Mobilization of intracellular Ca2+ by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

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

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelin-1 (ET-1) increases intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca2+ mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca2+ response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10-10 to 10-8 M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca2+]i. At 10-8 M, ET-1 caused a large, transient increase in [Ca2+]i (>1 µM) followed by a sustained elevation in [Ca2+]i (<200 nM). The ET-1-induced increase in [Ca2+]i was attenuated (<80%) by extracellular Ca2+ removal; by verapamil, a voltage-gated Ca2+-channel antagonist; and by ryanodine, an inhibitor of Ca2+ release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca2+]i, but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca2+]i indicated that depolarization preceded the rise in [Ca2+]i. These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca2+ influx through voltage-gated Ca2+ channels and Ca2+ release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.

calcium-induced calcium release; vascular smooth muscle; potassium chloride; L-type calcium channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIN (ET)-1, the most potent vasoconstrictor known to date, is a 21-amino acid peptide secreted by the vascular endothelium. It has been shown that ET-1 is synthesized in pulmonary arterial endothelial cells, and its secretion can be induced by several stimuli including transforming growth factor-beta (22), thrombin (35), hypoxia (23), and shear stress (24). After its release from the endothelium, ET-1 contracts smooth muscle by binding to either ETA or ETB receptors, both of which are abundantly present in the pulmonary vasculature (26). Exogenous ET-1 constricts the pulmonary vasculature of several species including dog (2), cat (27, 40), guinea pig (18), rat (5, 25, 30, 41), rabbit (31), and human (33).

The mechanisms mediating the pressor effects of ET-1 in the pulmonary circulation remain in debate. ET-1 depolarizes rat intrapulmonary arterial smooth muscle cells (PASMCs) (1, 37, 41) via inhibition of voltage-gated K+ (KV) channels (37, 41) and/or activation of Cl- channels (19, 37), which may enhance Ca2+ influx through voltage-gated Ca2+ channels and contribute to the ET-1-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in the lung. This is supported by observations that Ca2+-channel antagonists inhibit ET-1-induced pulmonary vasoconstriction (18, 31, 41). However, in some studies, blockade of voltage-gated Ca2+ channels had no inhibitory effect on the pulmonary vascular contractile response to ET-1, whereas removal of extracellular Ca2+ abolished the response, suggesting activation of other Ca2+ influx pathways (2, 25). Direct measurement of intracellular Ca2+ has only served to further complicate the picture. Two internal stores, ryanodine (caffeine) sensitive and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] sensitive, have been identified in vascular smooth muscle (3, 43), both of which have been demonstrated to be activated by ET-1, resulting in Ca2+ oscillations (1, 19). A biphasic rise in Ca2+ due to both release and influx (42) has also been described.

Understanding the mechanisms by which ET-1 causes constriction in the pulmonary vasculature is important because recent studies (6, 7, 10, 11) implicate ET-1 as an important modulator of pulmonary vascular tone and suggest that ET-1 may be involved in the pathogenesis of hypoxic pulmonary hypertension. We hypothesized that ET-1 would increase [Ca2+]i in pulmonary vascular smooth muscle by a mechanism involving membrane depolarization, Ca2+ influx through voltage-dependent Ca2+ channels, and Ca2+ release from intracellular stores. In this study, we used Ca2+ microfluorescence and whole cell patch-clamp techniques in rat PASMCs to 1) quantify ET-1-induced increases in [Ca2+]i, 2) determine the relative contributions of Ca2+ influx and Ca2+ release from caffeine-sensitive and -insensitive stores to the increase in [Ca2+]i induced by ET-1, and 3) determine the temporal relationship between membrane depolarization and the rise in [Ca2+]i.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and Culture of PASMCs

Single PASMCs were obtained as previously described (41). 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 removed en bloc and transferred to a petri dish of HEPES-buffered salt solution (HBS) containing (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 smooth muscle cell basal medium (Clonetics) supplemented with 0.5% fetal calf serum, streptomycin, and penicillin for 24-48 h.

Intracellular Ca2+ 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 gassed with 21% O2-5% CO2 and maintained 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 onto the PASMCs under examination via a ×40 fluorescence oil-immersion objective (Fluor 40, Nikon). Light emitted from the cell was returned through the objective and split with a dichroic mirror, and fluorescence was 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). 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 the data were collected on-line 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. (16): [Ca2+]i = Kd × B × (R - Rmin)/(R - Rmax), where and 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. F405,background and F495,background were determined by measuring the fluorescence values after the addition of 10 mM Mn2+ to quench the dye.

Membrane Potential Measurements

Membrane potential measurements were made with patch-clamp techniques in the whole cell configuration with an Axopatch-ID amplifier (Axon Instruments, Foster City, CA). Cells were exposed to ET-1 via a rapid-exchange system with a multibarrel pipette connected to a common orifice positioned 100-200 µm from the myocyte studied. Complete solution exchange was achieved in <1 s. Pipette potential and capacitance were canceled, and access resistance was electronically compensated. Current-clamp protocols were applied by using pClamp software (Axon Instruments). Data were filtered at 5 kHz, digitized with a Digidata 1200 analog-to-digital converter (Axon Instruments), and analyzed with pClamp software. The membrane potential was recorded under current-clamp mode (current = 0), and cells with unstable or depolarized membrane potentials were discarded.

Experimental Protocols

Effects of ET-1 or KCl on [Ca2+]i. Baseline fluorescence was measured for 15 min before the cells were exposed to the agonists. Cells with an unstable [Ca2+]i were discarded. The effect of ET-1 and KCl (100 mM) on [Ca2+]i was determined by measuring fluorescence during 10 min of exposure to 10-10, 10-9, or 10-8 M ET-1 or during perfusion with 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.

Contribution from extra- and intracellular Ca2+. To determine the effect of Ca2+-channel antagonists, Ca2+ removal, and depletion of intracellular Ca2+ stores on the change in [Ca2+]i induced by ET-1, the PASMCs were equilibrated and stable fluorescence was monitored for 15 min. PASMCs were then perfused with PSS containing verapamil (10-5 M), a voltage-gated Ca2+-channel antagonist; ryanodine (10-5 M), an inhibitor of caffeine-sensitive Ca2+ release; or thapsigargin (10-7 M), a Ca2+-ATPase inhibitor that depletes intracellular stores 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 for 15 min before exposure to ET-1 (10-8 M) for 10 min. ET-1 and the antagonists were then washed out, and fluorescence was measured for an additional 5 min.

Ca2+ release from caffeine-sensitive stores. To determine whether treatment of PASMCs with thapsigargin depleted both the ryanodine (caffeine)-sensitive and -insensitive intracellular Ca2+ stores, PASMCs were exposed to caffeine (10 mM) for 30 s to induce Ca2+ release. After washout of caffeine for 5 min, PASMCs were pretreated with thapsigargin (10-7 M) for 10 min before reexposure to caffeine. A rapid-exchange system, as described in Membrane Potential Measurements, was used to introduce caffeine to the PASMCs under study.

Exposure to ET-1 during simultaneous measurement of membrane potential and [Ca2+]i. To measure membrane potential and [Ca2+]i simultaneously, 100 µM Indo 1 (pentapotassium salt) was introduced into the cell through the patch pipette (tip resistance 3-5 MOmega ) into an internal solution containing (in mM) 74 gluconic acid, 40 KCl, 6 NaCl, 5 MgATP, 2 disodium phosphocreatine, and 10 HEPES. Membrane potential and fluorescence were measured for 0.5 min before, 3 min during, and 1.5 min after exposure to ET-1 (10-8 M).

Drugs and Chemicals

ET-1 was obtained from American Peptides (Sunnyvale, CA). Indo 1-AM dye was obtained from Molecular Probes (Eugene, OR). Ryanodine, caffeine, and thapsigargin were obtained from Peptides International (Louisville, KY). Verapamil and all other chemicals were obtained from Sigma (St. Louis, MO). Stock solutions of ET-1 (10-5 M; distilled water), ryanodine (10-2 M; DMSO), and thapsigargin (10-2 M; DMSO) were divided into aliquots and stored at 0°C until used. All other drugs were made fresh in distilled water on the day of the experiment.

Data Analysis

Data are expressed as means ± SE; n is the number of cells tested. Baseline and sustained phases 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 the response. Data were compared with Student's t-test (paired or unpaired as applicable). A P value of <0.05 was accepted as significant. All experiments were conducted in cells from a minimum of three different animals.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of ET-1 on [Ca2+]i

In PASMCs, resting [Ca2+]i under control conditions was 109.5 ± 15.2 nM (n = 20). Exposure to ET-1 (10-8 M) caused a biphasic rise in [Ca2+]i, consisting of a rapid, transient, large-magnitude peak followed by a decline to steady-state levels that remained elevated above baseline and persisted throughout exposure to ET-1 (Fig. 1). The magnitude of the transient phase was concentration dependent, with the peak rise in [Ca2+]i (Delta [Ca2+]peak) increasing from 96.8 ± 62.8 to 273 ± 109.4 and 814.9 ± 225.2 nM for 10-10, 10-9, and 10-8 M ET-1, respectively. The elevation in [Ca2+]i during the sustained phase (Delta [Ca2+]plat) was similar in magnitude for all concentrations tested (58.8 ± 32.0, 55.2 ± 11, and 69.4 ± 17.7 nM for 10-10, 10-9, and 10-8 M ET-1, respectively). The increase in [Ca2+]i began 92.8 ± 11.8, 64.5 ± 7.6, and 68.6 ± 3.6 s after exposure to 10-10, 10-9, and 10-8 M ET-1, respectively, and the peak response lasted 69.2 ± 22.2, 102.8 ± 26.4, and 99.2 ± 20.2 s for 10-10, 10-9, and 10-8 M ET-1, respectively. In contrast to the biphasic increase in [Ca2+]i observed in the presence of ET-1, KCl (100 mM) caused only a sustained increase in [Ca2+]i, similar in magnitude to that for the Delta [Ca2+]plat observed in response to ET-1 (105.4 ± 26 nM), with no transient phase. In all cases, washout of the agonist resulted in a gradual return of [Ca2+]i to near baseline levels.


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Fig. 1.   Traces representing change in intracellular Ca2+ concentration ([Ca2+]i) induced by indicated concentrations of endothelin (ET)-1 (B-D) or KCl (A). Traces are averages of all cells tested (n = 7 in D; n = 4 in B and C; n = 5 in A). Bottom: average peak (left) and sustained (right) changes in [Ca2+]i (Delta [Ca2+]i) in response to ET-1 and KCl. Letters correspond to traces in A-D. * Significant difference from D, P < 0.05.

Role of Extracellular Ca2+

To determine whether the ET-1-induced increase in [Ca2+]i required Ca2+ influx through voltage-dependent (L-type) channels, ET-1 (10-8 M) was applied 5 min after exposure to verapamil (10-5 M), an L-type Ca2+-channel antagonist, was begun. Exposure to verapamil reduced baseline [Ca2+]i from 108.8 ± 53.5 to 65.6 ± 17.7 nM (n = 6), but the decrease was not significant. When PASMCs were exposed to ET-1 (10-8 M) in the presence of verapamil, the Delta [Ca2+]peak was 227 ± 42.4 nM (n = 3), which was significantly reduced compared with the transient [Ca2+]i response induced by ET-1 in the absence of verapamil. In addition, the ET-1-induced Delta [Ca2+]plat was abolished in PASMCs pretreated with verapamil (Fig. 2). To further evaluate the role of extracellular Ca2+ influx to the ET-1-induced rise in [Ca2+]i, ET-1 was applied to cells superfused with Ca2+-free extracellular solution. Removal of extracellular Ca2+ significantly reduced the baseline [Ca2+]i from 141.1 ± 12.5 to 55.3 ± 10.9 nM (P < 0.05; n = 5), reduced the Delta [Ca2+]peak to 228.7 ± 81 nM (P < 0.01; n = 7), and abolished the Delta [Ca2+]plat (P < 0.005), results similar to those observed when ET-1 was applied in the presence of verapamil. After washout of ET-1, restoring Ca2+ to the external solution resulted in a sustained rise in [Ca2+]i.


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Fig. 2.   Left: effect of Ca2+-channel blockade with verapamil (VER; n = 6 cells; top) and of removal of extracellular Ca2+ (n = 5 cells; bottom) on increase in [Ca2+]i induced by ET-1. Traces are averages of all cells tested. Right: average peak (top) and sustained (bottom) Delta [Ca2+]i in response to ET-1 under control conditions, in presence of VER, and after removal of extracellular Ca2+. * Significant difference from control value, P < 0.01.

Role of Intracellular Ca2+ Release

Because removal of extracellular Ca2+ reduced but did not abolish the Delta [Ca2+]peak of the ET-1-induced rise in [Ca2+]i, the contribution of release from the intracellular stores to the change in [Ca2+]i in response to ET-1 was examined. PASMCs were pretreated for 10 min with ryanodine (10-5 M) to inhibit Ca2+ release from ryanodine (caffeine)-sensitive intracellular stores (20). Exposing PASMCs to ryanodine had no effect on baseline [Ca2+]i (n = 9). In the presence of ryanodine, subsequent application of ET-1 (10-8 M) appeared, qualitatively, to induce a Delta [Ca2+]peak; however, this value was not significantly different from the Delta [Ca2+]plat (326.5 ± 160 vs. 140.5 ± 59.3 nM; P = 0.06; n = 5), which was unaffected by pretreatment with ryanodine (Fig. 3). In contrast, pretreatment with ryanodine had no effect on the rise in [Ca2+]i induced by KCl (n = 4; Fig. 3), with the Delta [Ca2+]plat 118 ± 20.1 and 177.3 ± 60.6 nM in the absence and presence, respectively, of ryanodine.


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Fig. 3.   Effect of ryanodine (RYN) on change in [Ca2+]i induced by ET-1 (top) or KCl (middle). Traces are averages of all cells tested with ET-1 (n = 7 for control; n = 5 for RYN) and KCl (n = 5 for control; n = 4 for RYN). Bottom: average Delta [Ca2+]i in response to ET-1 and KCl in absence (control) and presence of RYN. * Significant difference from control value, P < 0.05.

The role of Ca2+ release from Ins(1,4,5)P3-sensitive or caffeine-insensitive intracellular Ca2+ stores was evaluated by pretreating the cells with thapsigargin (10-7 M), which is thought to deplete Ins(1,4,5)P3-sensitive Ca2+ stores by inhibiting Ca2+-ATPase-dependent resequestration (43). Unlike ryanodine, thapsigargin significantly increased baseline [Ca2+]i from 129.3 ± 43.3 to 197.0 ± 48.1 nM (P < 0.05; n = 6). The Delta [Ca2+]peak in response to ET-1 was abolished (P < 0.005; Fig. 4) in the presence of thapsigargin; hence only the Delta [Ca2+]plat remained. The finding that the Delta [Ca2+]peak was abolished by pretreatment with thapsigargin suggested that, in addition to depleting Ins(1,4,5)P3-sensitive stores, ryanodine (caffeine)-sensitive stores might also be depleted. Examining the ability of thapsigargin to inhibit caffeine-induced release of intracellular Ca2+ tested this possibility. Rapid application of 10 mM caffeine via a fast-perfusion system caused a reproducible, rapid, and reversible increase in [Ca2+]i, with an average Delta [Ca2+]peak of 448 ± 169.2 nm (Fig. 5). Pretreatment with thapsigargin markedly attenuated the caffeine-induced rise in [Ca2+]i to 107 ± 66.2 nM (P < 0.05).


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Fig. 4.   Effect of intracellular Ca2+ store depletion with thapsigargin (THAPS; middle) on ET-1-induced increase in [Ca2+]i (top). Traces represent average of all cells tested with ET-1 (n = 7 for control; n = 6 for THAPS). Bottom: average peak and sustained Delta [Ca2+]i in response to ET-1 in presence and absence of THAPS. * Significant difference from control value, P < 0.05.



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Fig. 5.   Effect of THAPS (middle) on ability of caffeine (top) to induce Ca2+ release from intracellular stores. Traces are average of all cells tested (n = 5 for caffeine; n = 7 for THAPS). Bottom: average Delta [Ca2+]i in response to caffeine in absence and presence of THAPS. * Significant difference from control value, P < 0.01.

Temporal Relationship Between ET-1-Induced Changes in Membrane Potential and [Ca2+]i

We (41) previously demonstrated that the effect of ET-1 on membrane potential occurs 14 ± 6 s after application of ET-1. Because the ET-1-induced increase in [Ca2+]i appeared to occur much later, at 68.6 ± 3.6 s after exposure to 10-8 M ET-1, we verified the temporal relationship between the ET-1-induced depolarization and the rise in [Ca2+]i by simultaneously measuring membrane potential and [Ca2+]i under whole cell current-clamp conditions during exposure to ET-1 (10-8 M). Application of ET-1 caused a significant depolarization of the membrane potential from -41.2 ± 4.4 to -16.2 ± 4.1 mV (P < 0.01; n = 3; Fig. 6), which is consistent with previously published values (1, 37, 41) and well below the depolarization induced by KCl (to 8.9 ± 2.3 mV; n = 6). The change in [Ca2+]i was similar to that described in Effect of ET-1 on [Ca2+]i, with Delta [Ca2+]peak = 1,083.8 ± 436.4 nM and Delta [Ca2+]plat = 224.3 ± 83.5 nM. In all cells studied, depolarization preceded the rise in [Ca2+]i by an average of 27.9 ± 18 s (17.0 ± 3.8 s for membrane potential vs. 44.9 ± 17.6 s for rise in [Ca2+]i; n = 3).


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Fig. 6.   Membrane potential (Em) in absence and presence of ET-1 (top) and time to onset for change in Em and [Ca2+]i in response to ET-1 (bottom) in cells where Em and [Ca2+]i were simultaneously recorded. Results are averages from 3 cells. * Significant difference from control value, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, exposing rat PASMCs to ET-1 caused concentration-dependent increases in [Ca2+]i consisting of a transient peak followed by a decline to steady-state levels that remained significantly elevated above baseline during exposure to ET-1 and returned to normal levels after washout of the drug. Several mechanisms appear to participate in the increase in [Ca2+]i induced by ET-1, including Ca2+ influx through voltage-gated Ca2+ channels and release of Ca2+ from both ryanodine- and Ins(1,4,5)P3-sensitive intracellular stores.

The biphasic ET-1-induced increase in [Ca2+]i we observed is consistent with the Ca2+ transients evoked by similar concentrations of ET-1 in other vascular smooth muscle (9, 12, 13, 15, 28, 32, 34, 42) and is qualitatively similar to the contraction induced by ET-1 in isolated perfused lungs (18). The concentration dependence of the peak ET-1-induced rise in [Ca2+]i correlates with studies (5, 30) in isolated pulmonary arteries demonstrating graded contraction in response to ET-1 at concentrations between 10-10 and 10-7 M. It is unclear why the sustained phase of the ET-1-induced Ca2+ transient was not concentration dependent; however, because ET-1-induced inhibition of KV channels increases only 20%, from 10-10 to 10-8 M (41), the difference in activation of voltage-gated Ca2+ channels at the concentrations tested may be slight and not reflected in the global [Ca2+]i.

Short-duration (<3 min) exposure to ET-1 caused oscillations in [Ca2+]i in PASMCs isolated from rat main and intrapulmonary arteries (1, 19); however, ET-1-induced oscillations in [Ca2+]i were not observed in this study. The reason for this difference is unknown because the same species, a similar concentration of agonist, and a similar size pulmonary artery were used. The lack of oscillations cannot be accounted for by sampling error. The previously observed oscillations were ~4 s in duration and occurred ~10 s apart, well within the detection capability of our system (sampling rate = 2 s).

Our data indicate that Ca2+ from both extra- and intracellular sources contributed to the ET-1-induced increase in [Ca2+]i. ET-1 initiated Ca2+ influx through voltage-gated Ca2+ channels because blockade of these channels with verapamil significantly reduced both the peak and sustained increases in [Ca2+]i. Furthermore, compared with verapamil, removal of extracellular Ca2+ caused no additional reduction in the ET-1-induced increase in [Ca2+]i, suggesting that Ca2+ influx occurs entirely through voltage-gated Ca2+ channels. These results contrast with the inability of dihydropyridine Ca2+-channel blockers to prevent the sustained phase of the ET-1-induced Ca2+ transient in systemic vascular smooth muscle cells (13, 34). In light of these findings, it is likely that activation of nonselective cation or receptor-operated Ca2+ channels contributes to the Ca2+ influx and sustained elevated [Ca2+]i observed in systemic vascular smooth muscle, whereas in PASMCs, Ca2+ influx through voltage-gated Ca2+ channels plays a predominant role.

It is intriguing that KCl and ET-1 caused similar sustained elevations in [Ca2+]i despite the more positive membrane potential achieved with KCl. Based solely on membrane depolarization, KCl should cause a greater Ca2+ influx and, therefore, a bigger sustained increase in [Ca2+]i than ET-1. The fact that the sustained increase in [Ca2+]i was the same in response to KCl and ET-1 may be due to an increased Ca2+ current at a given membrane potential in the presence of ET-1 (15), thereby inducing current comparable in magnitude to KCl, albeit at a lower membrane potential.

Because removal of extracellular Ca2+ and pretreatment with verapamil reduced the peak ET-1-induced Ca2+ transient to a similar extent as did pretreatment with ryanodine (>70%), it is likely that the portions of the peak ET-1-induced Ca2+ transient dependent on Ca2+ influx and sensitive to ryanodine are linked through a common pathway, possibly the Ca2+-induced Ca2+-release (CICR) mechanism. Ryanodine receptors contain Ca2+ binding sites (8), allowing increased [Ca2+]i to initiate release from these stores (4). CICR has been demonstrated to occur in systemic vascular smooth muscle (4, 21) and, on the basis of our findings, appears to be operative in the pulmonary vasculature.

Given that ET-1 appears to induce CICR in PASMCs, it is reasonable to expect that Ca2+ influx induced by high extracellular K+ levels would also result in CICR. Unlike ET-1, however, KCl induced only a sustained increase in [Ca2+]i with no transient peak. Furthermore, in the presence of ryanodine, the response to KCl was not significantly different from that observed in untreated PASMCs. These findings suggest that KCl does not cause CICR. The lack of CICR in response to KCl may indicate that CICR requires phosphorylation of the release channel, which enhances ryanodine receptor channel activity in cardiac and skeletal muscle (17, 29, 44). CICR is also critically dependent on the spatial relationship between active voltage-gated Ca2+ channels and ryanodine receptors (39). Enhancement of voltage-gated Ca2+ currents by ET-1 (15), possibly due to the increased open probability of the channels or the availability of otherwise inactive channels closely coupled to ryanodine receptors, may further enhance the CICR process.

Although the transient peak in [Ca2+]i observed in response to ET-1 appears to be heavily dependent on Ca2+ release from ryanodine-sensitive stores, pretreating cells with ryanodine attenuated but did not abolish this response. In contrast, thapsigargin, an inhibitor of Ca2+-ATPase reuptake mechanisms, completely abolished the peak component of the Ca2+ transient, suggesting that the residual Ca2+ peak observed in the presence of ryanodine was due to release from Ins(1,4,5)P3-sensitive intracellular stores. Although ET-1-induced Ca2+ transients are generally attributable to Ca2+ release from Ins(1,4,5)P3-sensitive stores in systemic smooth muscle (28, 32, 45) and, in a previous study (19), in PASMCs, the results from the present study indicate that, in our PASMCs, the contribution from Ins(1,4,5)P3-sensitive stores may be less prominent.

Thapsigargin depletes Ins(1,4,5)P3-sensitive stores in vascular smooth muscle by inhibiting Ca2+ resequestration into the sarcoplasmic reticulum (43). Controversy exists, however, as to whether the ryanodine- and Ins(1,4,5)P3-sensitive stores are distinct and whether thapsigargin specifically depletes the Ins(1,4,5)P3-sensitive stores. In our hands, thapsigargin markedly attenuated the rise in [Ca2+]i associated with exposure to caffeine. In addition, thapsigargin abolished both the ryanodine-sensitive and -insensitive portions of the transient Ca2+ peak, implying that both stores were depleted.

Exposure to ET-1 is often associated with membrane depolarization (1, 38, 41, 45). We (41) previously demonstrated that ET-1 depolarizes PASMCs in part through protein kinase C-dependent inhibition of KV channels. Depolarization induced by ET-1 in this study was similar to that previously reported by our laboratory (41) and others (1, 37), although oscillations in the membrane potential superimposed on the graded depolarization have also been reported. ET-1-induced depolarization was not initiated by activation of Ca2+-activated Cl- channels (1, 19, 38) or inhibition of KV channels secondary to the rise in [Ca2+]i (14, 36) because simultaneous measurement of membrane potential and [Ca2+]i revealed that depolarization preceded the increase in [Ca2+]i in all cells tested. These data suggest that depolarization may be an initiating event in ET-1 signal transduction in PASMCs.

In summary, the results of this study indicate that pulmonary vascular smooth muscle cell contraction in response to ET-1 involves elevation of [Ca2+]i levels through a complex mechanism involving multiple Ca2+ signaling pathways. Our data suggest that membrane depolarization, possibly through protein kinase C-dependent inhibition of KV channels, activates Ca2+ influx through voltage-gated Ca2+ channels, initiating Ca2+ release primarily from ryanodine-sensitive intracellular stores (CICR). Additionally, Ca2+ release from Ins(1,4,5)P3-sensitive stores also contributes to the elevation of [Ca2+]i observed in response to ET-1.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51912 (to J. T. Sylvester), HL-09543 (to L. A. Shimoda), and HL-52652 (to J. S. K. Sham).


    FOOTNOTES

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.

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}welch.jhu.edu).

Received 16 March 1999; accepted in final form 8 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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