGTA and 10 mM Ca2+, respectively. B = F495,EGTA/F495,Ca, where F495,EGTA and F495,Ca are the fluorescence values of EGTA and Ca2+, respectively, at 495 nm, and the dissociation constant (Kd) of indo 1 is 288 nM (60). F405,bg and F495,bg were measured by quenching indo 1 with MnCl2.

Cell length was measured alone in the studies of vasoconstrictor agents or simultaneously with [Ca2+]i in the hypoxia experiments. The myocytes were monitored continuously with red light with a charge-coupled device camera TV system (Dage, Michigan City, IN). Images were captured and stored every 2 min with a video frame grabber (Data Translation, Marlborough, MA). Cell length was measured off-line with image analysis software (Mocha, Jandel Scientific, San Rafael, CA).

Measurement of KV Currents

The myocytes were bathed in modified Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. They were whole cell voltage clamped with patch pipettes (tip resistance of 3-5 MOmega ) containing an internal solution of the following composition (in mM): 74 potassium gluconate, 40 KCl, 6 NaCl, 5 MgATP, 2 disodium phosphocreatine, 10 HEPES, and 10 EGTA, pH 7.2. High concentrations of EGTA and ATP were included in the pipette solution to inhibit Ca2+-activated K+ (KCa) and ATP-dependent K+ currents. These conditions mimic those used previously by others (2, 64) showing a large reduction in the KV channel during hypoxia. Membrane currents were recorded with an Axopatch-1D amplifier (Axon Instruments) and filtered at 5 kHz. Junction potential, cell capacitance, and access resistance (80% compensation) were compensated for electronically. Voltage-clamp protocols were applied, digitized, and analyzed with the pClamp software (Axon Instruments).

Control of PO2

PO2 of the superfusate was controlled by an open laminar flow cell chamber (51), which prevented contact of the superfusate with atmospheric air by means of a laminar counterflow argon column. Krebs bicarbonate solutions were bubbled with an argon-balanced gas mixture containing 16% O2 plus 5% CO2 (normoxia), 5% O2 plus 5% CO2 (moderate hypoxia), 0% O2 plus 5% CO2 (severe hypoxia), or 0% O2 plus 5% CO2 plus 0.3 mM sodium dithionite (anoxia). The superfusate was delivered to the cell chamber at a flow rate of 2.5 ml/min by a peristaltic pump via stainless steel tubing. With this system, PO2 values measured inside the chamber with a microelectrode (Microelectrode, Bedford, NH) were 117 ± 1, 36 ± 2, 8 ± 2, and 0 mmHg during normoxia, moderate hypoxia, severe hypoxia, and anoxia, respectively. The temperature of the chamber was maintained at 35°C with a heat exchanger coupled to a circulator.

Experimental Protocols

Protocol 1: Contractile responses to vasoconstricting agents. The myocytes were equilibrated under control conditions for 30 min and then exposed to continuous superfusion of KCl (10 mM), phenylephrine (PE; 10 µM), 5-HT (10 µM), or A-23187 (10 µM) for 15 min. Each myocyte was exposed to only one vasoconstricting agent. Cell length was measured before and during 15 min of vasoconstrictor exposure. This time was always sufficient for achievement of maximal responses.

Protocol 2: Hypoxic responses in PASMCs and FASMCs. In this and the following protocols, [Ca2+]i and cell length were measured simultaneously. After equilibration under normoxic conditions for 30-35 min for deesterification and 10 min of baseline [Ca2+]i and cell length recording, proximal and distal PASMCs and FASMCs were exposed to severe hypoxia for 15 min followed by 30 min of normoxia. The cells were then exposed sequentially to 4-AP (10 mM), a blocker of KV channels, and KCl (100 mM) for 5 min, allowing 30 min for recovery between exposures. To determine the O2 dependence of the hypoxic responses, the same protocol was applied to additional distal PASMCs exposed to moderate hypoxia or anoxia instead of severe hypoxia. Cells were discarded if significant changes occurred during the baseline recording period.

Protocol 3: Hypoxic responses and KV channels. After equilibration under normoxic conditions, distal PASMCs were exposed to 10 mM 4-AP for 10 min followed by 15 min of severe hypoxia in the continued presence of 4-AP. After 30 min of recovery, KCl (100 mM) was given to verify the responsiveness of the cell. Control experiments were performed in a separate group of myocytes that were exposed to 4-AP without subsequent exposure to hypoxia. In a set of separate experiments, the effect of hypoxia on KV currents was examined. The myocytes were held at holding potential of -70 mV and depolarized to test potentials ranging from -60 to +30 mV in +10-mV step increments for 1 s once every 10 s. Current-voltage relationships generated before and during severe hypoxia were constructed for comparison.

Protocol 4: Hypoxia and Ca2+sensitivity of cell shortening. After equilibration under normoxic conditions, distal PASMCs were exposed to either continued normoxia or severe hypoxia. Seven minutes after the onset of exposure, the Ca2+ ionophore 4-bromo A-23187 was administered at 5-min intervals in progressively increasing concentrations (0.3, 1, and 3 µM).

Protocol 5: ET-1 and hypoxic responses. After equilibration under normoxic conditions for 45 min, distal PASMCs were first exposed to ET-1 (10-10 M) for 10 min followed by 15 min of severe hypoxia in the continued presence of ET-1. After 30 min of recovery, KCl (100 mM) was given to verify the myocyte responsiveness.

Chemicals and Drugs

Papain, collagenase, BSA, and other chemicals were purchased from Sigma (St. Louis, MO). Indo 1-AM was purchased from Molecular Probes; 4-bromo A-23187 was purchased from Calbiochem (La Jolla, CA). Stock solutions of indo 1-AM (1 mM) and 4-bromo A-23187 (10 mM) were made up fresh in dimethyl sulfoxide and diluted 1:400 and 1:1,000, respectively, in Krebs-Ringer bicarbonate solution. 4-AP was prepared fresh, with pH adjusted to 7.4 before use. High KCl solution was prepared by equimolar replacement of NaCl.

Data Analysis

Data are expressed as means ± SE. Changes in [Ca2+]i during hypoxia and anoxia or drug challenges were determined from the averaged [Ca2+]i transients measured in a group of cells. For statistical analysis, values of individual Ca2+ transients were averaged over a period of 1 min. One- or two-factor analysis of variance (ANOVA) with repeated measures was used to test for the significance of changes in the variables occurring over time. When significant variance ratios were obtained by one-factor ANOVA, Dunnett's test was used to identify the means that were significantly different from baseline. Student's t-test for paired analysis was also used for some comparisons. Values were considered significantly different when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Contractile Responses to Vasoconstricting Agents

Averaged resting cell length was 77.8 ± 2.0 µm in distal PASMCs (n = 107), 55.8 ± 1.2 µm in proximal PASMCs (n = 114), and 80.4 ± 2.4 µm in FASMCs (n = 81). Proximal PASMCs were significantly shorter than the other myocytes. Maximal concentrations of KCl (100 mM), PE (10 µM), 5-HT (10 µM), and A-23187 (10 µM) caused significant contractions in all three cell types (Table 1). Within the cell types, there were no differences among contractile responses induced by the four agonists. Among the cell types, contractile responses expressed as absolute shortening (Delta L) also were not different; however, when expressed as the ratio of Delta L to initial resting length (Delta L/L0), contractions induced by PE and A-23187 were greater in proximal PASMCs than in FASMCs and A-23187-induced contractions were greater in proximal PASMCs than in distal PASMCs.

                              
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Table 1.   Effects of KCl, PE, 5-HT, and 4-bromo A-23187 on cell length in FASMCs and proximal and distal PASMCs

Hypoxic Responses in PASMCs and FASMCs

Figure 1 shows the time course of PO2, [Ca2+]i, and normalized cell length (L/L0) in FASMCs (n = 7) and proximal (n = 10) and distal (n = 12) PASMCs exposed to severe hypoxia for 15 min. During the normoxic control period, PO2 was stable at levels > 100 mmHg, and [Ca2+]i averaged 73.1 ± 9.2, 75.3 ± 10.9, and 66.5 ± 8.2 nM in FASMCs, proximal PASMCs, and distal PASMCs, respectively. During hypoxia, PO2 fell rapidly in all myocytes, achieving levels < 10 mmHg within 5 min. In FASMCs, [Ca2+]i (75.6 ± 13.0 nM) and cell length (Delta L/L0 = -1 ± 1%) did not change during hypoxia. In proximal PASMCs, [Ca2+]i (84.3 ± 16.9 nM) did not change, but cell length (Delta L/L0 = -3 ± 1%) decreased slightly (P < 0.05). In distal PASMCs, hypoxia caused a small but significant increase in [Ca2+]i that began 2-3 min after the onset of hypoxia, was sustained during the 15-min challenge, and reversed on return to normoxia (P < 0.02). The maximum mean [Ca2+]i achieved was 92.1 ± 15.2 nM at 14 min of exposure. This [Ca2+]i response was accompanied by a small but significant decrease in cell length (Delta L/L0 = -4 ± 1%; P < 0.005) that did not reverse on reoxygenation. In contrast to the small effects of severe hypoxia, application of KCl at the end of the experiments caused a marked increase in [Ca2+]i and decreases in cell length in all three cell types (Fig. 2). Maximum [Ca2+]i values achieved during KCl exposure were 243 ± 12, 196 ± 57, and 191 ± 40 nM in FASMCs, proximal PASMCs, and distal PASMCs, respectively. These changes in [Ca2+]i were accompanied by Delta L/L0 values of -17 ± 4, -12 ± 5, and -11 ± 4%, respectively.


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Fig. 1.   Effects of severe hypoxia on intracellular Ca2+ concentration ([Ca2+]i) and normalized cell length [ratio of length to initial resting length (L/L0)] in femoral arterial smooth muscle cells (FASMCs; A) and proximal (B) and distal (C) pulmonary arterial smooth muscle cells (PASMCs). PO2 and [Ca2+]i transients are averages ± SE (dotted lines) of traces from FASMCs (n = 7), proximal PASMCs (n = 10), and proximal PASMCs (n = 12). Values are means ± SE for L/L0. P values comparing before and during hypoxia were obtained by repeated-measures 1-way ANOVA. NS, no significant difference before and during hypoxia.



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Fig. 2.   Effects of KCl on [Ca2+]i and L/L0 in FASMCs (A) and proximal (B) and distal (C) PASMCs after exposure to hypoxia. [Ca2+]i transients are averages ± SE (dotted lines) of traces from the same groups of myocytes used in Fig. 1. Values are means ± SE for L/L0.

O2 Dependence of Hypoxic Responses

Two other groups of distal PASMCs were exposed to moderate hypoxia (n = 12) or anoxia (n = 13). As shown in Fig. 3A, moderate hypoxia had no effect on [Ca2+]i, which averaged 59.1 ± 8.4 and 69.0 ± 13.9 nM before and during hypoxia, respectively; however, moderate hypoxia did cause a slight decrease in cell length (Delta L/L0 = -2.2 ± 0.8%; P < 0.02). In comparison, anoxia increased [Ca2+]i from 53.0 ± 6.7 to 108.7 ± 29.9 nM (P < 0.01) and decreased L/L0 by 8.8 ± 3.0% (P < 0.02). These changes in [Ca2+]i and L/L0 were greater than those induced by severe hypoxia and were equivalent to 44 and 50%, respectively, of the response elicited by 100 mM KCl in a separate group of distal PASMCs (Fig. 3). On reoxygenation after anoxia, [Ca2+]i was only partially reversed, and the cells continued to shorten. Figure 3B shows the relationship between the mean change in (Delta ) [Ca2+]i and Delta L/L0 to mean PO2 measured in distal PASMCs exposed to moderate hypoxia, severe hypoxia, and anoxia. As PO2 fell from >100 to 0 mmHg, Delta [Ca2+]i increased and Delta L/L0 decreased in a curvilinear manner, with the greatest changes in both variables occurring at PO2 < 10 mmHg.


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Fig. 3.   Effects of different PO2 values on [Ca2+]i and L/L0 in distal PASMCs. A: time courses of PO2, [Ca2+]i transient, and L/L0 obtained from cells exposed to moderate hypoxia (n = 12), severe hypoxia (n = 12), and anoxia (n = 13). A separate group of cells (n = 11) were exposed to KCl (normoxia) for comparison. PO2 and [Ca2+]i transients are averages ± SE (dotted lines). Values are means ± SE for L/L0. B: change in (Delta ) [Ca2+]i and L/L0 are plotted against PO2 to illustrate O2 dependence of hypoxic responses. Values are means ± SE.

Hypoxia and Ca2+Sensitivity of Cell Shortening

To examine the effect of hypoxia on the Ca2+sensitivity of cell shortening, distal PASMCs were exposed at 5-min intervals to progressively increasing concentrations (0.3, 1, and 3 µM) of the Ca2+ ionophore 4-bromo A-23187 during normoxia or severe hypoxia. Under both conditions, 4-bromo A-23187 caused significant concentration-dependent increases in [Ca2+]i and decreases in L/L0. The relationship between mean Delta [Ca2+]i and mean L/L0 obtained in this manner during normoxia did not differ from that obtained during hypoxia (Fig. 4).


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Fig. 4.   Effects of hypoxia on Ca2+ sensitivity of cell shortening in distal PASMCs. Myocytes were exposed to increasing concentrations of 4-bromo A-23187 during normoxia and hypoxia. n, No. of cells. Mean (±SE) L/L0 is plotted against mean (±SE) Delta [Ca2+]i at each concentration. There was no significant difference between the 2 curves.

Hypoxic Responses and KV Channels

To examine whether inhibition of KV channel activates PASMCs, 4-AP was applied to proximal and distal PASMCs. Inhibition of KV channels with 10 mM 4-AP increased [Ca2+]i in 24 of 33 distal PASMCs and 6 of 14 proximal PASMCs. The Delta [Ca2+]i was significantly greater in distal than in proximal PASMCs, averaging 34.9 ± 8.9 and 14.2 ± 3.1 nM, respectively (P < 0.05), suggesting a greater role for KV channels in controlling the resting membrane potential in distal PASMCs. Among individual distal PASMCs, there was no significant correlation between Delta [Ca2+]i induced by 4-AP and Delta [Ca2+]i induced by severe hypoxia or anoxia (r = 0.103 and 0.496, respectively).

To examine whether hypoxia inhibits KV channels, KV currents were recorded with whole cell patch-clamp techniques. Cell capacitance of distal PASMCs (n = 20) was 21 ± 2 pF. Depolarization to test potentials ranging between -30 and +30 mV activated KV currents, which were characterized by low noise and slow inactivation kinetics. Hypoxia caused a small reversible inhibition of KV currents in whole cell patch-clamped distal PASMCs (Fig. 5A). After exposure to severe hypoxia, the current-voltage relationship of the KV current was shifted downward significantly (P < 0.01 by two-factor repeated-measures ANOVA), with an average inhibition of the KV current of 22 ± 10% at -20 mV and 17 ± 7% at +20 mV.


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Fig. 5.   Effect of hypoxia on outward K+ current in distal PASMCs. A: representative traces from myocytes depolarized from -70 mV to various test potentials before (control), during (hypoxia), and after (recovery) hypoxic exposure. B: averaged voltage-dependent K+ (KV) current-voltage relationships in 6 cells under control and hypoxic conditions. The difference between the 2 current-voltage relationships is significant (P < 0.01).

To further investigate whether KV channels are required for hypoxic responses, distal PASMCs were first exposed to 4-AP for 10 min to inhibit KV channels followed by 15 min of severe hypoxia in the continued presence of 4-AP (Fig. 6). 4-AP caused L/L0 to decrease 4.4 ± 1.2% (P < 0.05) and [Ca2+]i to increase from 85.2 ± 17.8 to 108.7 ± 17.0 nM. Subsequent hypoxia caused a further significant reduction in cell length (10.0 ± 2.4%; P < 0.05) and raised [Ca2+]i to 140.5 ± 25.6 nM. Compared with myocytes that were exposed to 4-AP only, the reduction in L/L0 was significantly greater after exposure to severe hypoxia (P < 0.05 by two-factor repeated-measures split-plot ANOVA). Although the [Ca2+]i responses of the two groups were not significantly different, [Ca2+]i in the hypoxic group was persistently higher during the hypoxic period. Moreover, 5 of 13 cells exhibited both a clear increase in [Ca2+]i and a marked cell shortening on exposure to hypoxia in the presence of 4-AP. Therefore, inhibition of KV channels did not abolish the hypoxic responses in distal PASMCs.


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Fig. 6.   Effects of inhibition of KV channels with 4-aminopyridine (4-AP) on the hypoxic responses in distal PASMCs. Shown are time courses of averaged PO2 and [Ca2+]i transients and mean (±SE) L/L0 of distal PASMCs first exposed to 10 mM 4-AP for 10 min followed by either 15 min of exposure to severe hypoxia in the continued presence of 4-AP (n = 11 cells) or continued exposure to 4-AP but without hypoxic challenge (n = 12 cells). L/L0 of the 2 groups was significantly different between the onset (12 min) and end (28 min) of hypoxic exposure (P = 0.035).

ET-1 Priming and Hypoxic Responses

To examine the interaction between ET-1 and hypoxia, distal PASMCs were exposed to a low concentration (10-10 M) of ET-1 for 10 min followed by 15 min of severe hypoxia in the presence of ET-1 (Fig. 7). At this concentration, ET-1 had no effect on [Ca2+]i or L/L0 during normoxia. [Ca2+]i was 97 ± 13 and 98 ± 17 nM and cell length was 65 ± 4 and 63 ± 4 µm before and 10 min after exposure, respectively, to ET-1. However, the contraction induced by severe hypoxia after ET-1 priming was markedly potentiated (Delta L/L0 = -24.0 ± 9.6%; P < 0.05; n = 6 cells); it was significantly greater than that in PASMCs without priming (Delta L/L0 = -4 ± 1%; P < 0.05) and comparable to the contractions induced by 100 mM KCl (Delta L/L0 = -17 ± 6%; Fig. 3B). In contrast, the increase in [Ca2+]i induced by hypoxia in cells primed with ET-1 was not significantly different from that in myocytes without ET-1 priming. Except for one myocyte primed with ET-1, a large Ca2+ transient was elicited at the onset of hypoxia, leading to a spike in mean [Ca2+]i at the beginning of hypoxic exposure (Fig. 7); however, this increase did not achieve significance, and spikes were not seen in other cells.


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Fig. 7.   Effects of endothelin (ET)-1 priming on hypoxic responses in distal PASMCs. Shown are time courses of averaged Delta [Ca2+]i and mean (±SE) L/L0 obtained from distal PASMCs (n = 7) that were first exposed to 10-10 M ET-1 for 10 min followed by 15 min of exposure to severe hypoxia. [Ca2+]i transients and L/L0 of control myocytes (same as in Fig. 2) obtained during hypoxia without ET-1 priming are superimposed for comparison.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was designed to examine the direct effects of hypoxia on porcine PASMCs. We found that 1) hypoxia caused a small contraction in proximal and distal PASMCs but not in FASMCs and small increases in [Ca2+]i in distal PASMCs but not in proximal PASMCs or FASMCs; 2) the magnitude of hypoxic responses in distal PASMCs was greatest at PO2 < 10 mmHg; 3) severe hypoxia caused a slight inhibition of KV channels in distal PASMCs; 4) inhibition of KV channels in distal PASMCs with 4-AP did not always abolish the hypoxic response; and 5) hypoxic contraction, but not [Ca2+]i responses, in distal PASMCs were significantly enhanced in the presence of 10-10 M ET-1, which by itself did not alter cell length or [Ca2+]i. These results suggest that hypoxia can directly stimulate contraction and increase [Ca2+]i in PASMCs from vessels that exhibit a predominantly endothelium-dependent hypoxic response. However, these responses are small and cannot be fully explained by inhibition of KV channels of PASMCs, and their full expression may rely critically on factors derived from the endothelium.

Our contraction experiments demonstrated that porcine FASMCs and proximal and distal PASMCs shortened significantly on exposure to KCl, PE, 5-HT, or A-23187, indicating that receptor-linked transduction pathways and pathways acting through membrane depolarization and Ca2+ influx were intact in these cells. The magnitude of contraction (12-38%) was comparable to that previously observed in pulmonary and systemic arterial myocytes under similar conditions (33, 66).

Hypoxia caused contraction in distal and proximal PASMCs but not in FASMCs and elevated [Ca2+]i in distal PASMCs but not in proximal PASMCs or FASMCs. The lack of hypoxic contraction and [Ca2+]i response in FASMCs is consistent with previous observations in systemic arterial myocytes (32, 33, 56). These results cannot be explained by functional impairment because, as stated above, FASMCs responded significantly to vasoactive agonists acting through a variety of mechanisms. Our finding that distal PASMCs were more responsive to 4-AP and hypoxia and less responsive to A-23187 than proximal PASMCs is consistent with several reports suggesting that myocyte characteristics vary with location and size of the pulmonary artery from which they are obtained. For example, in the cat, HPV occurred in myocytes or segments from pulmonary arteries < 600 µm; however, myocytes or segments from arteries > 800 µm failed to contract and even relaxed during hypoxia (32, 33, 56). Cornfield et al. (10) showed that hypoxia increased [Ca2+]i in cultured PASMCs from distal but not from main pulmonary arteries of fetal lambs. Similarly, Archer et al. (3) found that hypoxia produced a sustained contraction in small pulmonary resistance vessels from the rat but elicited an initial contraction, which was followed by relaxation, in large conduit vessels. Differences in hypoxic responses were attributed to a differential distribution of phenotypically distinct myocytes. Proximal cells were generally larger and expressed mainly KCa channels, whereas distal cells were usually shorter with predominantly KV channels (1, 3). The differences we observed in the 4-AP responses between proximal and distal PASMCs are consistent with their findings; the discrepancy between cell length observed in our study could be related to species. However, the significance of the phenotypical difference between proximal and distal PASMCs remains unsettled because consistent hypoxic responses have also been described in proximal pulmonary arteries of intact pigs (21) and segments or cells from main pulmonary arteries of rats (47, 65) and the hypoxic responses of rat main and distal pulmonary arteries were found to be quantitatively similar (30).

Hypoxic contractions in pulmonary myocytes were accompanied by either no significant change (proximal PASMCs) or only small changes (distal PASMCs) in [Ca2+]i, suggesting that hypoxia may have altered myofilament sensitivity to Ca2+. As shown in Fig. 4, the relationships between Delta [Ca2+]i and L/L0 elicited by exposure to varying concentrations of the calcium ionophore 4-bromo A-23187 were similar during normoxia and hypoxia. If the magnitude of myocyte shortening in these experiments was determined primarily by the strength of myofilament contraction, these results suggest that hypoxia did not alter Ca2+ sensitivity.

To further characterize the hypoxic responses in distal PASMCs, we determined the effect of different PO2 values on [Ca2+]i and L/L0 (Fig. 3). Although decreasing PO2 from >100 to 0 mmHg elicited proportional decreases in L/L0 and increases in [Ca2+]i, the changes in both variables were small and occurred mainly at a PO2 < 10 mmHg. These findings differ from the larger contractions and increases in [Ca2+]i observed in PASMCs of cats, dog, fetal lambs, and rats at a PO2 of 25-50 mmHg (10, 15, 33, 38, 43, 47) and the robust responses to mild or moderate hypoxic challenges observed in intact or isolated perfused pig lungs (52, 54).

Previous studies in isolated porcine pulmonary arteries suggested that the endothelium plays an important role in the hypoxic responses of this tissue. Holden and McCall (22) reported vigorous contraction of porcine main pulmonary arteries after prolonged (4- to 6-h) preconditioning of the tissue at a PO2 of 40 mmHg, but the responses were greatly attenuated after endothelial denudation. Kovitz et al. (29) could not elicit endothelium-independent hypoxic contraction in porcine pulmonary arteries with IDs of 1-12 mm. Recently, Liu et al. (31) found that HPV occurred in porcine pulmonary arteries with IDs of 0.2-0.3 mm only if the endothelium was intact. In contrast, Ogata et al. (40) reported that contractions of small porcine pulmonary arteries (2-mm OD) to hypoxia (PO2 = 15 mmHg) were enhanced by removal of the endothelium. We cannot explain this discrepancy. Our observation that PASMCs obtained from distal porcine pulmonary arteries with IDs of 0.2-0.7 mm did not respond to moderate hypoxia and exhibited only small responses to severe hypoxia suggests that myocytes from arteries that exhibit predominantly endothelium-dependent hypoxic responses can respond directly to hypoxia, but additional factors may be required for the full expression of HPV.

It has been proposed that hypoxia causes pulmonary vasoconstriction by reducing activities of smooth muscle KV channels, resulting in membrane depolarization and Ca2+ influx via L-type Ca2+ channels (2, 59, 64). This hypothesis is based on findings in pulmonary arterial myocytes that 1) hypoxia caused membrane depolarization (3, 19, 44, 64), 2) hypoxia inhibited KV currents (2, 3, 64), 3) pharmacological inhibition of KV channels caused membrane depolarization and an increase in [Ca2+]i (3, 49, 63), and 4) hypoxic responses were blocked by Ca2+ channel antagonists and potentiated by Ca2+ channel agonists (19, 36, 37, 46, 53). Given the small hypoxic contractions and [Ca2+]i responses we observed in distal PASMCs, we performed two experiments to determine whether KV channels played a similar role in these cells.

To determine whether hypoxia inhibited KV channels, we measured voltage-current relationships in distal PASMCs using whole cell patch-clamp techniques. Because the internal solution was Ca2+ free and contained high concentrations of ATP and EGTA, outward currents resulted primarily from KV channels rather than from ATP-dependent K+ or KCa channels. As shown in Fig. 5, hypoxia caused only a slight inhibition of these currents (e.g., 17 ± 7% at +20 mV), consistent with the small magnitude of its effects on cell length and [Ca2+]i. In comparison, hypoxia caused a >50% inhibition of KV currents measured at +40 mV under similar conditions in PASMCs from rats (2, 64).

To determine whether blockade of KV channels prevents HPV, we examined the effect of 4-AP, an inhibitor of KV channels, alone and in combination with hypoxia on cell length and [Ca2+]i in distal PASMCs. Hypoxia caused a further significant reduction in L/L0 in the presence of 4-AP because a clear divergence in L/L0 in 4-AP-treated and untreated cells was observed starting at the onset of hypoxia (Fig. 6). Moreover, the additional 3% reduction in L/L0 in myocytes exposed to hypoxia is comparable to the hypoxic contraction elicited in the absence of 4-AP (Fig. 1). The small hypoxic [Ca2+]i responses of distal PASMCs did not achieve a significant difference compared with those in control myocytes. However, Delta [Ca2+]i was persistently higher in myocytes during the hypoxic challenge, and clear hypoxia-induced responses were observed in several 4-AP-treated cells. Because 10 mM 4-AP completely inhibits KV channels (2, 49), these results suggest that in distal PASMCs, hypoxic contraction can be elicited via mechanism(s) other than the inhibition of KV channels, although a contributing role of KV channels in the responses cannot be excluded.

The inability of 4-AP to inhibit HPV in some distal PASMCs, in fact, is consistent with several previous studies. In isolated perfused lungs and distal and proximal pulmonary arteries isolated from rats, complete inhibition of KV channels with 10 mM 4-AP caused significant enhancement instead of inhibition of HPV (3, 20). In rat PASMCs, intracellular dialysis of an antibody against a KV channel subtype (Kv1.5) inhibited KV currents and caused membrane depolarization but failed to inhibit the hypoxia-induced [Ca2+]i response (15). These findings suggest that hypoxic responses could be activated even when KV channels were unavailable. Moreover, several studies suggested that initiation of HPV depends on Ca2+ release from intracellular stores. For example, hypoxia-induced elevation in [Ca2+]i in PASMCs could be abolished or significantly reduced by inhibiting Ca2+ release from the sarcoplasmic reticulum (SR) with ryanodine or depleting SR Ca2+ stores with thapsigargin or caffeine (15, 26, 47, 56). Removal of extracellular Ca2+ inhibited the sustained but not the transient phase of the hypoxic Ca2+ responses (15, 47), and a hypoxia-induced increase in [Ca2+]i preceded membrane depolarization after KCa and Cl- channels were blocked (43). Because intracellular Ca2+ is known to inhibit KV channels (16, 17), it has been proposed alternatively that hypoxia first activates Ca2+ release from the SR, leading to the inhibition of KV channels, membrane depolarization, and Ca2+ influx via L-type Ca2+ channels (15, 43).

Besides direct effects on PASMCs, hypoxia may elicit HPV by stimulating or inhibiting release of factors derived from endothelium. It has been suggested that an unidentified endothelium-derived constricting factor(s) is responsible for the late-phase contraction induced by hypoxia in isolated rat and porcine arteries (14, 29, 45, 58). ET-1, a potent pulmonary vasoconstrictor, is an attractive candidate for this role because it is synthesized by pulmonary endothelial cells and released in greater quantity during hypoxia (28, 41). ET-1 antagonists can prevent acute HPV in isolated proximal and distal intrapulmonary porcine arteries (31, 50) and in intact animals (7, 23, 24, 42, 57, 61) including the pig (23, 24).

In this context, our observation that a low concentration of ET-1 (10-10 M) did not alter length or [Ca2+]i in distal PASMCs during normoxia but markedly enhanced contractile responses to hypoxia (Fig. 7) suggests that even basal release of ET-1 from endothelial cells may have significant synergistic effects on the direct contractile response of PASMCs to hypoxia. Similar results were observed in porcine pulmonary arterial microvessels (150- to 200-µm ID) in that hypoxia caused significant vasoconstriction only after they were primed with 10-10 M ET-1 (Liu Q and Sylvester JT, unpublished data). This synergism is consistent with the findings of Turner and Kozlowski (55) that an initial priming of rat PASMCs with ET-1 (10-10 M) allowed the expression of membrane depolarization induced by hypoxia (55). They proposed that an initial "priming" depolarization induced by ET-1 may confer a sensitivity to hypoxia by activating delayed rectifier KV channels, which can then be closed by hypoxia, leading to further depolarization. Moreover, threshold concentrations of ET-1 (3 × 10-10 and 10-9 M) were found to potentiate vasoconstriction induced by norepinephrine and 5-HT in coronary arteries (62). However, the potentiation of HPV by ET-1 in this study might not be due to a greater increase in [Ca2+]i because the Ca2+ response to hypoxia was not significantly enhanced in the presence of ET-1 despite a large Ca2+ spike that occurred at the onset of hypoxia in one cell. Enhancement of contraction without alteration in [Ca2+]i suggests that ET-1 increased myofilament Ca2+ sensitivity. This effect of ET-1 has been previously demonstrated in systemic vessels (5, 39) and is consistent with the findings that the endothelial mediator of late-phase hypoxic contraction in isolated pulmonary arteries may act by increasing myofilament Ca2+ sensitivity via a protein kinase C-independent mechanism (45, 58). Our results, however, did not prove that ET-1 is the unidentified endothelial factor responsible for endothelium-dependent HPV or that it is the only such factor because prestimulation with other agonists has been shown to enhance HPV in both isolated lungs and vessels (35, 46), and some investigators (12, 14) have not been able to demonstrate the involvement of ET-1 in HPV in isolated vessels. Therefore, the exact mechanism(s) through which ET-1 priming enhances hypoxic vasoconstriction remains to be elucidated.

In conclusion, our results indicate that hypoxia can increase [Ca2+]i and cause contraction in freshly isolated porcine distal PASMCs; however, the small magnitude of these direct effects and their low sensitivity to a reduced PO2 suggest that they do not account completely for HPV, which may require synergistic interactions between endothelium and smooth muscle for the full expression.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-52652 (to J. S. K. Sham) and HL-51912 (to J. T. Sylvester) and an American Heart Association Established Investigator Award (to J. S. K. Sham).


    FOOTNOTES

Address for reprint requests and other correspondence: J. S. K. Sham, Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: jsks{at}welchlink.welch.jhu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 19 March 1999; accepted in final form 3 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Albarwani, S, Heinert G, Turner JL, and Kozlowski RZ. Differential K+ channel distribution in smooth muscle cells isolated from the pulmonary arterial tree of the rat. Biochem Biophys Res Commun 208: 183-189, 1995[ISI][Medline].

2.   Archer, SL, Huang J, Henry T, Peterson D, and Weir EK. A redox-based O2 sensor in rat pulmonary vasculature. Circ Res 73: 1100-1112, 1993[Abstract].

3.   Archer, SL, Huang JM, Reeve HL, Hampl V, Tolarova S, Michelakis E, and Weir EK. Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ Res 78: 431-442, 1996[Abstract/Free Full Text].

4.   Bennie, RE, Packer CS, Powell DR, Jin N, and Rhoades RA. Biphasic contractile response of pulmonary artery to hypoxia. Am J Physiol Lung Cell Mol Physiol 261: L156-L163, 1991[Abstract/Free Full Text].

5.   Bruce, L, and Nixon GF. Increased sensitization of the myofilaments in rat neonatal portal vein: a potential mechanism. Exp Physiol 82: 985-993, 1997[Abstract].

6.   Burke-Wolin, T, and Wolin MS. H2O2 and cGMP may function as an O2 sensor in the pulmonary artery. J Appl Physiol 66: 167-170, 1989[Abstract/Free Full Text].

7.   Chen, SJ, Chen YF, Meng QC, Durand J, DiCarlo VS, and Oparil S. Endothelin-receptor antagonist bosentan prevents and reverses hypoxic pulmonary hypertension in rats. J Appl Physiol 79: 2122-2131, 1995[Abstract/Free Full Text].

8.   Chen, SJ, Chen YF, Opgenorth TJ, Wessale JL, Meng QC, Durand J, DiCarlo VS, and Oparil S. The orally active nonpeptide endothelin A-receptor antagonist A-127722 prevents and reverses hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in Sprague-Dawley rats. J Cardiovasc Pharmacol 29: 713-725, 1997[ISI][Medline].

9.   Clapp, LH, and Gurney AM. Outward currents in rabbit pulmonary artery cells dissociated with a new technique. Exp Physiol 76: 677-693, 1991[Abstract].

10.   Cornfield, DN, Stevens T, McMurtry IF, Abman SH, and Rodman DM. Acute hypoxia increases cytosolic calcium in fetal pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 265: L53-L56, 1993[Abstract/Free Full Text].

11.   Cornfield, DN, Stevens T, McMurtry IF, Abman SH, and Rodman DM. Acute hypoxia causes membrane depolarization and calcium influx in fetal pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 266: L469-L475, 1994[Abstract/Free Full Text].

12.   Douglas, SA, Vickery-Clark LM, and Ohlstein EH. Endothelin-1 does not mediate hypoxic vasoconstriction in canine isolated blood vessels: effect of BQ-123. Br J Pharmacol 108: 418-421, 1993[ISI][Medline].

13.   Fishman, AP. Hypoxia on the pulmonary circulation. How and where it acts. Circ Res 38: 221-231, 1976[ISI][Medline].

14.   Gaine, SP, Hales MA, and Flavahan NA. Hypoxic pulmonary endothelial cells release a diffusible contractile factor distinct from endothelin. Am J Physiol Lung Cell Mol Physiol 274: L657-L664, 1998[Abstract/Free Full Text].

15.   Gelband, CH, and Gelband H. Ca2+ release from intracellular stores is an initial step in hypoxic pulmonary vasoconstriction of rat pulmonary artery resistance vessels. Circulation 96: 3647-3654, 1997[Abstract/Free Full Text].

16.   Gelband, CH, and Hume JR. [Ca2+]i inhibition of K+ channels in canine renal artery. Novel mechanism for agonist-induced membrane depolarization. Circ Res 77: 121-130, 1995[Abstract/Free Full Text].

17.   Gelband, CH, Ishikawa T, Post JM, Keef KD, and Hume JR. Intracellular divalent cations block smooth muscle K+ channels. Circ Res 73: 24-34, 1993[Abstract].

18.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

19.   Harder, DR, Madden JA, and Dawson C. Hypoxic induction of Ca2+-dependent action potentials in small pulmonary arteries of the cat. J Appl Physiol 59: 1389-1393, 1985[Abstract/Free Full Text].

20.   Hasunuma, K, Rodman DM, and McMurtry IF. Effects of K+ channel blockers on vascular tone in the perfused rat lung. Am Rev Respir Dis 144: 884-887, 1991[ISI][Medline].

21.   Herold, CJ, Wetzel RC, Robotham JL, Herold SM, and Zerhouni EA. Acute effects of increased intravascular volume and hypoxia on the pulmonary circulation: assessment with high-resolution CT. Radiology 183: 655-662, 1992[Abstract].

22.   Holden, WE, and McCall E. Hypoxia-induced contractions of porcine pulmonary artery strips depend on intact endothelium. Exp Lung Res 7: 101-112, 1984[ISI][Medline].

23.   Holm, P, Liska J, Clozel M, and Franco-Cereceda A. The endothelin antagonist bosentan: hemodynamic effects during normoxia and hypoxic pulmonary hypertension in pigs. J Thorac Cardiovasc Surg 112: 890-897, 1996[Abstract/Free Full Text].

24.   Holm, P, Liska J, and Franco-Cereceda A. The ETA receptor antagonist, BMS-182874, reduces acute hypoxic pulmonary hypertension in pigs in vivo. Cardiovasc Res 37: 765-771, 1998[ISI][Medline].

25.   Horio, T, Kohno M, Yokokawa K, Murakawa K, Yasunari K, Fujiwara H, Kurihara N, and Takeda T. Effect of hypoxia on plasma immunoreactive endothelin-1 concentration in anesthetized rats. Metabolism 40: 999-1001, 1991[ISI][Medline].

26.   Jabr, RI, Toland H, Gelband CH, Wang XX, and Hume JR. Prominent role of intracellular Ca2+ release in hypoxic vasoconstriction of canine pulmonary artery. Br J Pharmacol 122: 21-30, 1997[Abstract].

27.   Jin, N, Packer CS, and Rhoades RA. Pulmonary arterial hypoxic contraction: signal transduction. Am J Physiol Lung Cell Mol Physiol 263: L73-L78, 1992[Abstract/Free Full Text].

28.   Kourembanas, S, Marsden PA, McQuillan LP, and Faller DV. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J Clin Invest 88: 1054-1057, 1991[ISI][Medline].

29.   Kovitz, KL, Aleskowitch TD, Sylvester JT, and Flavahan NA. Endothelium-derived contracting and relaxing factors contribute to hypoxic responses of pulmonary arteries. Am J Physiol Heart Circ Physiol 265: H1139-H1148, 1993[Abstract/Free Full Text].

30.   Leach, RM, Robertson TP, Twort CH, and Ward JP. Hypoxic vasoconstriction in rat pulmonary and mesenteric arteries. Am J Physiol Lung Cell Mol Physiol 266: L223-L231, 1994[Abstract/Free Full Text].

31.   Liu, Q, Fan HB, and Sylvester JT. Hypoxic contraction of isolated porcine pulmonary arterioles: role of endothelin-1 (Abstract). Circulation 98: 665S, 1998.

32.   Madden, JA, Dawson CA, and Harder DR. Hypoxia-induced activation in small isolated pulmonary arteries from the cat. J Appl Physiol 59: 113-118, 1985[Abstract/Free Full Text].

33.   Madden, JA, Vadula MS, and Kurup VP. Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 263: L384-L393, 1992[Abstract/Free Full Text].

34.   McDonnell, TJ, Westcott JY, Czartolomna J, and Voelkel NF. Role of peptidoleukotrienes in hypoxic pulmonary vasoconstriction in rats. Am J Physiol Heart Circ Physiol 259: H751-H758, 1990[Abstract/Free Full Text].

35.   McMurtry, IF. Angiotensin is not required for hypoxic constriction in salt solution-perfused rat lungs. J Appl Physiol 56: 375-380, 1984[Abstract/Free Full Text].

36.   McMurtry, IF. BAY K 8644 potentiates and A23187 inhibits hypoxic vasoconstriction in rat lungs. Am J Physiol Heart Circ Physiol 249: H741-H746, 1985[Abstract/Free Full Text].

37.   McMurtry, IF, Davidson AB, Reeves JT, and Grover RF. Inhibition of hypoxic pulmonary vasoconstriction by calcium antagonists in isolated rat lungs. Circ Res 38: 99-104, 1976[Abstract].

38.   Murray, TR, Chen L, Marshall BE, and Macarak EJ. Hypoxic contraction of cultured pulmonary vascular smooth muscle cells. Am J Respir Cell Mol Biol 3: 457-465, 1990[ISI][Medline].

39.   Nishimura, J, Moreland S, Ahn HY, Kawase T, Moreland RS, and van Breemen C. Endothelin increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized rabbit mesenteric artery. Circ Res 71: 951-959, 1992[Abstract].

40.   Ogata, M, Ohe M, Katayose D, and Takishima T. Modulatory role of EDRF in hypoxic contraction of isolated porcine pulmonary arteries. Am J Physiol Heart Circ Physiol 262: H691-H697, 1992[Abstract/Free Full Text].

41.   Ohlstein, EH, Arleth A, Ezekiel M, Horohonich S, Ator MA, Caltabiano MM, and Sung CP. Biosynthesis and modulation of endothelin from bovine pulmonary arterial endothelial cells. Life Sci 46: 181-188, 1990[ISI][Medline].

42.   Oparil, S, Chen SJ, Meng QC, Elton TS, Yano M, and Chen YF. Endothelin-A receptor antagonist prevents acute hypoxia-induced pulmonary hypertension in the rat. Am J Physiol Lung Cell Mol Physiol 268: L95-L100, 1995[Abstract/Free Full Text].

43.   Post, JM, Gelband CH, and Hume JR. [Ca2+]i inhibition of K+ channels in canine pulmonary artery. Novel mechanism for hypoxia-induced membrane depolarization. Circ Res 77: 131-139, 1995[Abstract/Free Full Text].

44.   Post, JM, Hume JR, Archer SL, and Weir EK. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol Cell Physiol 262: C882-C890, 1992[Abstract/Free Full Text].

45.   Robertson, TP, Aaronson PI, and Ward JP. Hypoxic vasoconstriction and intracellular Ca2+ in pulmonary arteries: evidence for PKC-independent Ca2+ sensitization. Am J Physiol Heart Circ Physiol 268: H301-H307, 1995[Abstract/Free Full Text].

46.   Rodman, DM, Yamaguchi T, O'Brien RF, and McMurtry IF. Hypoxic contraction of isolated rat pulmonary artery. J Pharmacol Exp Ther 248: 952-959, 1989[Abstract].

47.   Salvaterra, CG, and Goldman WF. Acute hypoxia increases cytosolic calcium in cultured pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 264: L323-L328, 1993[Abstract/Free Full Text].

48.   Schnader, J, Undem B, Adams GK, III, Peters SP, Adkinson NF, and Sylvester JT. Effects of hypoxia on leukotriene activity and vasomotor tone in isolated sheep lungs. J Appl Physiol 68: 2457-2465, 1990[Abstract/Free Full Text].

49.   Shimoda, LA, Sylvester JT, and Sham JS. Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1. Am J Physiol Lung Cell Mol Physiol 274: L842-L853, 1998[Abstract/Free Full Text].

50.   Shimoda, T, Booth G, Liu Q, Shimoda L, Sham J, and Sylvester JT. Endothelium-dependent hypoxic contraction of pig proximal intrapulmonary arteries: role of endothelin (Abstract). Am J Respir Crit Care Med 153: A724, 1998.

51.   Stern, MD, Silverman HS, Houser SR, Josephson RA, Capogrossi MC, Nichols CG, Lederer WJ, and Lakatta EG. Anoxic contractile failure in rat heart myocytes is caused by failure of intracellular calcium release due to alteration of the action potential. Proc Natl Acad Sci USA 85: 6954-6958, 1988[Abstract].

52.   Sylvester, JT, Harabin AL, Peake MD, and Frank RS. Vasodilator and constrictor responses to hypoxia in isolated pig lungs. J Appl Physiol 49: 820-825, 1980[Abstract/Free Full Text].

53.   Tolins, M, Weir EK, Chesler E, Nelson DP, and From AH. Pulmonary vascular tone is increased by a voltage-dependent calcium channel potentiator. J Appl Physiol 60: 942-948, 1986[Abstract/Free Full Text].

54.   Tucker, A, McMurtry IF, Reeves JT, Alexander AF, Will DH, and Grover RF. Lung vascular smooth muscle as a determinant of pulmonary hypertension at high altitude. Am J Physiol 228: 762-767, 1975[ISI][Medline].

55.   Turner, JL, and Kozlowski RZ. Relationship between membrane potential, delayed rectifier K+ currents and hypoxia in rat pulmonary arterial myocytes. Exp Physiol 82: 629-645, 1997[Abstract].

56.   Vadula, MS, Kleinman JG, and Madden JA. Effect of hypoxia and norepinephrine on cytoplasmic free Ca2+ in pulmonary and cerebral arterial myocytes. Am J Physiol Lung Cell Mol Physiol 265: L591-L597, 1993[Abstract/Free Full Text].

57.   Wang, Y, Coe Y, Toyoda O, and Coceani F. Involvement of endothelin-1 in hypoxic pulmonary vasoconstriction in the lamb. J Physiol (Lond) 482: 421-434, 1995[Abstract].

58.   Ward, JP, and Robertson TP. The role of the endothelium in hypoxic pulmonary vasoconstriction. Exp Physiol 80: 793-801, 1995[Abstract].

59.   Weir, EK, and Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB J 9: 183-189, 1995[Abstract/Free Full Text].

60.   Wier, WG, Egan TM, Lopez-Lopez JR, and Balke CW. Local control of excitation-contraction coupling in rat heart cells. J Physiol (Lond) 474: 463-471, 1994[Abstract].

61.   Willette, RN, Ohlstein EH, Mitchell MP, Sauermelch CF, Beck GR, Luttmann MA, and Hay DW. Nonpeptide endothelin receptor antagonists. VIII: attenuation of acute hypoxia-induced pulmonary hypertension in the dog. J Pharmacol Exp Ther 280: 695-701, 1997[Abstract/Free Full Text].

62.   Yang, ZH, Richard V, von Segesser L, Bauer E, Stulz P, Turina M, and Luscher TF. Threshold concentrations of endothelin-1 potentiate contractions to norepinephrine and serotonin in human arteries. A new mechanism of vasospasm? Circulation 82: 188-195, 1990[Abstract].

63.   Yuan, XJ. Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary arterial myocytes. Circ Res 77: 370-378, 1995[Abstract/Free Full Text].

64.   Yuan, XJ, Goldman WF, Tod ML, Rubin LJ, and Blaustein MP. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am J Physiol Lung Cell Mol Physiol 264: L116-L123, 1993[Abstract/Free Full Text].

65.   Yuan, XJ, Tod ML, Rubin LJ, and Blaustein MP. Contrasting effects of hypoxia on tension in rat pulmonary and mesenteric arteries. Am J Physiol Heart Circ Physiol 259: H281-H289, 1990[Abstract/Free Full Text].

66.   Zhang, F, Carson RC, Zhang H, Gibson G, and Thomas HM, III. Pulmonary artery smooth muscle cell [Ca2+]i and contraction: responses to diphenyleneiodonium and hypoxia. Am J Physiol Lung Cell Mol Physiol 273: L603-L611, 1997[Abstract/Free Full Text].


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