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(Hypertension. 1997;29:1322-1328.)
© 1997 American Heart Association, Inc.

Articles

Alterations in Calcium Stores in Aortic Myocytes From Spontaneously Hypertensive Rats

Steyner de F. Côrtes; Virgínia Soares Lemos; ; Jean-Claude Stoclet

From Laboratoire de Pharmacologie et Physiopathologie Cellulaires, Université Louis Pasteur de Strasbourg, URA CNRS 600, Faculté de Pharmacie, Illkirch, France.

Correspondence to J.-C. Stoclet, Laboratoire de Pharmacologie et Physiopathologie Cellulaires, Université Louis Pasteur de Strasbourg, URA CNRS 600, Faculté de Pharmacie, BP 24.74, route du Rhin, F-67041 Illkirch, France.

	Abstract

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Abstract The aim of the present work was to further characterize intracellular calcium stores released by angiotensin II (Ang II) in spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) vascular smooth muscle cells (VSMCs) and to study their alterations associated with proliferation. Intracellular Ca²⁺ concentration was monitored by image analysis in aortic myocytes loaded with fura 2. In the presence of extracellular Ca²⁺, sensitivity to Ang II in proliferating VSMCs was not different in the two strains, but it increased 10-fold in confluent VSMCs from SHR compared with those from WKY. In Ca²⁺-free medium, Ca²⁺ release induced by thapsigargin (10 µmol/L) was significantly greater (about twofold) in SHR than WKY, in both proliferating and confluent cultures, with responses during proliferation being 0.7-fold smaller. Responses to Ang II were abolished after exposure of the cells to thapsigargin. In proliferating cultures, ryanodine (10 µmol/L) did not modify the rises in intracellular Ca²⁺ concentration induced by Ang II in VSMCs from both strains. Conversely, in confluent cultures, ryanodine reduced Ang II (100 nmol/L)–induced Ca²⁺ release to the same level as in proliferating cultures, and it suppressed the difference between SHR and WKY. These results show that the ryanodine-sensitive Ca²⁺ release induced by Ang II is enhanced in VSMCs from SHR at confluence and is impaired dur-ing proliferation. Thus, they suggest that differences in Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum may participate in increased responsiveness of VSMCs to Ang II in SHR and in phenotypic modulation of vascular myocytes during proliferation.

Key Words: ion transport • rats, inbred WKY • ryanodine • angiotensin II • intracellular • cell proliferation • rats, spontaneously hypertensive

	Introduction

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Defects in calcium handling in arterial smooth muscle cells are among the candidate cellular mechanisms for the multifactorial pathogenesis of hypertension.^{1 2 3 4 5} [Ca²⁺]_i is elevated in various cells from patients with hypertension and from SHR. Because [Ca²⁺]_i controls contraction, its elevation may account for the increased responses of VSMCs to vasoconstrictor stimuli⁶ and maintenance of myogenic tone^{2 3 4 5 7} that characterize hypertension. Recently, it was suggested that alterations in Ca²⁺-dependent signal transduction in SHR might also explain the increased VSMC growth observed in this strain.⁸

The above abnormalities were seen in arteries^{1 2 3} and primary or early passaged cultures of VSMCs^{5 7 8} from SHR. In these VSMCs, the responses produced by Ang II, including contraction,⁹ an increase in [Ca²⁺]_i,^{5 10} and proliferation,¹¹ were found to be augmented. This enhanced responsiveness has been attributed to increased Ang II receptor density,¹² enhanced activation of phospholipases C and D,^{13 14} and altered calcium storage mechanisms.¹⁵

In previous work,¹⁶ we observed that enhanced increases in [Ca²⁺]_i elicited by Ang II in confluent and postconfluent VSMC cultures from SHR compared with those from WKY disappeared in proliferating cultures. In addition, Ang II–and ionomycin-releasable calcium stores were markedly impaired in proliferating VSMC cultures, whereas Ang II binding and inositol 1,4,5-trisphosphate (IP₃) production in response to Ang II were unchanged.^{16 17} These results led to the hypothesis that alterations in intracellular calcium stores were involved both in the enhanced responses to Ang II of VSMCs from SHR and in the decreased responses to Ang II associated with proliferation. To further investigate this hypothesis, we characterized calcium stores in aortic myocytes from SHR and WKY in single-passaged cultures using the sarcoplasmic reticular Ca²⁺-ATPase inhibitor thapsigargin¹⁸ and ryanodine, which acts on the CICR channel.¹⁹ With an image-analysis technique, [Ca²⁺]_i was monitored in single myocytes loaded with fura 2. The results suggest that alterations in ryanodine-sensitive Ca²⁺ release are involved both in the enhanced responses to Ang II of aortic myocytes from SHR and in decreased responses to Ang II during proliferation.

	Methods

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Materials
Hanks' balanced salt solution (HBSS), Ham's F-10, and Eagle's minimum essential medium (MEM) culture media were from Eurobio, collagenase from Boehringer-Mannheim, elastase from Biosys, fetal calf serum and HEPES from GIBCO, and glutamine and EGTA from Merck. Ang II, thapsigargin, caffeine, ascorbic acid, proline, and bovine serum albumin were from Sigma Chemical Co. Fura 2–acetoxymethyl ester (fura 2-AM) was from Molecular Probes and ryanodine from Latoxan.

Cell Isolation and Culture
Male 9- to 10 week-old SHR and WKY, bred in our Institute from progenitors provided by Iffa-Credo (St Germain-sur-l'Arbresle, France), were used in this study. Systolic pressure was determined in some conscious, restrained rats by the tail-cuff plethysmographic method (PE-300, Narco Biosystems Inc). Mean systolic pressure was significantly higher (P<.001) in SHR (189±6 mm Hg, n=16) than WKY (124±8 mm Hg, n=15). VSMCs were obtained by enzymatic digestion of aortic media and prepared according to Corriu et al.¹⁷ Briefly, the aortas were dissected, and the adventitia was stripped off mechanically. They were longitudinally opened, and the endothelium was removed. The tissue was then minced into small pieces, which were incubated in HBSS containing collagenase (87.5 U/mL per aorta) and elastase (4 U/mL per aorta) for 120 minutes at 37°C. The resultant cell suspension was centrifuged and resuspended in culture medium (MEM with 10% fetal calf serum, supplemented with 0.05 mmol/L ascorbic acid, 0.01 mmol/L proline, and 2 mmol/L glutamine). The supernatant containing freshly dissociated VSMCs was plated at a density of 10⁴ cells/cm². The cultures were passaged once with trypsin (0.05%). The cells were counted and diluted to the desired plating density (as described above). The cells were kept in a humidified, 37°C incubator gassed with 95% air/5% CO₂. The medium was changed every 3 days. Proliferating VSMCs in log growth phase were obtained 3 to 5 days later and confluent VSMCs between 8 and 10 days.

[Ca²⁺]_i Measurements
[Ca²⁺]_i levels were determined in single cells as previously described.²⁰ Briefly, VSMC suspensions obtained from primary cultures (as indicated above) were plated into culture dishes in which the bottom was replaced by a thin (0.07-mm) glass coverslip. They were incubated with fura 2-AM (5 µmol/L) for 30 minutes at 37°C. Cells were then placed on a Diaphot microscope (Nikon) and washed continuously for 30 minutes in balanced salt solution (BSS) (mmol/L: NaCl 135, KCl 5, MgCl₂ 1, CaCl₂ 1.25, glucose 10, HEPES 20; pH 7.4). Fluorescent measurements were made at 30°C. Fura 2 fluorescence was excited alternatively at 340 and 380 nm, with emitted fluorescence being filtered by a 490- to 530-nm band-pass filter (Nikon). Drugs were applied by pressure ejection from fine-tipped glass pipettes placed 200- to 300-µm from the target cells. Some experiments were performed in Ca²⁺-free BSS (mmol/L: NaCl 135, KCl 5, MgCl₂ 1, glucose 10, HEPES 20, and EGTA 1; pH 7.4). [Ca²⁺]_i values were calculated as previously described.²¹ Cells were considered responsive to a given drug when the [Ca²⁺]_i increased by at least 50 nmol/L on exposure to the drug.

Data Analysis
Results are expressed as mean±SEM; n denotes the number of studied cells. MANOVA was used for statistical analysis of concentration-response curves to Ang II. Statistical analysis for single concentrations of Ang II, thapsigargin, and ryanodine was done with the Mann-Whitney test. Values of P<.05 were considered significant. The concentration producing 50% of response (EC₅₀) was calculated by nonlinear curve fitting, and the area under the curve was measured with GraphPad software and expressed in arbitrary units.

	Results

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Basal [Ca²⁺]_i Values
Basal [Ca²⁺]_i values found in proliferating and con-fluent cultures, in normal and Ca²⁺-free BSS, are given in Table 1. There was no significant difference between the groups, except in confluent cultures from SHR in normal BSS, in which [Ca²⁺]_i was significantly increased. These results are in accordance with those of our previous work.¹⁶

View this table: [in this window] [in a new window]   Table 1. Basal [Ca2+]i in Vascular Smooth Muscle Cells From WKY and SHR in the Presence and Absence of Extracellular Ca2+

Effect of Ang II in Proliferating and Confluent Cultures From WKY and SHR
In single-cell studies, it was possible to quantify the proportion of responsive VSMCs (rise in [Ca²⁺]_i 50 nmol/L) and amplitude of [Ca²⁺]_i elevations in responsive cells as a function of Ang II concentration (Fig 1). In proliferating cultures, no significant difference was found between the two strains with respect to the proportion of responsive cells (Fig 1A) and the amplitude of their response (Fig 1C), which both increased with Ang II concentration. However, even at the highest Ang II concentration (1000 nmol/L), the proportion of responsive cells did not reach 100%, and their responses remained weak during proliferation.

View larger version (29K): [in this window] [in a new window]   Figure 1. Effect of Ang II on [Ca2+]i in proliferating and confluent cultures from WKY and SHR. A and B show the proportion of responsive cells to the different Ang II concentrations in proliferating (A) and confluent (B) cultures from WKY (open bars) and SHR (solid bars). C and D represent the rise in [Ca2+]i (peak-basal values) induced by Ang II in proliferating (C) and confluent (D) cultures from WKY () and SHR (). Results are expressed as mean±SEM of at least 20 cells. Basal [Ca2+]i values were not significantly different from those shown in Table 1. **P<.01 for comparison of entire concentration-effect curves.

At all tested Ang II concentrations, there was a marked increase in both the proportion of responsive cells (MANOVA: P<.01) and amplitude of responses (MANOVA: P<.05 for WKY and P<.001 for SHR) in confluent compared with proliferating cultures. At confluence, almost all VSMCs from both strains responded to Ang II at concentrations greater than or equal to 10 nmol/L, whereas only half of studied VSMCs were responsive at 1 nmol/L (Fig 1B). The concentration-response curve to Ang II was shifted to the left in confluent VSMCs from SHR compared with WKY (Fig 1D), the response to all studied concentrations being significantly higher in VSMCs from SHR than in those from WKY (MANOVA: P<.01). Although it seems evident that the EC₅₀ value of Ang II was lower in SHR cultures, a precise determination of this parameter was not possible in WKY VSMCs (as it is not certain that the maximal response was reached with 1000 nmol/L Ang II in this case and as larger concentrations produced lower responses due to rapid desensitization). However, using the response at 1000 nmol/L as the maximal response, the apparent EC₅₀ values to Ang II were 80 nmol/L for WKY and 7 nmol/L for SHR.

Effect of Thapsigargin
As illustrated in Fig 2, thapsigargin (10 µmol/L) induced a gradual and sustained response in proliferating and confluent cultures from both WKY and SHR in Ca²⁺-free BSS. The responses to thapsigargin reached their maximum after approximately 2 minutes and gradually declined close to the basal values approximately 5 minutes after the beginning of the response. In both culture phases, incubation of VSMCs with thapsigargin during 10 minutes abolished the response to subsequent infusion of Ang II (100 nmol/L) in the two strains (not shown), showing that thapsigargin depleted the Ang II–releasable calcium store. Furthermore, the area under the curve of responses to thapsigargin was significantly higher (P<.001) than that for Ang II in proliferating and confluent cultures from the two strains (Fig 2 and Table 2).

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of thapsigargin (10 µmol/L) on proliferating and confluent cultures from WKY and SHR. A and E show representative curves obtained with thapsigargin in proliferating cultures from WKY (A) and SHR (E). B and F show representative curves to thapsigargin in confluent cultures from WKY (B) and SHR (F). C and G represent mean±SEM of increases in [Ca2+]i (peak-basal values) induced by thapsigargin in proliferating (open columns) and confluent (shaded columns) cultures from WKY (C) and SHR (G) obtained in at least 15 cells. D and H represent mean±SEM of the area under the [Ca2+]i curve in proliferating (open columns) and confluent (shaded columns) cultures from WKY (D) and SHR (H) obtained from 10 responsive cells. *P<.05, **P<.01 vs values in proliferating cultures; P<.05, P<.01, P<.001 vs values from WKY cultures, using Mann-Whitney test. Basal [Ca2+]i values were not significantly different from those shown in Table 1. BSS indicates balanced salt solution.

View this table: [in this window] [in a new window]   Table 2. Effect of Ryanodine (10 µmol/L) on Angiotensin II (100 nmol/L)–Induced Ca2+ Release in Responsive Aortic Myocytes in Confluent and Proliferating Cultures From WKY and SHR

In proliferating cultures, the proportion of responsive cells was 74±3% and 76±5% for WKY and SHR, respectively. During this culture phase, the peak [Ca²⁺]_i values and the area under the curve of the responses to thapsigargin were both significantly higher in VSMCs from SHR than in those from WKY (Fig 2).

In confluent cultures, almost all cells responded to thapsigargin (97±2% and 98±3% for WKY and SHR, respectively). Both the proportion of responsive cells (P<.01) and the responses to thapsigargin (peak effect and area under the curve) were significantly higher than observed during proliferation (Fig 2). During this culture phase, the responses to thapsigargin were significantly higher in VSMCs from SHR than in those from WKY (Fig 2).

Effect of Ryanodine
The effect of ryanodine on Ang II–induced Ca²⁺ release was studied in Ca²⁺-free medium. Exposure of VSMCs to 10 µmol/L ryanodine did not modify resting [Ca²⁺]_i values, whatever the strain and the proliferation state (not shown). In these experiments, cells were exposed to 100 nmol/L Ang II injected 10 minutes after addition of 10 µmol/L ryanodine to the bath. We chose the Ang II concentration to induce significant responses in both proliferating and confluent cultures from the two strains so that we could observe differences between confluent VSMCs cultures from SHR and WKY (Fig 1).

Fig 3 shows representative traces of responses to Ang II in Ca²⁺-free medium in the presence or absence of ryanodine. The mean values of peak responses and of the area under the curve are given in Fig 4 and Table 2, respectively. In the absence of extracellular Ca²⁺, Ang II elicited transient responses whose peaks were not different from the peak values obtained in normal Ca²⁺ BSS (Fig 1). The proportion of responsive cells was also the same in the absence and presence of extracellular Ca²⁺ (not shown).

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of ryanodine (Ryan, 10 µmol/L) on elevations in [Ca2+]i induced by Ang II (100 nmol/L) in proliferating and confluent cultures from WKY and SHR. Top panels, Responses obtained in proliferating (A and B) and confluent (C and D) cultures from WKY; bottom panels, responses in proliferating (E and F) and confluent (G and H) cultures from SHR. Ryanodine was applied 10 minutes before Ang II and it did not induce any change in resting [Ca2+]i level by itself. BSS indicates balanced salt solution.

View larger version (22K): [in this window] [in a new window]   Figure 4. Effect of ryanodine (10 µmol/L) on mean peak responses to Ang II (100 nmol/L) in proliferating and confluent cultures from WKY and SHR. Results are mean±SEM of rises in [Ca2+]i (peak-basal values) elicited by Ang II in the absence (open columns) and presence (solid columns) of ryanodine in proliferating (A and C) and confluent (B and D) cultures from WKY (A and B) and SHR (C and D) of at least 20 cells. Basal [Ca2+]i values are not significantly different from those shown in Table 1 in the absence or presence of ryanodine. **P<.01, ***P<.001 vs control values; P<.001 vs WKY values.

In proliferating cultures, ryanodine had no significant effect on the responses to Ang II in both strains (Figs 3 and 4). By contrast, in confluent cultures, ryanodine decreased Ang II–induced rises in [Ca²⁺]_i by 45±1% and 65±4% in WKY and SHR VSMCs, respectively (Figs 3 and 4, Table 2). The ryanodine-resistant component of the Ang II–induced Ca²⁺ transients was not different in responsive cells, whatever the strain or proliferative state (Figs 3 and 4, Table 2).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings reported here are that ryanodine (10 µmol/L) abolished the enhanced release of Ca²⁺ ions elicited by Ang II in SHR aortic myocytes and ryanodine-sensitive stores were absent in proliferating cultures. In addition, the results confirm that Ang II released Ca²⁺ from thapsigargin-sensitive calcium stores and that these stores were increased in SHR compared with WKY myocytes. Finally, it was shown that the thapsigargin-sensitive stores were impaired in proliferating cultures.

VSMCs after a single passage were used here to avoid the dedifferentiation that occurs after several pas-sages.^{22 23} The expression of markers of VSMC differentiation, including smooth muscle -actin and the more specific myosin heavy chain, was previously reported in primary and single-passaged confluent cultures of rat aortic myocytes.²² VSMCs also display contractile responses in these conditions, whereas these responses disappear after the second passage.²⁴ It was previously shown in our laboratory that smooth muscle -actin expression and increases in [Ca²⁺]_i elicited by Ang II, vasopressin, and ATP are impaired during the growing phase of cultures but are restored at confluence in primary and single-passaged cultures.^{16 17} In addition, the Ang II–induced [Ca²⁺]_i increases in confluent single-passaged cultures are not different from those obtained in postconfluent primary cultures.¹⁶ Finally, in confluent cultures, SHR VSMCs were found to be more sensitive to the contractile effects of Ang II and norepinephrine⁹ and to the [Ca²⁺]_i-elevating action of Ang II (this study) than WKY VSMCs. Altogether, these results provide evidence that in the chosen experimental conditions, confluent VSMCs expressed structural and functional characteristics representative of those expressed in the original vessel and of their alterations in arteries from SHR, despite the fact that VSMC cultures are not entirely growth-arrested at confluence, especially in SHR.²⁵

We previously reported that alterations in responses to Ang II of rat myocytes during the proliferative phase of cultures¹⁷ and in confluent cultures from SHR¹⁶ were not due to alterations in receptor subtype (Ang II type 1 in all cases) or affinity for the peptide. Impairment of the Ang II–induced rise in [Ca²⁺]_i during proliferation was also not due to impaired coupling to phospholipase C and IP₃ production,¹⁷ but it was associated with a marked decrease in Ang II and ionomycin-releasable calcium stores.¹⁶ Conversely, enhanced responses to Ang II found in confluent and postconfluent cultures from SHR were associated with enhanced Ang II– and ionomycin-releasable calcium stores.¹⁷ We therefore hypothesized that modifications in calcium stores might be involved in the altered responses to Ang II in SHR.

The present study gives further support to this hypothesis and provides further information on the stores. However, this does not preclude the possibility that an increase in Ang II type 1 receptor density, as previously found in postconfluent SHR cultures,¹⁶ could also contribute to the 10-fold increase in sensitivity to Ang II found here, together with other described alterations in transduction mechanisms^{13 14} and in calcium entry pathways.^{7 26 27 28 29}

The single-cell analysis reported here shows that in both strains, proliferation was associated not only with a decreased amplitude of the response to Ang II and decreased proportion of responsive cells but also with a marked heterogeneity of individual VSMCs with respect to Ang II sensitivity. This is shown by the finding that in proliferating cultures, the proportion of responsive cells increased from 25% to 75% when the Ang II concentration was raised from 10 to 1000 nmol/L. By contrast, the proportion of responsive cells increased from 50% to practically 100% when the Ang II concentration was raised from 1 to 10 nmol/L in confluent cultures. This heterogeneity in proliferating cultures may be linked to myocytes being in different phases of the cell cycle. An influence of cell cycle on the proportion of VSMCs responsive to Ang II was previously suggested by Masuo et al,³⁰ who first reported growth-associated alterations in VSMC [Ca²⁺]_i responses elicited by various mechanisms. The heterogeneity in sensitivity to a low concentration of Ang II (1 nmol/L) remaining when cultures reached confluence might also be associated with the presence of a minor proportion of proliferating cells, as a result of the well-known deficient contact inhibition of VSMC growth. Since binding studies could not be performed on single cells, VSMC heterogeneity in receptor density or affinity cannot be entirely excluded. However, the proportion of VSMCs responsive to thapsigargin declined in proliferating cultures to the same extent as the proportion of cells responsive to the largest concentration of Ang II (about 75%), suggesting that impairment of sarcoplasmic reticular calcium stores was involved in the heterogeneity of [Ca²⁺]_i responses of VSMCs associated with proliferation.

The results obtained here with thapsigargin are consistent with those previously obtained with ionomycin in identical experimental conditions.¹⁶ Ionomycin-sensitive stores are larger than thapsigargin-sensitive stores, as ionomycin is able to release Ca²⁺ from all intracellular compartments, whereas thapsigargin selectively inhibits sarcoplasmic reticular Ca²⁺-ATPase. In proliferating cultures, 100% of the cells responded to ionomycin, whereas only 75% were responsive to thapsigargin or Ang II (1000 nmol/L). However, calcium stores released by the two agents were both larger in confluent than in proliferating VSMCs, and they were enhanced in SHR compared with WKY independent of the proliferative state.

In VSMCs, Ang II type 1 receptors are believed to cause Ca²⁺ release from the sarcoplasmic reticulum via an IP₃-mediated mechanism.³¹ The present results are consistent with this view, as Ang II–induced Ca²⁺ release was abolished after store depletion by thapsigargin. Ang II–releasable calcium stores probably represent only a part of thapsigargin-sensitive stores, as the area under the [Ca²⁺]_i curve (representing total Ca²⁺ release) was smaller after exposure to Ang II than to thapsigargin (P<.001). Furthermore, in proliferating cultures, thapsigargin-released Ca²⁺ was larger in responsive VSMCs from SHR than those from WKY, whereas Ang II–released Ca²⁺ was not.

Ryanodine at 10 µmol/L binds to CICR channels and blocks them open.³² It thereby causes Ca²⁺ release and depletion of stores responsible for CICR. In the absence of extracellular Ca²⁺, ryanodine produced no increase in resting [Ca²⁺]_i, probably because released Ca²⁺ was rapidly extruded, but the proportion of ryanodine-impaired Ca²⁺ release over total release induced by Ang II shows the relative participation of CICR. However, in proliferating cultures, no ryanodine-inhibited component was found in Ang II–responsive cells, suggesting that CICR did not participate in the response to Ang II in this case. Differential loss of various Ca²⁺ release mechanisms may correspond to the heterogeneity of myocytes in proliferating cultures with respect to cell cycle. It was recently reported that expression of voltage-dependent calcium channels is cell cycle–dependent in rat aortic myocytes in primary culture.³³ Further experiments are needed to clarify the role of the different Ca²⁺-release and other Ca²⁺-handling mechanisms in VSMCs during the cell cycle and the consequence of their alterations in SHR. However, increased responses to thapsigargin found here in VSMCs from SHR are consistent with the observation that VSMCs from SHR are more sensitive to inhibition of proliferation by thapsigargin than VSMCs from WKY.³⁴ It was recently reported that activation by Ang II of mitogen-activated protein kinase, a critical signal transduction element in proliferation, is more dependent on [Ca²⁺]_i in quiescent VSMCs from SHR than from WKY.⁸ As rises in [Ca²⁺]_i produced by Ang II were found to be increased in confluent VSMC cultures from SHR but not in proliferating ones, the alterations in Ca²⁺ release mechanisms found here in SHR VSMCs might be involved in the transition from the quiescent to the proliferating state rather than during the mitotic cycle itself.

Higher sarcoplasmic reticular Ca²⁺-ATPase 2a and 2b isoform expression previously reported in VSMCs from SHR¹⁵ may perhaps account for the increased responses to thapsigargin and enhanced calcium stores found here. However, ryanodine abolished the alterations in Ang II–induced Ca²⁺ release found here in SHR VSMCs and with the proliferating state, suggesting that enhanced (in the SHR) or impaired (in proliferating VSMCs) CICR accounted for the major part of the alterations.

Neylon et al³⁵ demonstrated the presence of all three members of the ryanodine receptor gene family in aortic VSMCs from WKY. In VSMCs, the presence or absence of ryanodine receptor has been shown to be influenced by cell growth and differentiation.^{32 36} Several investigators have described that caffeine- or ryanodine-sensitive stores are larger in aortic segments from SHR than in those from WKY.^{10 37} However, it was recently suggested that the sarcoplasmic reticular calcium release channels in aortic VSMCs from SHR were not abnormal with respect to activation by caffeine and ryanodine.³⁸ The results obtained in the present work show the participation of ryanodine-sensitive calcium stores in Ang II responses in confluent but not in proliferating cultures from both strains. This difference between proliferating and confluent cultures could be explained by differential expression of ryanodine receptors depending on the growth state of cells, as suggested in previous work.^{32 36} In addition, the present results show that in confluent cultures, the participation of ryanodine-sensitive calcium stores in Ang II–induced responses was markedly increased in VSMCs from SHR compared with those from WKY. This observation might explain the increased responses to Ang II in VSMCs from SHR, as ryanodine suppressed the difference between the two strains. In agreement with this hypothesis, increased buffering capacity of sarcoplasmic reticulum was recently suggested in femoral arteries from SHR to explain the enhanced contractile effect of ryanodine.³⁹ This supports the view that the present findings may be relevant to smooth muscle contractile function in SHR arteries. Together with previously described enhanced calcium entry,¹ augmented Ca²⁺ release from sarcoplasmic reticulum may contribute to increased responsiveness of vascular smooth muscle to vasoconstrictor stimuli in SHR.

In conclusion, the decreased responses to Ang II of VSMCs in proliferating cultures might be due to disappearance of the ryanodine-sensitive calcium stores involved in CICR. In confluent cultures of VSMCs from both SHR and WKY, CICR participates importantly in Ang II–induced [Ca²⁺]_i rises, but this mechanism is markedly enhanced in SHR, resulting in increased [Ca²⁺]_i responses.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II [Ca2+]i = intracellular Ca2+ concentration CICR = Ca2+-induced Ca2+ release SHR = spontaneously hypertensive rat(s) VSMC = vascular smooth muscle cell WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This study was partially supported by grants from the French Ministry of Research (ACC SV9-95) and from "La Fondation Recherche et Partage." S. de F. Côrtes and V. Soares Lemos were supported by Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil). The authors wish to express their appreciation to Dr Kenneth Takeda for critical review of the manuscript.

Received August 15, 1996; first decision September 19, 1996; accepted November 14, 1996.

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