Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21224
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ABSTRACT |
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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
(1010 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
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INTRODUCTION |
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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- (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.
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METHODS |
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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 × (RMembrane 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 10Contribution 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
(105 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 (107
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 M) 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 (10Data 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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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
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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
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Role of Intracellular Ca2+ Release
Because removal of extracellular Ca2+ reduced but did not abolish the
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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 (107 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
[Ca2+]peak in response to ET-1
was abolished (P < 0.005; Fig. 4)
in the presence of thapsigargin; hence only the
[Ca2+]plat remained. The finding
that the
[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
[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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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
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DISCUSSION |
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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 1010 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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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