L-type Ca2+ channels, resting [Ca2+]i, and ET-1-induced responses in chronically hypoxic pulmonary myocytes

Larissa A. Shimoda, James S. K. Sham, Tenille H. Shimoda, and J. T. Sylvester

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca2+ channels contributes to 1) maintenance of resting intracellular Ca2+ concentration ([Ca2+]i), 2) increased [Ca2+]i in response to ET-1 (10-8 M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca2+]i in PASMCs from intrapulmonary arteries of rats exposed to 10% O2 for 21 days was 293.9 ± 25.2 nM (vs. 153.6 ± 28.7 nM in normoxia). Resting [Ca2+]i was decreased after extracellular Ca2+ removal but not with nifedipine (10-6 M), an L-type Ca2+ channel antagonist. After CH, the ET-1-induced increase in [Ca2+]i was reduced and was abolished after extracellular Ca2+ removal or nifedipine. Removal of extracellular Ca2+ reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca2+]i in PASMCs from chronically hypoxic rats does not require activation of L-type Ca2+ channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca2+]i.

intracellular calcium concentration; endothelin-1; chronic hypoxia; pulmonary hypertension; contraction; voltage-gated calcium channels; smooth muscle; pulmonary artery smooth muscle cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PROLONGED EXPOSURE to decreased alveolar oxygen tension results in the development of pulmonary hypertension that is characterized by vascular remodeling and sustained vasoconstriction. The remodeling is due to pulmonary arterial smooth muscle cell (PASMC) proliferation and extension of smooth muscle into previously nonmuscular arterioles (33, 40), whereas active vasoconstriction is evidenced by an acute reduction in pulmonary arterial pressure in response to vasodilators (19, 21, 26, 35, 38). Despite extensive study, however, identification of the exact mechanisms underlying hypoxic pulmonary vasoconstriction and the subsequent development of pulmonary hypertension due to chronic hypoxia (CH) has remained elusive.

Abnormalities in PASMCs are likely to contribute to the pathogenesis of chronic hypoxic pulmonary hypertension. The PASMC contraction and proliferation associated with CH may be caused by an elevation in intracellular Ca2+ concentration ([Ca2+]i) because both acute hypoxic vasoconstriction and in vitro smooth muscle proliferation are associated with a rise in [Ca2+]i (8, 9, 22, 27, 30) and can be prevented by voltage-gated, or L-type, Ca2+ channel antagonists (22, 30). L-type Ca2+ channel activity is controlled in large part by the membrane potential. Under normoxic conditions, the major regulator of resting membrane potential in PASMCs appears to be voltage-gated K+ (KV) channels because inhibition of these channels causes depolarization, activation of L-type Ca2+ channels, and increased [Ca2+]i (50). A reduction in K+ channel proteins and KV current as well as in membrane depolarization is observed in PASMCs after CH (39, 43, 46-48), fueling speculation that inhibition of K+ channels and PASMC depolarization may contribute to the development of chronic hypoxic pulmonary hypertension by increasing the availability of cytosolic Ca2+.

In vivo, basal [Ca2+]i and PASMC tone can be influenced by vasoconstrictor and vasodilator factors from other cells. A change in synthesis of these factors may contribute to the active contraction and cellular changes observed with CH. Although several vasoactive factors have been proposed to be involved in the pathogenesis of chronic hypoxic pulmonary hypertension, recent attention has focused on the role of the endothelium-derived contracting factor endothelin (ET)-1. ET-1 levels are markedly increased during CH (7, 10, 12), and ET receptor antagonists prevent and partially reverse the development of pulmonary hypertension secondary to CH (5, 7, 10). Furthermore, pulmonary arteries appear to be hyperresponsive to exogenous ET-1 after chronic exposure to hypoxia (11, 25, 29, 39). Under normoxic conditions, ET-1 increases [Ca2+]i (4, 44) and causes depolarization, in part due to inhibition of KV and/or Ca2+-activated K+ channels (39, 41, 42). However, in vivo exposure to CH appears to alter ET-1 signal transduction mechanisms so that the ability of ET-1 to inhibit KV currents is lost (43), suggesting that alterations in other parts of the signal transduction pathway may be responsible for the enhanced ET-1 vasoreactivity.

The fact that Ca2+ signaling plays a vital role in many cell functions including proliferation and contraction emphasizes the importance of understanding the mechanisms regulating Ca2+ homeostasis under both physiological and pathophysiological conditions. Based on the findings that PASMCs are depolarized after exposure to CH and that ET-1 signal transduction and reactivity may be altered by CH, we determined whether activation of L-type Ca2+ channels plays a major role in 1) maintenance of resting [Ca2+]i, 2) increased [Ca2+]i in response to ET-1, and 3) ET-1-induced contraction in pulmonary arterial smooth muscle from chronically hypoxic rats. Using Ca2+ microfluorescence techniques in intrapulmonary arterial myocytes and isometric tension recording in intrapulmonary arteries isolated from normoxic and chronically hypoxic rats, we determined 1) the effect of CH on resting [Ca2+]i, 2) the contribution of extracellular influx through L-type Ca2+ channels to the maintenance of resting [Ca2+]i and baseline tension, 3) the effect of CH on the ability of ET-1 to increase [Ca2+]i, and 4) the contribution from extracellular Ca2+ sources to the ET-1-induced increase in [Ca2+]i and isometric tension.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CH

Male Wistar rats (150-250 g) were placed in a hypoxic chamber and exposed to either normoxia or normobaric hypoxia for 17-21 days as previously described (43). The chamber was continuously flushed with either room air or a mixture of room air and N2 (10 ± 0.5% O2) to maintain a low CO2 concentration (<0.5%). Chamber O2 and CO2 concentrations were continuously monitored (OM-11 oxygen analyzer and LB-2 gas analyzer, Sensormedics, Anaheim, CA). The rats were exposed to room air for 10 min twice a week to clean the cages and replenish the food and water supplies.

Isolation and Culture of PASMCs

Single PASMCs were obtained as previously described (42). Briefly, male Wistar rats (150-250 g) were injected with heparin and anesthetized with pentobarbital sodium (130 mg/kg ip). The rats were exsanguinated, and the heart and lungs were removed en bloc and transferred to a petri dish containing HEPES-buffered saline solution (HBS) composed of (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with 5 M NaOH. Intrapulmonary arteries (~300- to 800-µm OD) were isolated and cleaned of connective tissue. The endothelium was disrupted by gently rubbing the luminal surface with a cotton swab. The arteries were allowed to recover for 30 min in cold (4°C) HBS followed by 20 min in reduced-Ca2+ HBS (20 µM CaCl2) at room temperature. The tissue was digested at 37°C for 20 min in reduced-Ca2+ HBS containing collagenase (type I, 1,750 U/ml), papain (9.5 U/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM). After digestion, single smooth muscle cells were dispersed by gentle trituration with a wide-bore transfer pipette in Ca2+-free HBS. The cell suspension was filtered and placed on 25-mm glass coverslips, and the cells were transiently cultured in Ham's F-12 medium with L-glutamine (Mediatech) supplemented with 0.5% fetal calf serum, 1% streptomycin, and 1% penicillin for 24-48 h.

[Ca2+]i Measurements

[Ca2+]i in single PASMCs was measured with the membrane-permeant (acetoxymethyl ester) form of the Ca2+-sensitive fluorescent dye indo 1 (indo 1-AM). Transiently cultured PASMCs were incubated with 5 µM indo 1-AM for 30 min at room temperature (22°C) under an atmosphere of 21% O2-5% CO2. The cells were then washed for 30 min with physiological saline solution (PSS) containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 11 glucose, 1.2 KH2PO4, and 2 CaCl2 that was gassed with 21% O2-5% CO2 at 37°C to remove extracellular indo 1-AM and allow complete deesterification of the cytosolic indo 1-AM. Ratiometric measurement of fluorescence from indo 1-AM was performed on an inverted microscope (Nikon Diaphot) workstation. The collimated light beam from a 75-W xenon arc lamp was filtered by an interference filter at 365 nm and focused on the PASMC under examination via a ×40 fluorescence oil-immersion objective (Fluor 40, Nikon). Light emitted from the cell was returned through the objective, split with a dichroic mirror, and detected at 405 (F405) and 495 (F495) nm by two photomultiplier tubes. The emission signals were amplified with a dual-emission fluorometer (Biomedical Instrumentation Group, University of Pennsylvania, Philadelphia, PA). Photobleaching of indo 1-AM dye was minimized by using a neutral density filter (ND-3, Omega Optics) and an electronic shutter (Vincent Associates). The shutter was opened for 35 ms every second, and the fluorescence signals during the open period were integrated with a sample-and-hold circuit. The protocols were executed, and data were collected online with a Labmaster analog-to-digital interface (DMA TL-1) and the pClamp software package. [Ca2+]i was calculated with the equation described by Grynkiewicz et al. (15)
[Ca<SUP><IT>2+</IT></SUP>]<SUB>i</SUB><IT>=K</IT><SUB>d</SUB><IT>×</IT>B<IT>×</IT>(R<IT>−</IT>R<SUB>min</SUB>)<IT>/</IT>(R<IT>−</IT>R<SUB>max</SUB>)
where Kd is the dissociation constant (288 nM); B is the ratio of F495,EGTA to F495,Ca, where F495,EGTA and F495,Ca are the fluorescence values corresponding to the minimum and maximum Ca2+ levels, respectively; R is the ratio of (F405 - F405,background) to (F495 - F495,background), where F405,background and F495,background are the background fluorescence values measured at 405 and 495 nm, respectively; and Rmin and Rmax are the minimum and maximum fluorescence ratios, respectively. Calibration to determine F495,EGTA and F495,Ca was performed in vivo by perfusing PASMCs with PSS containing 10 mM CaCl2 to determine the value corresponding to maximum Ca2+ fluorescence followed by perfusion with PSS containing 0 mM CaCl2 and 10 mM EGTA to determine minimum Ca2+ fluorescence. F495,background and F405,background were determined by measuring F405 and F495 after the addition of 10 mM Mn2+ to quench the dye.

Isolated Arterial Ring Segments

Intrapulmonary arteries (300- to 800-µm OD) were obtained as described in Isolation and Culture of PASMCs; placed in oxygenated modified Krebs solution containing (in mM) 118 NaCl, 4.7 KCl, 0.57 MgSO4, 1.18 KH2PO4, 25 NaHCO3, 10 dextrose and 2.5 CaCl2 at 25°C; cleaned of connective tissue; and cut into ring segments 4 mm in length. The endothelium was disrupted by gently rubbing the lumen with a modified cotton swab, and the arterial rings were suspended between two stainless steel stirrups in organ chambers filled with modified Krebs solution for isometric tension recording. The solution was gassed with 16% O2-5% CO2 to maintain a pH of 7.4. One stirrup was anchored in the chamber, and the other was connected to a strain gauge (model FT03, Grass Instruments, Quincy, MA) attached to a micrometer for the continuous measurement of isometric tension (model 7E polygraph, Grass Instruments). The arteries were adjusted to a resting tension of 2 g in 0.5-g steps over a period of 40 min. Preliminary experiments revealed that contractile responses to KCl (80 mM) were maximal at these resting tensions. The arteries were exposed to 80 mM KCl to establish viability and maximum contraction and to phenylephrine (3 × 10 -7 M) followed by acetylcholine (10-6 M) to verify disruption of endothelium integrity. Vessels that did not contract with KCl or dilate with acetylcholine were discarded. To confirm that endothelial denudation did not seriously damage smooth muscle, contractile responses to KCl in rings subjected to denudation were required to be >= 80% of the responses measured in control rings with endothelium.

Experimental Protocols

Effect of CH on resting [Ca2+]i. Baseline fluorescence was measured for 10 min in PASMCs from either normoxic or chronically hypoxic rats. Cells with unstable [Ca2+]i levels were discarded. To determine the effect of an L-type Ca2+ channel antagonist or extracellular Ca2+ removal on resting [Ca2+]i, PASMCs from chronically hypoxic rats were subsequently perfused with PSS containing nifedipine (10-6 M) or with Ca2+-free PSS containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 11 glucose, 1.2 KH2PO4, and 1 EGTA while fluorescence was monitored for an additional 10 min.

Effect of ET-1 and KCl on [Ca2+]i. PASMCs were equilibrated, and stable fluorescence was monitored for 15 min. The effect of ET-1 and KCl (100 mM) on [Ca2+]i was determined by measuring fluorescence during 10 min of exposure to 10-8 M ET-1 or high-K+ PSS containing (in mM) 23 NaCl, 100 KCl, 1.2 MgSO4, 25 NaHCO3, 11.1 glucose, and 1.2 KH2PO4. After 10 min, the agonists were washed out, and fluorescence was measured for an additional 5 min. In separate experiments, the effect of an L-type Ca2+ channel antagonist on the change in [Ca2+]i induced by ET-1 or KCl was determined by perfusing the PASMCs for 15 min with PSS containing nifedipine (10-6 M) before exposure to ET-1 or KCl for 10 min. ET-1 or KCl and the antagonist were then washed out, and fluorescence was measured for an additional 5 min. Similarly, the effect of extracellular Ca2+ removal on ET-1-induced increase in [Ca2+]i was determined by perfusing stable PASMCs with Ca2+-free PSS for 15 min before exposure to ET-1 for 10 min. ET-1 was then washed out with Ca2+-containing PSS, and fluorescence was measured for an additional 5 min.

Role of L-type Ca2+ channels in KCl- and ET-1-induced contraction. To determine whether exposure to CH augments KCl- or ET-1-induced contraction, the tension generated in response to these agonists was measured in intrapulmonary arterial segments from both normoxic and chronically hypoxic rats. The tension was monitored for 20 min before (for baseline tension measurements) and 30 min after exposure to either ET-1 or KCl. The tension generated in response to both KCl and ET-1 is expressed as a percentage of the contraction induced by the initial exposure to 80 mM KCl. To determine the functional role of Ca2+ influx through L-type Ca2+ channels in the pressor response to KCl or ET-1, arterial segments from normoxic and chronically hypoxic rats were challenged with agonists in the absence and presence of the L-type Ca2+ channel antagonist nifedipine (10-6 M). The role of Ca2+ influx in the pressor response to ET-1 was determined by comparing the tension developed in response to ET-1 in arteries bathed in Ca2+-containing or Ca2+-free extracellular solution.

Ca2+ release from caffeine-sensitive stores. To determine whether intracellular stores were depleted in PASMCs from chronically hypoxic rats, fluorescence was measured while the PASMCs were exposed to caffeine (10 mM) for 30 s to induce Ca2+ release. A rapid exchange system as previously described (42) was used to introduce caffeine to the PASMCs under study.

Effect of ET-1 on membrane potential. Membrane potential measurements were made with whole cell patch-clamp techniques under current-clamp mode in PASMCs from normoxic and chronically hypoxic rats. The effect of ET-1 (10-8 M) on the membrane potential was determined by recording the membrane potential for 1 min before, 2 min during, and 2 min after exposure to ET-1.

Drugs

ET-1 was obtained from American Peptides (Sunnyvale, CA). Indo 1-AM dye was obtained from Molecular Probes (Eugene, OR). Nifedipine was obtained from Calbiochem (La Jolla, CA). Caffeine was obtained from Peptides International (Louisville, KY). All other chemicals were obtained from Sigma (St. Louis, MO). ET-1 was made up in a stock solution (10-5 M; distilled water), divided into aliquots, and stored at 0°C until used. All other drugs were made fresh in distilled water or ethanol (nifedipine) on the day of the experiment.

Data Analysis

Data are expressed as means ± SE; n is the number of cells or arteries tested. Two or three arterial segments were obtained from each rat. Protocols were performed on rings from different animals, and each rat served as its own control. Therefore, in experiments measuring isometric tension, the number of arteries tested also refers to the number of animals for each protocol. Baseline and sustained phase of agonist-induced [Ca2+]i were determined for each cell from the average of 20 data points; peak [Ca2+]i was determined from the average of 5 data points including the absolute maximum of response. Data were compared with Student's t-test (paired or unpaired as applicable). A value P < 0.05 was accepted as significant. Data expressed as percentages underwent arcsine transformation before comparison.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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Effect of CH on Resting [Ca2+]i

Average resting [Ca2+]i in PASMCs from normoxic rats was 172.8 ± 20.9 nM (n = 19). Resting [Ca2+]i was significantly elevated to 269.9 ± 17.5 nM in PASMCs from rats exposed to 17-21 days of hypoxia (P < 0.01; n = 35; Fig. 1). To determine whether Ca2+ influx from extracellular sources was required for the maintenance of resting [Ca2+]i in PASMCs from normoxic and chronically hypoxic rats, PASMCs were perfused with Ca2+-free PSS. In PASMCs from normoxic rats, removal of extracellular Ca2+ had no effect on resting [Ca2+]i (132.7 ± 36.6 to 151.2 ± 50.6 nM; n = 4). In contrast, in PASMCs from chronically hypoxic rats, when extracellular Ca2+ was removed, resting [Ca2+]i rapidly decreased from 267.8 ± 68.5 to 174.2 ± 47.1 nM (n = 6). To determine whether Ca2+ influx was due to activation of L-type Ca2+ channels, PASMCs were exposed to nifedipine (10-6 M), an L-type Ca2+ channel antagonist. The addition of nifedipine had no effect on resting [Ca2+]i in PASMCs from either normoxic (136.8 ± 34.5 to 141.8 ± 41.4 nM; n = 4) or chronically hypoxic (249.8 ± 17.2 to 251.5 ± 16.5 nM; n = 8; Fig. 2) rats.


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Fig. 1.   Average resting intracellular Ca2+ concentration ([Ca2+]i) measured in pulmonary arterial smooth muscle cells (PASMCs) from normoxic (n = 19 cells from 10 animals) and chronically hypoxic (n = 35 cells from 21 animals) rats. * Significant difference from PASMCs from normoxic rats, P < 0.01.



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Fig. 2.   Composite traces illustrating the effects of nifedipine (Nif; n = 4 cells from 4 animals for normoxia and 8 cells from 5 animals for chronic hypoxia; top) and the removal of extracellular Ca2+ (n = 4 cells from 3 animals for normoxia and 6 cells from 5 animals for chronic hypoxia; middle) on resting [Ca2+]i in PASMCs from normoxic (A) and chronically hypoxic (B) rats. Bottom: average [Ca2+]i before (open bars) and after (hatched bars) removal of extracellular Ca2+ or addition of nifedipine. * Significant difference from control value, P < 0.05.

Effect of Nifedipine on KCl-Induced Rise in [Ca2+]i

To confirm that the concentration of nifedipine used was sufficient to inhibit Ca2+ influx through L-type Ca2+ channels, PASMCs from chronically hypoxic rats were exposed to 100 mM KCl in the absence and presence of nifedipine (10-6 M). KCl caused a sustained increase in [Ca2+]i, from 201.1 ± 25.3 to 233.1 ± 29.3 nM (P < 0.01; n = 5; Fig. 3), which resembled that previously reported in PASMCs from normoxic rats (44). Pretreating PASMCs with nifedipine for 10 min before exposure to KCl was begun completely abolished the KCl-induced increase in (Delta ) [Ca2+]i (Delta [Ca2+]i = 83.26 ± 19.19 vs. 1.9 ± 7 nM; P < 0.001; n = 5).


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Fig. 3.   A: composite traces demonstrating the effect of nifedipine on the KCl-induced increase in [Ca2+]i in PASMCs from chronically hypoxic rats. B: average change in (Delta ) [Ca2+]i induced by KCl (100 mM) in the absence (n = 5 cells from 4 animals) and presence (n = 5 cells from 3 animals) of nifedipine. * Significant difference from control value, P < 0.01.

Effect of CH on ET-1-Induced Rise in [Ca2+]i

In PASMCs from normoxic rats, ET-1 (10-8 M) caused a significant increase in [Ca2+]i consisting of a transient, large-amplitude peak (Delta Capeak2+) followed by a decline to a steady-state level (Delta Caplat2+) that remained elevated above baseline (Fig. 4). The average Delta Capeak2+ was 1,156.7 ± 540.1 nM (n = 8), whereas Delta Caplat2+ was 70.8 ± 24.6 nM. In PASMCs from chronically hypoxic rats, the ET-1-induced Delta Capeak2+ was markedly reduced to 185 ± 35.7 nM (P < 0.01; n = 8), whereas Delta Caplat2+ was 54.2 ± 12 nM, similar to the change in [Ca2+]i observed in PASMCs from normoxic rats. The contribution of extracellular Ca2+ influx through L-type Ca2+ channels to the ET-1-induced rise in [Ca2+]i in PASMCs from both normoxic and chronically hypoxic rats was determined by pretreating PASMCs with nifedipine or removing extracellular Ca2+. In normoxic PASMCs, nifedipine significantly reduced the peak change in [Ca2+]i induced by ET-1 to 308.5 ± 62.7 nM (n = 4), and no sustained elevation was observed. Removal of extracellular Ca2+ reduced the peak change in [Ca2+]i to 239.7 ± 16.8 nM (n = 4). When PASMCs from chronically hypoxic rats were exposed to nifedipine (10-6 M) 10 min before ET-1 challenge, the ET-1-induced change in [Ca2+]i was completely abolished (n = 6; Fig. 5). Similarly, when PASMCs were bathed in Ca2+-free extracellular solution before exposure to ET-1, no change in [Ca2+]i was observed in response to ET-1 (n = 3).


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Fig. 4.   Composite traces illustrating the effect of endothelin (ET)-1 on [Ca2+]i in PASMCs from normoxic (n = 8 cells from 5 animals; A) and chronically hypoxic (n = 8 cells from 6 animals; C) rats. B and D: average peak and sustained change, respectively, in Delta [Ca2+]i induced by ET-1 in PASMCs from both groups of rats. * Significant difference from normoxic value, P < 0.01.



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Fig. 5.   Composite traces indicating the effects of nifedipine (top), an L-type Ca2+ channel antagonist, and perfusion with Ca2+-free physiological salt solution (middle) on the ET-1-induced rise in [Ca2+]i in PASMCs from normoxic (A) and chronically hypoxic (B) rats. Bottom: average sustained Delta [Ca2+]i induced by ET-1 (10-8 M) after either exposure to nifedipine (n = 4 cells from 4 animals for normoxia and 6 cells from 5 animals for chronic hypoxia) or removal of extracellular Ca2+ (n = 4 cells from 3 animals for normoxia and 3 cells from 3 animals for chronic hypoxia). *Significant difference from normoxic value, P < 0.001.

Effect of CH on Caffeine-Induced Ca2+ Release

Because the ET-1-induced Delta Capeak2+, which we (44) have previously demonstrated to be primarily due to Ca2+-induced Ca2+ release from caffeine-sensitive intracellular stores, was absent in PASMCs from chronically hypoxic rats, we examined whether caffeine could still induce Ca2+ release in PASMCs from chronically hypoxic rats. Exposure to 10 mM caffeine caused a significant increase in [Ca2+]i of 399.5 ± 131.9 nM (n = 5; Fig. 6) in normoxic PASMCs. Similarly, caffeine increased [Ca2+]i in PASMCs from chronically hypoxic rats by 338.8 ± 108.2 nM (n = 8).


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Fig. 6.   Composite traces illustrating that caffeine caused a significant increase in [Ca2+]i in PASMCs from both normoxic (n = 5 cells from 4 animals; A) and chronically hypoxic (n = 8 cells from 6 animals; B) rats. C: average Delta [Ca2+]i induced by caffeine in PASMCs from normoxic (open bar) and chronically hypoxic (solid bar) rats.

Effect of ET-1 on Membrane Potential

We (42) previously demonstrated that ET-1 causes depolarization in PASMCs from normoxic rats, in part, by inhibiting KV channels. We have also demonstrated that after exposure to CH, the effect of ET-1 on KV current was markedly reduced (43). Because the ET-1-induced increase in [Ca2+]i was abolished in the presence of nifedipine in PASMCs from chronically hypoxic rats, we examined the ability of ET-1 to depolarize PASMCs after exposure to CH. Consistent with the previous findings, ET-1 (10-8 M) caused a significant depolarization in membrane potential, from -38 to -24 mV, in PASMCs from normoxic rats (P < 0.01; n = 4; Fig. 7). The resting membrane potential was significantly depolarized in PASMCs from chronically hypoxic rats, again confirming the previous observations (43). When exposed to ET-1 (10-8 M; n = 7), however, no further change in membrane potential was observed.


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Fig. 7.   Representative traces illustrating the effect of ET-1 on membrane potential in PASMCs from normoxic (A) and chronically hypoxic (B) rats. C: effect of chronic hypoxia on the average change in membrane potential (Em) induced by ET-1 (n = 4 cells from 3 animals for normoxia and 7 cells from 4 animals for chronic hypoxia). * Significant difference from control value, P < 0.01. § Significant difference from normoxic value, P < 0.01.

Effect of Nifedipine and Extracellular Ca2+ Removal on Tension

To determine the role of Ca2+ influx through L-type Ca2+ channels in the maintenance of baseline tension, the tension in rat intrapulmonary arteries was set to 2 g and measured for 20 min in arteries under control conditions, after removal of extracellular Ca2+, and in the presence of nifedipine (10-6 M). In arterial segments from either normoxic or hypoxic rats maintained under control conditions, tension was maintained near 100% of the original tension (Fig. 8). In arterial segments from normoxic rats, nifedipine had no effect on baseline tension (n = 3 each). Removal of extracellular Ca2+ caused a small, transient decrease in tension (to 98.5 ± 2.8%), which returned to 100% of baseline within 3 min (n = 3 each). In arterial segments from chronically hypoxic rats, removal of extracellular Ca2+ decreased tension from 100.3 ± 0.3 to 91.7 ± 1.7% (P < 0.01; n = 4 each). In the presence of nifedipine, tension decreased slightly but significantly (from 99.3 ± 0.5 to 96.1 ± 0.9%; P < 0.01; n = 10 each) after 20 min.


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Fig. 8.   Effects of nifedipine (top) and removal of extracellular Ca2+ (middle) on baseline tension in intrapulmonary arterial segments from normoxic (A) and chronically hypoxic (B) rats. Bottom: average percentage of original baseline tension under control conditions, after addition of nifedipine (n = 3 vessels from 3 rats for normoxia and 10 vessels from 10 rats for chronic hypoxia), and after removal of extracellular Ca2+ (n = 3 vessels from 3 rats for normoxia and 4 vessels from 4 rats for chronic hypoxia). * Significant difference from control value, P < 0.05. § Significant difference from nifedipine value (P < 0.01).

Effect of Nifedipine on KCl-Induced Tension

To determine the effect of L-type Ca2+ channel blockade on the contraction induced by KCl, tension was measured in rat intrapulmonary arterial segments from normoxic and chronically hypoxic rats during exposure to KCl (80 mM). Maximum tension generated in response to KCl was not different in rings from normoxic and chronically hypoxic rats (1.07 ± 0.04 and 1.17 ± 0.05 g, respectively). In intrapulmonary arterial rings from both normoxic and chronically hypoxic rats, the maximum tension generated in response to KCl was significantly reduced in the presence of nifedipine by 78.4 ± 5.2 (n = 6) and 85.4 ± 3.4% (n = 6), respectively (Fig. 9).


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Fig. 9.   Composite data indicating the effect of nifedipine on tension generated in response to KCl in intrapulmonary arterial segments from normoxic (A) and chronically hypoxic (B) rats. C: average percent inhibition of maximum tension by nifedipine in arterial segments from normoxic and chronically hypoxic rats (n = 6 arteries each).

Effect of Nifedipine and Extracellular Ca2+ Removal on ET-1-Induced Tension

Because pretreatment with nifedipine or perfusion with a Ca2+-free extracellular solution completely prevented any change in [Ca2+]i on exposure to ET-1, the effect of L-type Ca2+ channel blockade and removal of extracellular Ca2+ on the tension generated in response to ET-1 was examined. The maximum tension generated in response to ET-1 appeared to be greater in intrapulmonary arterial ring segments from chronically hypoxic rats (n = 9) than in rings from normoxic rats (n = 6), although this difference did not reach significance (94.9 ± 12.8 and 68.2 ± 9.8%, respectively, of maximum KCl tension; P = 0.078). In arterial segments from normoxic rats, nifedipine reduced the maximum tension generated in response to ET-1 by 45.5 ± 9.1% (n = 3), whereas removal of extracellular Ca2+ reduced maximum tension by 63.6 ± 5.9% (n = 3). Nifedipine inhibited maximum ET-1-induced tension by 12.2 ± 7.1% in arteries from chronically hypoxic rats (n = 9; Fig. 10), whereas removal of extracellular Ca2+ reduced the maximum tension generated in response to ET-1 to 48.5 ± 8.5% of the maximum tension generated in the presence of extracellular Ca2+ (n = 4).


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Fig. 10.   Average maximum tension generated in intrapulmonary arterial rings from normoxic (A) and chronically hypoxic (B) rats in response to ET-1 in the absence and presence of nifedipine (10-6 M; n = 3 rats for normoxia and 9 rats for chronic hypoxia; top) and with and without extracellular Ca2+ (n = 3 rats for normoxia and 4 rats for chronic hypoxia; middle). Bottom: percent inhibition of maximum ET-1-induced tension in arterial segments with nifedipine and after removal of extracellular Ca2+. * Significant difference from normoxic value, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we found that chronic exposure to hypoxia resulted in an elevation in resting [Ca2+]i in PASMCs. Although the elevation in resting [Ca2+]i required an influx of Ca2+ from extracellular sources, exposure to nifedipine, which prevented the KCl- and ET-1-induced increases in [Ca2+]i, had no effect on resting [Ca2+]i, suggesting that elevated resting [Ca2+]i in PASMCs from chronically hypoxic rats is maintained via mechanisms independent of L-type Ca2+ channel activation. Exposure to CH also reduced the ability of ET-1 to increase [Ca2+]i. The small rise in [Ca2+]i observed in response to ET-1 in PASMCs from chronically hypoxic rats was due entirely to Ca2+ influx through L-type Ca2+ channels because no change in [Ca2+]i was observed in response to ET-1 in PASMCs pretreated with nifedipine or bathed in Ca2+-free PSS. Although blockade of L-type Ca2+ channels abolished the change in [Ca2+]i induced by ET-1, the ability of ET-1 to contract pulmonary vascular smooth muscle in the presence of nifedipine was only slightly reduced, even though removal of extracellular Ca2+ caused a significant reduction in ET-1-induced tension.

Several studies (39, 43, 46) have confirmed that exposure to CH causes pulmonary vascular smooth muscle cell depolarization, a phenomenon initially reported by Suzuki and Twarog (47). Furthermore, pulmonary vascular contraction in response to acute reductions in oxygen tension has been shown to be associated with depolarization, L-type Ca2+ channel activation, and Ca2+ influx (3, 8, 9, 16, 27). Based on these findings, it seemed likely that sustained hypoxic vasoconstriction in the pulmonary vasculature may result from increased [Ca2+]i secondary to activation of L-type Ca2+ channels resulting from depolarization. Indeed, we found that the resting [Ca2+]i in PASMCs isolated from chronically hypoxic rats was significantly greater than that measured in PASMCs obtained from their normoxic counterparts. We further demonstrated that removal of extracellular Ca2+ reduced the resting [Ca2+]i to levels similar to those observed in PASMCs from normoxic rats. However, the results from our study also indicate that although dependent on Ca2+ influx, maintenance of resting [Ca2+]i in PASMCs from chronically hypoxic rats did not require activation of L-type Ca2+ channels because an antagonist of these channels had no effect on the resting [Ca2+]i. The concentration of nifedipine used was sufficient to completely block the change in [Ca2+]i induced by KCl as well as the KCl-induced increase in tension in arterial segments; thus the lack of reduction in resting Ca2+ by nifedipine cannot be attributed to incomplete channel blockade.

Similar to the results obtained from an examination of resting [Ca2+]i, resting tension in intrapulmonary arterial segments from chronically hypoxic rats was reduced only slightly with the addition of nifedipine but was markedly reduced when bathing the arterial segments in Ca2+-free extracellular solution. Neither nifedipine nor removal of extracellular Ca2+ had an effect on baseline tension in arteries from normoxic rats. These results suggest that the elevation in [Ca2+]i during CH is functionally coupled to active contraction under resting conditions in this preparation and provide further evidence that Ca2+ influx but not L-type Ca2+ channel activation is important in the physiological responses of the pulmonary vasculature to CH.

Our findings stand in contrast to the results observed in studies (8, 9) that examined the effect of L-type Ca2+ channel blockade on the increase in [Ca2+]i observed in response to acute reductions in oxygen tension. Furthermore, the increase in [Ca2+]i was maintained after removal of the hypoxic stimulus, suggesting that it was not due to vasoconstrictive factors released from the endothelium or other cell types. Rather, our results indicate that Ca2+ homeostasis was altered during chronic exposure to hypoxia by a mechanism not requiring activation of L-type Ca2+ channels. Another subtype of voltage-dependent Ca2+ channels has been identified in vascular smooth muscle. T-type Ca2+ channels are activated only from very negative holding potentials, however, and inactivate rapidly at potentials at or near those observed in our cells (23, 36). Based on their voltage dependence and the passive electrical properties of our PASMCs (resting membrane potential of -20 mV), it is unlikely that T-type Ca2+ channels would have been active under resting conditions in our cells. Because depolarization can alter the activation of Ca2+ exchange pathways other than L-type Ca2+ channels, including nonselective cation channels and the Na+/Ca2+ exchanger (18, 28, 31), further experiments will be necessary to evaluate the contributions of these transporters to the maintenance of PASMC resting [Ca2+]i during CH.

ET-1 gene expression and protein levels are markedly increased during chronic exposure to hypoxia (7, 10, 12), and ET receptor antagonists prevent and partially reverse the development of chronic hypoxic pulmonary hypertension (5, 7, 10). These findings strongly suggest an important role for ET-1 in the pathogenesis of hypoxic pulmonary hypertension. In vivo, PASMCs are continuously exposed to ET-1, and several studies (11, 25, 29, 39) suggest that pulmonary arteries from chronically hypoxic animals exhibit hyperreactivity to ET-1. In PASMCs from normoxic rats, ET-1 caused a biphasic change in [Ca2+]i similar to that previously reported by our laboratory (44). In contrast, exposure of PASMCs from chronically hypoxic rats to ET-1 resulted in only a small sustained increase in [Ca2+]i, with a markedly reduced transient peak in [Ca2+]i. We (44) previously demonstrated that the biphasic rise in [Ca2+]i in response to ET-1 in normoxic PASMCs is due to a complex signaling pathway involving 1) Ca2+ influx initiated by activation of L-type Ca2+ channels; 2) Ca2+ release from caffeine-sensitive, ryanodine-gated intracellular Ca2+ stores induced by Ca2+ influx (Ca2+-induced Ca2+ release); and 3) Ca2+ release from caffeine-insensitive, inositol 1,4,5-trisphosphate-gated stores secondary to inositol 1,4,5-trisphosphate production as a result of phospholipase C activation. In PASMCs from normoxic rats, both nifedipine and the removal of extracellular Ca2+ reduced the peak change in [Ca2+]i induced by ET-1 and eliminated the sustained phase, whereas in PASMCs from chronically hypoxic rats, the ET-1-induced rise in [Ca2+]i was abolished in the presence of nifedipine or after the removal of extracellular Ca2+. These results suggest that mechanisms activating Ca2+ release from intracellular stores in response to ET-1 are no longer operative in these cells after exposure to CH. To ensure that Ca2+ store depletion did not account for the lack of a transient peak in [Ca2+]i in response to ET-1, PASMCs from chronically hypoxic rats were exposed to caffeine to initiate Ca2+ release. The change in [Ca2+]i induced by caffeine in PASMCs from chronically hypoxic rats was similar in magnitude to that observed in PASMCs from normoxic rats, confirming that Ca2+ stores and Ca2+ release mechanisms were intact.

The ET-1-induced activation of L-type Ca2+ channels in PASMCs from chronically hypoxic rats does not appear to result from depolarization because ET-1 had no effect on the membrane potential in these cells. This is consistent with our previous findings that under normoxic conditions ET-1-induced depolarization of PASMCs is due, in part, to inhibition of KV channels (42) and that after exposure to CH, the effect of ET-1 on KV channels is markedly reduced (43). The activation of the L-type Ca2+ channels by ET-1 may instead be due to the ability of ET-1 to increase the open probability of Ca2+ channels independent of membrane potential because at a given holding potential, Ca2+ current in coronary arterial smooth muscle cells was markedly enhanced in the presence of ET-1 (14). Because the membrane potential in PASMCs from chronically hypoxic rats is significantly depolarized to a range where L-type Ca2+ channels may be activated, application of ET-1 may be able to induce Ca2+ influx through these channels in the absence of a change in membrane potential. Further experiments will be required to verify this possibility.

Measurement of isometric tension generated in response to ET-1 indicates that despite a markedly reduced rise in [Ca2+]i, the contractile response to ET-1 was still present. Although several previous studies (11, 25, 29, 39) demonstrated hyperreactivity in response to ET-1 after prolonged exposure to hypoxia, the maximum tension generated in response to ET-1 normalized to either maximum tension induced by KCl (which was not altered by CH) or to tissue wet weight (Shimoda, Sham, Shimoda, and Sylvester, unpublished observations) was not significantly different in endothelium-denuded intrapulmonary arterial segments from normoxic and chronically hypoxic rats. In pulmonary arterial smooth muscle from normoxic rats, blockade of L-type Ca2+ channels with nifedipine, which attenuated the ET-1-induced increase in [Ca2+]i, reduced the maximum tension induced by ET-1 by ~50% and removal of extracellular Ca2+ reduced the maximum tension generated by ET-1 by >60%. In contrast, although the ET-1-induced increase in [Ca2+]i was abolished in PASMCs from chronically hypoxic rats treated with nifedipine, the maximum tension generated in response to ET-1 was reduced by only 25%, suggesting that in pulmonary vascular smooth muscle chronically exposed to low oxygen, the primary mechanism by which ET-1 causes contraction is largely independent of a change in [Ca2+]i. However, given the fact that removal of extracellular Ca2+, which reduced resting [Ca2+]i, decreased the maximum ET-1-induced tension by >50%, it would appear that the elevation in resting [Ca2+]i is important in the generation of tension. One possible explanation for this observation is that ET-1 has been demonstrated to enhance the Ca2+ sensitivity of the contractile apparatus in vascular smooth muscle (14, 37). If ET-1 alters the Ca2+ sensitivity of the contractile apparatus, the elevation in resting [Ca2+]i alone may be sufficient to trigger generation of tension, resulting in contraction that does not require an additional change in [Ca2+]i on application of ET-1.

The signal transduction pathways responsible for ET-1-induced Ca2+-independent contraction are currently unknown but may involve protein kinase C-dependent activation of mitogen-activated protein kinase (17), which then phosphorylates the thin filament-associated contractile regulatory protein calponin (32). Unphosphorylated calponin binds to actin, inhibiting myosin Mg-ATPase; phosphorylation of calponin causes its release from the actin filament and allows cycling of cross bridges and development of tension (49). ET-1 has also been shown to induce phosphorylation of calponin (32), lending support to this theory. Other investigators (1, 2, 37) have proposed mechanisms involving protein kinase activation of myosin light chain kinase or inactivation of myosin light chain phosphatase. Further experiments will be necessary to confirm that ET-1 increases Ca2+ sensitivity in PASMCs from chronically hypoxic rats and to determine the signal transduction pathways involved in this process.

The mechanism by which CH alters Ca2+ homeostasis and ET-1 Ca2+ signaling is not clear. ET-1 receptor density changes with exposure to CH (24). Because contraction was maintained in response to ET-1, a simple reduction in receptor density cannot account for the reduced ability of ET-1 to increase [Ca2+]i. However, because the exact signal transduction pathways for each receptor subtype have not been completely elucidated, a shift in receptor subtype cannot be ruled out. Furthermore, the pulmonary vascular wall may be composed of multiple PASMC phenotypes with distinctly different growth responses to hypoxia (13). Based on our experiments, it is impossible to determine whether the changes in Ca2+ homeostasis and ET-1 Ca2+ signaling observed after CH are due to alterations in a single subtype of PASMCs or reflect the growth of a new phenotype. Other possible explanations for the loss of the Ca2+ release mechanism in response to ET-1 in the PASMCs from chronically hypoxic rats include desensitization of the ryanodine receptor by elevated resting [Ca2+]i or a loss of localization between the ryanodine receptor and the L-type Ca2+ channels. These possibilities need to be tested in future experiments.

In summary, our results indicate that after exposure to CH, Ca2+ influx through pathways not involving L-type Ca2+ channels leads to an elevated resting [Ca2+]i and maintenance of active tension and imply that there is an uncoupling of Ca2+ contraction mechanisms in response to ET-1 in the pulmonary vasculature. In vivo, elevated [Ca2+]i is associated with both pulmonary vascular contraction and proliferation (9, 22, 30), and contraction in response to elevated ET-1 levels may contribute to the sustained vasoconstriction observed during CH. Our findings that neither resting [Ca2+]i nor ET-1-induced contraction was dependent on L-type Ca2+ channel activation in lungs from chronically hypoxic rats are consistent with in vivo data indicating that L-type Ca2+ channel antagonists cannot prevent the development of hypertension secondary to CH (20, 34, 38) and that acute administration of vasodilators (19, 21, 26, 36) but not of Ca2+ channel antagonists (6, 20, 45) reduces pulmonary arterial pressure in patients with CH due to chronic obstructive pulmonary disease. Future experiments will be necessary to elucidate the exact mechanisms by which CH alters Ca2+ homeostasis and ET-1 signaling in the pulmonary vasculature.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51912 (to J. T. Sylvester) and HL-52652 (to J. S. K. Sham) and American Heart Association Scientist Development Grant 9930255N (to L. A. Shimoda).


    FOOTNOTES

Address for reprint requests and other correspondence: L. A. Shimoda, Division of Pulmonary and Critical Care Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: shimodal{at}jhmi.edu).

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

Received 21 June 1999; accepted in final form 7 June 2000.


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