Departments of 1 Medicine and 2 Physiology, Wayne State University School of Medicine and John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201
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ABSTRACT |
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Hypotonic swelling increases the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC). The source of this Ca2+ is not clear. To study the source of increase in [Ca2+]i in response to hypotonic swelling, we measured [Ca2+]i in fura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a 40.7-nM increase in [Ca2+]i that was not inhibited by EGTA but was inhibited by 1 µM thapsigargin. Prior depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores with vasopressin did not inhibit the increase in [Ca2+]i in response to hypotonic swelling. Exposure of 45Ca2+-loaded intracellular stores to hypotonic swelling in permeabilized VSMC produced an increase in 45Ca2+ efflux, which was inhibited by 1 µM thapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or 25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release of Ca2+ from the intracellular stores from a novel site distinct from the IP3-, ryanodine-, and nicotinic acid adenine dinucleotide phosphate-sensitive stores.
calcium; vascular smooth muscle cells; inositol 1,4,5-trisphosphate; ryanodine
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INTRODUCTION |
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INTRACELLULAR CALCIUM CONCENTRATION ([Ca2+]i) can increase in two ways in vascular smooth muscle cells (VSMC): 1) influx of Ca2+ through Ca2+ channels in the plasma membrane and 2) release of Ca2+ from the intracellular Ca2+ stores (1, 2, 28). Ca2+ is released from the intracellular stores via two known channels in VSMC. One is sensitive to inositol 1,4,5-trisphosphate (IP3), and the other is sensitive to ryanodine (17). The sensitivity of ryanodine channels varies greatly in different types of VSMC but is generally low. Ryanodine receptors are not present in all smooth muscle cell types, and their presence or absence is influenced by cell growth and differentiation status. Some smooth muscle cells, such as myometrium and A7r5 cells, do not express ryanodine receptors (28). A third channel, a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ channel, has been identified in sea urchin eggs, pancreatic acinar cells, and neuronal cells, but has not yet been identified in VSMC (9).
Various mechanical stimuli are known to cause an increase in [Ca2+]i (7, 30, 35). It is commonly believed that the increase in [Ca2+]i in response to mechanical stimulation is due to the influx of Ca2+ through stretch-activated channels in the plasma membrane (14, 33, 34). This belief is based on studies demonstrating that removal of extracellular Ca2+ inhibits the increase in [Ca2+]i induced by mechanical stimulation. However, removal of extracellular Ca2+ promotes Ca2+ efflux and depletes the intracellular Ca2+ stores. Therefore, release of Ca2+ from the intracellular stores in response to mechanical stimulation is not definitively ruled out by these studies. Thus the possibility remains that the increase in [Ca2+]i in response to mechanical stimulation in VSMC may result from the release of Ca2+ from the intracellular stores.
In bovine aortic endothelial cells, the increase in [Ca2+]i in response to mechanical stimulation produced by hypotonic swelling is not due to the opening of Ca2+ channels in the plasma membrane but is due to the release of Ca2+ from the intracellular stores independent of the IP3 and ryanodine channels (16). Therefore, it is possible that the increase in [Ca2+]i induced by hypotonic swelling in VSMC may also result from the release of Ca2+ from the intracellular stores independent of the known channels for Ca2+ release. The purpose of this study is to elucidate the mechanism(s) by which hypotonic swelling increases [Ca2+]i in VSMC.
Hypotonic swelling is a convenient, reproducible, reversible, and widely used model of mechanical stimulation; therefore, we studied cultured A7r5 cells, a VSMC line, and used hypotonic swelling as a model of mechanical stimulation (10, 15, 16, 23, 24, 27).
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MATERIALS AND METHODS |
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Materials. A7r5 cells were obtained from American Type Culture Collection (Rockville, MD). Fura 2 was obtained from Molecular Probes (Eugene, OR). A-23187 was obtained from Calbiochem (San Diego, CA). All other chemicals were purchased from Sigma (St. Louis, MO).
Culture of A7r5 cells. A7r5 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 0.9% nonessential amino acids. Cells were subcultured every 5 to 7 days and used for experiments 5 to 7 days after plating.
Measurement of [Ca2+]i. Confluent A7r5 cells were washed with isotonic solution containing 130 mM NaCl, 5.36 mM KCl, 26 mM HEPES, 0.8 mM MgSO4, 1.8 mM CaCl2, 1 mM NaH2PO4, 10 mM glucose, pH 7.4, and 300 mosmol/kgH2O and briefly treated with 0.25% trypsin and 0.53 mM EDTA to bring them into suspension. Cells were then washed to remove the trypsin and loaded with fura 2 by incubation in isotonic solution containing 2 µM fura 2-AM, 0.02% Pluronic F-127, and 0.1% bovine serum albumin for 2 h at room temperature. The cells were washed and reincubated in isotonic solution at room temperature for 30 min to allow hydrolysis of internalized fura 2-AM. The cells were then washed and kept in isotonic solution. Ca2+ measurements were made in isotonic solution at room temperature with constant stirring to maintain homogeneity of the suspension.
Fluorescence was measured with a spectrofluorimeter (SPEXFLUOROLOG II) at 510 nm with excitation wavelengths of 340 and 380 nm. The spectrofluorimeter has built-in software (DM3000) that converts fluorescence to [Ca2+]i values using the following formula
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Measurement of 45Ca2+ efflux from the intracellular Ca2+ stores in permeabilized cells. Confluent monolayers of cells grown on 12-well plates (Falcon, Lincoln Park, NJ) were permeabilized by incubating them with 25 µg/ml saponin, 125 mM KCl, 25 mM NaCl, 2 mM MgCl2, and 10 mM HEPES, pH 6.9, at room temperature for 10 min. After 10 min, saponin was removed, and 2 µCi/ml 45Ca2+, 3 mM ATP (to facilitate uptake of 45Ca2+ into the intracellular Ca2+ stores), and 1 µM ruthenium red (to prevent loading of 45Ca2+ into the mitochondria) were added for 10 min. No CaCl2 was added to the 45Ca2+ or saponin solutions to facilitate the selective uptake of the radioactive 45Ca2+ into the intracellular Ca2+ stores. The cells were then washed and incubated in isotonic solution that contained 125 mM KCl, 25 mM NaCl, 2 mM MgCl2, 0.2 mM CaCl2, 1 mM EGTA, 10 mM HEPES, pH 6.9, 150 nM free Ca2+, and 300 mosmol/kgH2O to mimic the intracellular milieu. This was collected and replaced with an equal volume of isotonic solution at 30-s intervals. In studies involving hypotonic swelling, cells were incubated with hypotonic solution that contained 35 mM KCl, 25 mM NaCl, 2 mM MgCl2, 0.2 mM CaCl2, 1 mM EGTA, 10 mM HEPES at pH 6.9, 150 nM free Ca2+, and 145 mosmol/kgH2O. The assay was terminated by adding 5 µM A-23187, a Ca2+ ionophore, to release the remaining 45Ca2+ in the intracellular Ca2+ stores. Results are expressed as rate constant (fraction of remaining 45Ca2+ released/30 s) (16).
Statistics. Differences between groups of data were determined using a Tukey-Kramer one-way ANOVA or the paired t-test as appropriate. Data are reported as means ± SE. P < 0.01 was considered statistically significant.
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RESULTS |
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Effect of hypotonic swelling on
[Ca2+]i.
[Ca2+]i increased from 62.8 nM in isotonic solution to 106.3 nM when the cells were placed in
hypotonic solution (Fig.
1A; P < 0.0001, n = 18). The increase in
[Ca2+]i occurred immediately and reached a
peak within 15 s. There was no change in
[Ca2+]i when the cells were left stirring in
isotonic solution in a time-controlled experiment. As the cells were
placed in solutions of progressively decreasing tonicity (253, 222, and
200 mosmol/kgH2O), there was a progressive increase in the
magnitude of increase in [Ca2+]i (Fig.
1B). The increase in [Ca2+]i in
hypotonic solution reverted to baseline when the cells were put back in
isotonic solution (Fig. 1C). Figure 1D depicts a
time-controlled experiment that demonstrates that the increase in
[Ca2+]i is sustained when the cells are
incubated in isotonic solution and then left in hypotonic solution.
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Response to blocking of Ca2+ influx. To study the role of influx of Ca2+ from the extracellular medium, the increase in [Ca2+]i in response to hypotonic solution was studied in the presence of 5.4 mM EGTA. Depletion of extracellular Ca2+ promotes efflux of Ca2+ and gradually depletes the intracellular Ca2+ stores. Hence, in this study, measurement of [Ca2+]i was done 5 s after EGTA was added so that [Ca2+]i was measured before the intracellular Ca2+ stores were depleted.
To verify that EGTA inhibits influx of Ca2+, the increase in [Ca2+]i in response to vasopressin was observed. In the absence of EGTA, there was a biphasic increase in [Ca2+]i in response to vasopressin, consisting of an initial rapid peak [that is known to be due to the release of Ca2+ from the intracellular Ca2+ stores (peak phase)] followed by a smaller sustained release of Ca2+ [that is known to be due to the influx of extracellular Ca2+ (plateau phase)] (38). The plateau phase of response to vasopressin was significantly inhibited after a 5-s preincubation with 5.4 mM EGTA, consistent with EGTA blockade of Ca2+ influx (126.7 ± 12.07 nM in the absence of EGTA and 30.76 ± 4.674 nM in the presence of EGTA) (Fig. 2A).
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Effect of depletion of intracellular Ca2+ stores. The increase in [Ca2+]i in response to hypotonic solution was studied in the presence of 1 µM thapsigargin, a Ca2+-ATPase inhibitor that depletes the intracellular Ca2+ stores by inhibiting the active uptake of Ca2+ into the storage compartments (36). Depletion of intracellular Ca2+ stores causes secondary influx of Ca2+ from the extracellular medium. To block this secondary influx of Ca2+, 5.4 mM EGTA was added to thapsigargin-treated cells before the addition of hypotonic solution or vasopressin.
Figure 3A depicts the change in [Ca2+]i when the cells are first incubated in isotonic solution and then in 10 nM vasopressin in the absence and presence of a 30-min preincubation with 1 µM thapsigargin. The increase in [Ca2+]i in response to vasopressin was completely inhibited by thapsigargin. This indicates that thapsigargin indeed depletes intracellular Ca2+ stores in these cells.
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Effect of depletion of IP3 stores.
When cells were stimulated with 10 nM vasopressin twice at an interval
of 30 s, there was no further increase in
[Ca2+]i in response to the second stimulation
with vasopressin, indicating that vasopressin-sensitive
(IP3) stores were depleted at that time (Fig.
4A). When the cells were
stimulated first with vasopressin and then 30 s later with
hypotonic solution, when vasopressin-sensitive (IP3) stores
were still depleted, [Ca2+]i increased by
50.2 ± 7.11 nM in response to hypotonic solution (Fig.
4B). This increase in response to hypotonic swelling is similar in magnitude to the response to hypotonic swelling in the
absence of prior stimulation with vasopressin. This suggests the
presence of two different stores: one sensitive to IP3 and the other to hypotonic swelling.
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Effect of direct swelling of the intracellular
Ca2+ stores.
To study whether direct swelling of the intracellular Ca2+
stores causes a release of Ca2+,
45Ca2+ efflux from the intracellular
Ca2+ stores was measured in saponin-permeabilized A7r5
cells. As shown in Fig. 5A,
45Ca2+ release was measured at 30-s intervals
in isotonic solution and then in hypotonic solution. The
45Ca2+ rate constant increased by 0.115 ± 0.009 in hypotonic solution. This suggests that direct swelling of the
intracellular Ca2+ stores with hypotonic solution causes a
release of Ca2+.
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Effect of blockade of IP3, ryanodine, and NAADP
channels on 45Ca2+ efflux in
response to hypotonic stimuli.
When 45Ca2+-loaded cells were exposed to 50 µg/ml of heparin, an inhibitor of IP3 channels, the
change in 45Ca2+ rate constant in response to
IP3 was 0.305 ± 0.025 in the absence and
0.0185 ± 0.0221 in the presence of heparin. Thus the
45Ca2+ efflux in response to IP3
was completely blocked in the presence of heparin. However, there was
no inhibition of the response to hypotonic solution in the presence of
heparin (Fig. 6).
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Change in ion concentration. The composition of the isotonic solution differs from that of the hypotonic solution only in that the concentration of KCl is 125 mM in isotonic solution and 35 mM in hypotonic solution. To investigate whether the release of 45Ca2+ in response to hypotonic solution is due to the swelling of the intracellular Ca2+ stores or to the exposure of the cells to a lower concentration of KCl, permeabilized cells were placed in standard isotonic solution and then in an isotonic solution that contained 35 mM KCl and 160 mM sucrose, with all other constituents remaining unchanged.
When the permeabilized cells were placed in isotonic solution and then in an isotonic solution that contained 35 mM KCl and 160 mM sucrose and all other constituents remained unchanged, there was no further increase in the rate of 45Ca2+ efflux (Fig. 7). This indicates that the increase in 45Ca2+ efflux in response to hypotonic solution is not due to a change in the concentration of KCl.
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DISCUSSION |
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These data demonstrate that hypotonic swelling results in a release of Ca2+ from the intracellular Ca2+ stores from a novel site independent of the IP3-, ryanodine-, and NAADP-sensitive stores. Our studies show that hypotonic swelling results in an increase in [Ca2+]i that is reversible, reproducible, and proportional to the degree of hypotonicity. This increase in [Ca2+]i was not blocked by inhibiting the influx of Ca2+ but was inhibited by depletion of the intracellular Ca2+ stores. Depletion of the IP3-sensitive stores did not affect the release of [Ca2+]i in response to hypotonic solution, indicating that the increase in [Ca2+]i was independent of the IP3-sensitive stores. There was an increase in 45Ca2+ efflux in response to hypotonic swelling despite the presence of inhibitors of the IP3, ryanodine, and NAADP channels. This further supports the existence of a yet unidentified novel channel that leads to a release of Ca2+ from the intracellular Ca2+ stores in response to mechanical stimulation produced by hypotonic swelling.
These results differ from previously reported results that show that the release of Ca2+ in response to mechanical stimulation is either due to influx of Ca2+ or due to both influx and release of Ca2+ from the intracellular Ca2+ stores (7, 35). In these studies, cells were preincubated for 10-60 min with EGTA. However, this duration of incubation with EGTA promotes Ca2+ efflux and depletes the intracellular Ca2+ stores as well. Therefore, these studies do not definitively exclude that the release of Ca2+ in response to mechanical stimulation is from the intracellular Ca2+ stores. In our study, measurement of Ca2+ in response to hypotonic swelling or vasopressin was done 5 s after EGTA was added so that the [Ca2+]i was measured when Ca2+ influx was inhibited but before the intracellular Ca2+ stores were depleted.
Our studies indicate that hypotonic swelling causes a 44-nM increase in [Ca2+]i. It has been shown that rises in [Ca2+]i of this magnitude increase the myogenic tone (19, 22). Thus hypotonic swelling can produce physiologically significant increases in [Ca2+]i.
The magnitude of increase in [Ca2+]i in response to hypotonic swelling may appear less than that which is produced by agonists such as vasopressin. However, in our studies we have used supraphysiological levels of vasopressin (10 nM vasopressin). This was done to deplete the vasopressin-sensitive stores (Fig. 4, A and B). The response to more physiological levels of vasopressin (1.5-6 pmol) is closer to that in response to hypotonic swelling (3). Also, response to other vasoagonists in the physiological concentration range is 40 nM. Hence, the increase in [Ca2+]i of 44 nM in response to hypotonic swelling may be physiologically significant.
Because our data demonstrate that the increase in [Ca2+]i in response to hypotonic swelling is due to a release from intracellular Ca2+ stores, which is distinct from the IP3-, ryanodine-, and NAADP-sensitive stores, it is possible that there is a yet unidentified mediator of Ca2+ release that is released in response to hypotonic swelling.
Conversely, it is possible that no messenger is needed for the release of Ca2+ from the intracellular Ca2+ stores in response to hypotonic swelling. For example, when cells swell in response to hypotonic swelling, it leads to stretching of the plasma membrane. It is possible that the cytoskeletal network, which is linked to both the plasma membrane and the sarcoplasmic reticulum, the physiologically important Ca2+ store, transmits mechanical force from the surface of the cells to the intracellular Ca2+ stores without a chemical mediator and results in a direct release of Ca2+ (11, 20, 37). The role of the cytoskeleton in release of Ca2+ needs to be further elucidated.
Furthermore, during hypotonic swelling, influx of water dilutes the cytoplasm and thus the intracellular Ca2+ stores are directly exposed to decreased tonicity of the cytoplasm (24). It has been shown that the endoplasmic reticulum, the physiologically important intracellular Ca2+ reservoir, exhibits hypotonic swelling (4). Therefore, it is possible that direct exposure of the intracellular Ca2+ stores to hypotonicity causes swelling of the intracellular Ca2+ stores, resulting in release of Ca2+ from the intracellular Ca2+ stores. Hence, in the hypotonic model of mechanical stimulation, the cytoskeletal involvement may not be required for the release of Ca2+ from the intracellular Ca2+ stores. However, during mechanical stimulation produced by direct stretch, the cytoskeleton may play a role in the transmission of mechanical force from the surface of the cells to the intracellular Ca2+ stores.
To study the direct effect of swelling of the intracellular Ca2+ stores, saponin-permeabilized cells were studied. Saponin selectively permeabilizes the plasma membrane and not the membranes of the intracellular organelles so that when saponin-permeabilized cells are incubated in hypotonic medium, osmotic forces are eliminated across the plasma membrane but not in the membranes limiting the intracellular Ca2+ stores (16). Hence, when saponin-permeabilized cells are incubated in hypotonic medium, it causes direct swelling of the intracellular Ca2+ stores.
The cells are permeabilized and hence the bathing fluid mimics the intracellular milieu. Therefore, the buffers used in these experiments have a high KCl concentration. Because the plasma membranes of the cells are permeabilized, the voltage-sensitive Ca2+ channels in the plasma membrane are not activated in the presence of the high KCl concentration.
The studies in permeabilized cells demonstrate that the release of 45Ca2+ in response to hypotonic swelling is not from the IP3-, ryanodine-, and NAADP-sensitive channels and is independent of the change in ion concentration in the medium. However, with our experimental design, we cannot rule out a direct independent effect of lower osmolality rather than swelling of the intracellular Ca2+ stores as an underlying cause for 45Ca2+ release.
Various methods have been devised to produce mechanical stimulation in
vitro including hypotonic swelling, stretching of Silastic membranes,
extrusion of buffer from a pipette, magnetic forces, flow-induced shear
stress, and application of suction with a pipette. These various
mechanical stimuli are not physiologically equivalent. For instance,
hypotonic swelling causes stretching of the plasma membrane due to
forces from within the cells, whereas direct stretch applies external
force on the cells. Also, hypotonic swelling and direct stretch
activate different ion channels in the plasma membrane. Hypotonic
swelling predominantly activates Cl channels (12,
39). However, hypotonic swelling also activates Ca2+-activated K+ channels in rabbit coronary
VSMC and stretch-activated cation channels in brain capillaries
(21, 29). On the other hand, direct stretch predominantly
causes opening of nonselective, Gd3+-sensitive cation
channels (6, 26, 32). However, application of direct
stretch also activates Ca2+-activated K+
channels in mesenteric and pulmonary artery smooth muscle cells and
Cl
channels in isolated human arterial myocytes (8,
18, 31). Although mechanical stimulation produced by hypotonic
swelling and direct stretch may not be entirely equivalent, hypotonic
swelling is a widely used, convenient, and reproducible model of
mechanical stimulation (10, 15, 16, 23, 24, 27).
Therefore, we chose to use hypotonic swelling as a model of mechanical stimulation.
Our studies in permeabilized cells are in agreement with those reported by Missiaen et al. (24), which demonstrate that the increase in 45Ca2+ efflux in response to hypotonic solution is independent of IP3 and ryanodine channels in permeabilized A7r5 cells. In a previous study, it was shown that in bovine aortic endothelial cells, hypotonic swelling causes a release of Ca2+ from the intracellular Ca2+ stores independent of the IP3-, ryanodine-, and NAADP-sensitive stores (16). Niggel et al. (25) showed that mechanical stimulation of C6 glioma cells by magnetic forces caused a release of Ca2+ from the intracellular Ca2+ stores independent of the IP3 and ryanodine channels. This further strengthens the evidence for a mechanosensitive Ca2+ store.
In summary, our studies show that mechanical stimulation produced by hypotonic swelling causes a release of Ca2+ from the intracellular Ca2+ stores from a novel site independent of the IP3-, ryanodine-, and NAADP-sensitive stores. Because VSMC are subjected to various mechanical stimuli such as shear stress and pulsatile stretch in vivo, this increase in [Ca2+]i in response to mechanical stimulation may play a physiologically important role (5).
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ACKNOWLEDGEMENTS |
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We thank Dr. James Sowers for support and guidance during this work.
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FOOTNOTES |
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This research was supported by funds from the Merit Review Entry Program of the Department of Veterans Affairs, the National Kidney Foundation of Michigan, and a Seed Money Award from the Department of Internal Medicine, Wayne State University.
A portion of this work has been published in abstract form (16a).
Address for reprint requests and other correspondence: M. J. Mohanty, HPB, Suite 908, 4160 John R. Road, Detroit, MI 48201 (E-mail: jenam{at}intmed.wayne.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 17 August 2000; accepted in final form 26 March 2001.
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