AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 277: H834-H841, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 2, H834-H841, August 1999

ANG II-induced Ca2+ increase in smooth muscle cells from SHR is regulated by actin and microtubule networks

Emmanuel Samain, Hèléne Bouillier, Claudine Perret, Michel Safar, and Georges Dagher

Institut National de la Santé et de la Recherche Médicale U337, Faculté Broussais-Hotel Dieu, 75006 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that the cytoskeletal network in vascular smooth muscle cells (VSMC) is critical to the signaling pathways from angiotensin (ANG) II-receptor subtype 1 (AT1) activation to intracellular Ca2+ (Ca2+i) release from internal stores and Ca2+ influx. This was tested in spontaneously hypertensive rats (SHR), in which differences were reported in cultured aortic VSMC Ca2+i regulation and G protein function compared with those in normotensive Wistar-Kyoto (WKY) rats. In cultured aortic VSMC, disorganization of actin filaments with cytochalasin D (2 µmol/l) decreased the ANG II-induced Ca2+i release from internal stores and the ANG II-induced Ca2+ influx in SHR in a reversible fashion, whereas it was without effect in WKY rats. On the other hand, blocking the dynamic state of the microtubule network significantly reduced ANG II-induced Ca2+i release from internal stores but was without effect on Ca2+ influx in either SHR or WKY rats. This study demonstrates for the first time that, in the SHR, actin filaments play a major role in linking AT1-receptor activation to both Ca2+i release mechanisms and capacitative Ca2+ influx. Furthermore, a functionally intact microtubule system is a necessary prerequisite for ANG II-induced Ca2+i release in both strains.

calcium ions; vascular smooth muscle cells; spontaneously hypertensive rat


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CYTOSKELETAL ELEMENTS are thought to play an important role in the segregation of signaling peptides on hormonal stimulation. Recent evidence suggests that signal transduction pathways involving G protein activation are associated with cytoskeletal elements (4-6, 13, 37, 49). Thus disruption of cytoskeletal structures by colchicine or cytochalasin in a variety of cell types alters the generation of second messengers such as inositol 1,4,5-trisphosphate (IP3) (23, 28, 32). On the other hand, a direct link between cytoskeletal structure and cell Ca2+ (Ca2+i) increase has been established. Thus, in platelets or permeabilized hepatocytes, IP3-dependent Ca2+i release requires an intact system of actin and microtubules (5, 18). Release of Ca2+i from intracellular stores is believed to occur by a mechanical link from IP3-sensitive channels at the plasma membranes to ryanodine-sensitive channels in the endoplasmic reticulum, mediated by the cytoskeleton (21). It has also been suggested (22) that actin filaments may be part of the Ca2+ pool itself, releasing bound Ca2+ when the actin filaments depolymerize.

Recently, several studies in vascular smooth muscle cells (VSMC) have suggested that the actin network is in a dynamic state of polymerization and depolymerization and that it is involved in the regulation of different cellular processes. Cytochalasins, known to alter actin polymerization, inhibit the contraction of aortic VSMC (2, 47, 51). This suggests that actin filaments are an integral component of signaling pathways regulating Ca2+ transport. The microtubule network is also in a dynamic state, converting rapidly between assembly and disassembly. This network plays a key role in the activation of Ca2+ channels (16) and IP3-induced Ca2+ release (5), suggesting a link between the microtubule network and Ca2+ transport.

Angiotensin (ANG) II receptors belong to the superfamily of the G protein-coupled receptors, and two subtypes, AT1 and AT2, have been identified. In VSMC, binding of ANG II to AT1 receptors is known to release Ca2+i from internal stores and to increase Ca2+ influx, thus resulting in transient Ca2+i increase. The signaling pathways involved in this event have been characterized, but little is known about the role of cytoskeletal elements in this phenomenon. Recent evidence suggests that the alpha q/alpha 11-subunit of the Gq/11 proteins are associated with both actin and tubulin (10).

In this study we have evaluated whether the integrity of the cytoskeletal network is necessary for the signaling pathways linking AT1 receptors to Ca2+i release from internal stores and Ca2+ influx. We have assessed whether this modulation is related to hypertension, in which a defect in Ca2+i increase induced by ANG II has been associated with an alteration in G protein signaling (9, 20, 35, 40). This alteration was reported to affect the function, but not the expression, of G protein subunits in genetic hypertension (12, 17).

The bulk of experiments suggests that in the spontaneously hypertensive rats (SHR), in contrast to the Wistar-Kyoto (WKY) rats, an intact actin network is required to link AT1 activation to the Ca2+i release mechanism and capacitative Ca2+ influx. Actin may be involved in the spatial interaction between phospholipase C and IP3 receptors. On the other hand, in both strains a functional microtubule network is necessary to link AT1-receptor activation to Ca2+ transport. Some of these results were previously presented in abstract form (36).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. VSMC were isolated from aortas of 5- to 6-wk-old male SHR [mean arterial pressure (MAP) = 136 ± 5 mmHg (mean ± SE), n = 25] and WKY rats (MAP = 98 ± 4 mmHg, n = 20) by enzymatic digestion, as previously described (31). In brief, aortas were incubated for 10 min in a dissecting solution containing DMEM (Eurobio) supplemented with glutamine (2 mmol/l), 0.1% BSA, penicillin (10 U/ml), streptomycin (100 mg/ml), and collagenase (295 U/ml). The adventitia was stripped off mechanically, and the endothelial layer was scraped off gently. The aorta media was then incubated at 37°C for 20 min in the dissecting solution, to which elastase (90 U/ml) and pronase (0.33 mg/ml) were added, and CaCl2 content was reduced to 0.85 mmol/l. The tissue was then mechanically dissected by gentle pipetting through a large-hole Pasteur pipette. The undigested tissue was incubated in a fresh dissecting medium for another 20 min. The overall procedure was repeated twice. At the end of the digestion procedure, CaCl2 was increased progressively in steps of 0.25 mmol/l to reach a final concentration of 1.6 mmol/l.

To obtain secondary cultures, isolated cells were seeded at 1.5-2 × 105 cells/ml into 25-cm2 flasks in DMEM supplemented with 10% FCS (Eurobio), 2 mmol/l of L-glutamine, 25 mmol/l of HEPES, pH 7.4, 10 U/ml of penicillin, and 100 mg/ml of streptomycin at 37°C, 5% CO2 in a humidified incubator. The medium was changed every 48 h. At confluence, secondary cultures were obtained by serial passages after the cells were harvested with 0.5 g/l trypsin and 0.2 g/l EDTA (Sigma) and reseeded in fresh DMEM containing 10% FCS.

Staining of filamentous actin and alpha -tubulin. To assess the effect of cytochalasin D (2 µmol/l) and nocodazole (5 µmol/l) on the organization of the cytoskeleton, the actin and microtubule networks were visualized using specific fluorescent probes. For this purpose, VSMC were grown on glass coverslips. At confluence, cells were made quiescent by incubation for 48 h in an FCS-free medium (0.5%) before the experiment. After being treated for 30 min with a specific agent, the cells were rinsed with Dulbecco's phosphate-buffered saline (PBS, Eurobio) medium with 1 mmol/l CaCl2 and 1 mmol/l MgCl2 and fixed with 3% paraformaldehyde in PBS solution. Cells were washed twice with PBS and incubated for 10 min in PBS supplemented with 50 mmol/l NH4Cl. Cells were then permeabilized by exposure for 5 min to 0.4% Triton X-100 in PBS. After being rinsed with PBS, cells were then incubated in the presence of rhodamine-phalloidin (Sigma) for 45 min at ambient temperature. After three washes with PBS, coverslips were mounted in 50% glycerol. For alpha -tubulin staining, fixed cells were incubated with a primary monoclonal antibody specific to alpha -tubulin (T-9026, Sigma) for 60 min. The primary antibody was visualized by using an FITC-conjugated goat anti-mouse secondary antibody.

Cell Ca2+ measurements. Ca2+i variations were assessed at the single-cell level using fluorescence imagery, as described previously (29). For this purpose, a portion of the cells was seeded on glass coverslips and, at confluence, made quiescent by incubation for 48 h in an FCS-free medium (0.5%) before the experiment. Cells were then loaded with fura 2 by incubation for 30 min at 37°C in a 5 µmol/l solution with the acetoxymethyl ester form of the dye in Na-HEPES solution consisting of (in mmol/l) 140 NaCl, 4.5 KCl, 0.8 MgCl2, 0.8 KH2PO4, 1.0 CaCl2, 5.6 glucose, and 10 HEPES, supplemented at this step with 0.1% BSA. The cells were then washed twice with fresh Na-HEPES solution, and the coverslip was mounted on the stage of a Nikon Diaphot microscope (Nikon, Tokyo, Japan) fitted with a cooled, integrating charge-coupled device (CCD) imaging system (Newcastle Photometric System, Newcastle, UK). The cells were superfused with Na-HEPES solution. Ca2+-free Na-HEPES solution was made without CaCl2 and with the addition of 1 mmol/l EGTA. ANG II, cytochalasin D, nocodazole, vinblastine, and Taxol (paclitaxel) were obtained from Sigma and dissolved in Na-HEPES solution before use. Cytochalasin D, nocodazole, vinblastine, and Taxol were added after cells were loaded with fura 2.

Cells were illuminated alternately at 350 and 380 nm, and the intensity of emitted light at wavelengths >520 nm was measured. Recordings were made from single cells over a 500-ms period at each excitation wavelength. The CCD imaging system allowed simultaneous measurements to be made from <= 16 cells in a field of view. The program displayed one image for 350-nm and another for 380-nm excitation wavelengths. The light intensity for each pixel in an area was summed for each wavelength, the background was subtracted, the ratio of the two values was calculated every 1.1 s, and the result was plotted against time. This ratio was displayed as a function of time for each experimental area. Because calibration procedures are prone to errors and believed by most experts to be unreliable (50), no attempt was made to accurately assess absolute free Ca2+i concentrations. Our study is concerned with relative changes in Ca2+i; therefore, the qualitative changes in Ca2+i are represented throughout by changes in the ratio of the emitted fluorescence at 350 and 380 nm (8).

Ca2+ influx measurement. Ca2+ influx was assessed according to the Ca2+ free-Ca2+ reintroduction protocol. To this end, cells were superfused with Ca2+-free medium supplemented with 1 mmol/l EGTA, and ANG II was added 1 min later. Ca2+ (1 mmol/l) was then reintroduced into the medium after 2 min, and the ensuing second Ca2+i peak (Ca2+ influx) was recorded. In parallel experiments, Ca2+i changes occurring in nonstimulated cells as a consequence of Ca2+ chelation and reintroduction were found to be negligible with a mean of 0.002 ratio units/min.

Assay of ANG II-receptor antagonists. The effect of 100 nmol/l CGP-48933 (Valsatran, Ciba-Geigy) and 100 nmol/l CI-996 (Parke-Davis), two specific AT1 antagonists, on agonist-evoked Ca2+i release was determined. For these experiments, cells from WKY rat and SHR aortas were preincubated for 5 min with one of the antagonists. ANG II (1 µmol/l) was then added and the Ca2+i variation assayed.

Data analysis. All individually shown experiments are representative of several separate experiments: fluorescent stainings are representative of 38 cells studied in 3 separate experiments; Ca2+ tracings are representative of 906 individual cells studied in 75 separate preparations. Comparison of mean values was done by ANOVA with multiple testing according to the Bonferroni method and by Student's unpaired t-test when appropriate. The results (means ± SE) are expressed as a percentage of the control values (ratio of treated cells per that of untreated cells).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of ANG II on cell Ca2+ in the presence of external Ca2+. As previously reported, steady-state values of Ca2+i were significantly different between the two strains (WKY: 60 ± 3 nmol/l, SHR: 95 ± 13 nmol/l, P < 0.05) (9). Exposure of VSMC from either strain to ANG II induced a transient increase in Ca2+i (Fig. 1A). In both strains, the maximal response was observed for concentrations of ANG II >0.5 µmol/l (Fig. 2), as reported in previous studies (9). Subsequently, the effect of ANG II was assayed at 1 µmol/l. The Ca2+i transient elicited by ANG II was characterized by three parameters: 1) the amplitude of Ca2+i release (peak minus basal values; Fig. 1A, b minus a); 2) the slope of Ca2+i increase (Fig. 1A, from a to b), and 3) the total Ca2+i mobilized (area under transient curve; Fig. 1A, a-c). Exposure of the cells to ANG II induced receptor desensitization that lasted for 30 min, as reported previously (1). Thus no repetitive determination of the effect of ANG II was performed in the same cell. ANG II-induced Ca2+i mobilization was not significantly different in cells from the third to the ninth passages in either strain [F = 0.26, P = nonsignificant (NS) with a 2-way ANOVA]. The ANG II-induced Ca2+i increase was greater in SHR than in WKY rats (Table 1). The steady-state value of Ca2+i reached after the addition of ANG II was higher than that before ANG II. An increase of 8.7 ± 0.8% was observed for WKY rats (n = 81, P <=  0.001) and 12.4 ± 1.2% for SHR (n = 79, P <=  0.001). Blockade of L-type Ca2+ channels with nifedipine (1 µmol/l) did not modify significantly the ANG II-induced Ca2+i mobilization in both WKY rats and SHR. The AT1 antagonists CGP-48933 (100 nmol/l) and CI-996 (100 nmol/l) both abolished the effect of ANG II on Ca2+i (>95% inhibition, P <=  0.001 for each compared with control values, results not shown), as previously reported (46).


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  		Fig. 1.   Effect of angiotensin (ANG) II (1 µmol/l) on intracellular Ca2+ (Ca2+i) increase in presence (A) and absence (B) of external Ca2+. Ratios of emission fluorescence (>520 nm) measured at excitation wavelengths of 350 and 380 nm are shown on ordinate. Abscissa shows time course of experiments (in s). A: original record of 1 of >400 experiments shows effect of ANG II in presence of external Ca2+. Three parameters were selected in subsequent analysis of Ca2+i transients: increase in amplitude [peak (b) minus basal (a) values] of Ca2+i released by ANG II; time course of Ca2+i increase (slope of Ca2+i increase from a to b); and amount of Ca2+i mobilized (area under each transient, a-c). B: ANG II (e) induced Ca2+i increase (f) in absence of external Ca2+ (0 Ca2+o). After a return to steady state (g), Ca2+ was reintroduced in medium (h), eliciting an increase in Ca2+ (i); the first rate of this increase was taken to represent Ca2+ influx. The new steady state reached (j) was higher than that observed before addition of ANG II (d).



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  		Fig. 2.   Effect of different concentrations of ANG II on amplitude of Ca2+i increase in cells from spontaneously hypertensive rats (SHR; ) and Wistar-Kyoto rats (WKY; triangle ). Results are expressed as means ± SD of 16-25 cells per concentration of ANG II.


                              
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  		Table 1.   ANG II-induced Ca2+i transient in vascular smooth muscle cells from WKY rats and SHR

Effect of ANG II on Ca2+i in the absence of external Ca2+ and on Ca2+ influx. To assess Ca2+ release from internal stores, cells were superfused with a Ca2+-free Na-HEPES medium and exposed to ANG II for 120 s (Fig. 1B). The Ca2+i transient peaked to lower values (Fig. 1B, f - e) than in the presence of external Ca2+. The response remained higher in SHR than in WKY rats (Table 1). In the absence of external Ca2+, no significant difference in steady-state values of Ca2+i was observed before and after the addition of ANG II in either strain (results not shown).

ANG II-induced Ca2+ influx was estimated from the initial rate of Ca2+i increase on reintroduction of external Ca2+ (Fig. 1B, from h to i). It reached a mean value of 0.0149 ± 0.0087 ratio units/s for WKY rats (n = 80) and 0.0122 ± 0.0092 ratio units/s for SHR (n = 167, P = NS). The blockade of L-type Ca2+ channels with nifedipine (1 µmol/l) did not significantly modify the Ca2+ influx in either strain (results not shown). In the WKY rats, the steady-state Ca2+i reached after reintroduction of external Ca2+ (Fig. 1B, from i to j) was 15.6 ± 0.9% higher than that observed before the addition of ANG II (n = 80), and in the SHR, the increase in baseline was 29 ± 1.7% (n = 338).

To assess the participation of the Na+/Ca2+ exchanger in ANG II-induced Ca2+ influx, cells were exposed to ANG II in Ca2+-free medium in the presence or absence of external Na+ [replaced with N-methyl-glucamine (NMG)]. Ca2+ influx observed on the reintroduction of 1 mmol/l Ca2+ in the solution was not significantly different in NMG-HEPES medium compared with that in Na-HEPES medium in both SHR and WKY rats (WKY: Na-HEPES, 0.0066 ± 0.0004 ratio units/s; NMG-HEPES, 0.0067 ± 0.0007 ratio units/s, n = 109, P = NS; SHR: Na-HEPES, 0.0080 ± 0.0008 ratio units/s; NMG-HEPES, 0.0066 ± 0.0007 ratio units/s, n = 103, P = NS). This suggests that the reverse mode of the Na+/Ca2+ exchanger is negligible, in accordance with findings of previous studies (41). In summary, ANG II-induced Ca2+ release from internal stores is higher in SHR than in WKY rats, whereas no difference could be observed in Ca2+ influx.

Effect of disorganizing the actin network on ANG II-induced Ca2+i increase. A common approach to assessment of the implication of actin filaments in cellular response is to hamper the organization of the network with the use of cytochalasin D at concentrations that do not induce cell shape modification. The immediate addition of cytochalasin D (0.2-2 µmol/l) to cells did not modify the ANG II-induced Ca2+i increase in either strain. Thereafter, cells were incubated at 37°C for 30 min in the presence of increasing concentrations of cytochalasin D (0.2-20 µmol/l). Pretreatment of cells with concentrations of 0.2-2 µmol/l did not modify cell shape, whereas, at concentrations of 5-20 µmol/l, cells from both strains exhibited a collapsed shape.

Pretreatment with cytochalasin D at concentration of 0.2 and 0.5 µmol/l did not modify the ANG II-induced Ca2+i transient in either strain (results not shown). In cells from SHR, pretreatment with cytochalasin D (2 µmol/l) significantly decreased the ANG II-induced Ca2+i transient in the presence of external Ca2+, whereas, in cells from WKY rats, a significant increase was observed (Table 2).

                              
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  		Table 2.   Effect of cytochalasin D and nocodazole on ANG II-induced Ca2+i transient in WKY rats and SHR

In the absence of external Ca2+, pretreatment with cytochalasin D (2 µmol/l) significantly reduced Ca2+i release from internal stores in SHR, whereas it was without effect in WKY rats (Table 2). Ca2+ influx elicited by ANG II was significantly reduced after pretreatment with cytochalasin D (2 µmol/l) in SHR (control: 0.0122 ± 0.0092 ratio unit/s; cytochalasin D: 0.0070 ± 0.0004 ratio units/s; n = 115, P <=  0.0001). Conversely, no effect could be observed in WKY rats (n = 172, P = NS).

Control experiments using FITC-labeled phalloidin showed that, in cells from both strains, cytochalasin D (2 µmol/l) disrupted the actin network, with the formation of patches scattered throughout the cytoplasm, without altering cell shape (Fig. 3). As expected, no differential effect could be observed on microtubules.


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  		Fig. 3.   Effect of cytochalasin D (2 µmol/l) on organization of actin filaments. Cells were fluorescently labeled for actin using rhodamine-conjugated phalloidin. A: actin network in WKY control cells. B: disorganization of actin network after 30-min incubation with cytochalasin D. C: actin network in SHR control cells. D: disorganization of actin network after 30-min incubation with cytochalasin D.

The actin network reorganized rapidly after the withdrawal of cytochalasin D. To assess this reversibility, cells pretreated with cytochalasin D (2 µmol/l for 30 min) were incubated in FCS-free DMEM medium for 2 h at 37°C, loaded with fura 2, and exposed to ANG II as described above. The reduction in response to ANG II induced by cytochalasin D in the SHR, observed in the presence and absence of external Ca2+, was completely reversed (results not shown, n = 45, P = NS).

One reason for the lack of effect of cytochalasin D on Ca2+i release from internal stores in WKY could be a lower sensitivity of actin in this strain to this compound. To verify this hypothesis, we assessed the effect of cytochalasin D at higher concentrations. Concentrations of 5, 10, and 20 µmol/l induced an inhibition of the ANG II-induced Ca2+i increase of 37, 50, and 55%, respectively (n = 30, P <=  0.03 for each) in the SHR, whereas no change in the response to ANG II was observed in the WKY rats despite the collapse in cell shape (n = 30, P = NS).

Effect of disorganizing the microtubule network on ANG II-induced Ca2+i increase. The implication of microtubules in cell response was assessed by disorganizing the microtubule network with nocodazole, vinblastine, or Taxol at concentrations that do not modify cell shape. The exposure of cells from SHR and WKY rats to nocodazole (5 µmol/l, 30 min at 37°C) resulted in the disorganization of the microtubule network, as visualized by immunofluorescence staining, without a change in cell shape (Fig. 4). Similar results were obtained from incubation with vinblastine (10 µmol/l, 20 min at 37°C), which depolymerizes microtubules using a mechanism different from that of nocodazole. As expected, no differential effect could be observed on the actin network with the use of these two agents.


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  		Fig. 4.   Effect of nocodazole (5 µmol/l) on organization of microtubules. Network was fluorescently visualized using a monoclonal antibody to alpha -tubulin followed by staining with FITC-conjugated anti-mouse IgG. A: microtubule network in WKY control cells. B: disorganization of network after 30-min incubation with nocodazole. C: microtubule network in SHR control cells. D: disorganization of network after 30-min incubation with nocodazole.

In the presence of external Ca2+, pretreatment with nocodazole for 30 min significantly reduced the ANG II-induced Ca2+i increase compared with the respective control in both SHR and WKY rats (Table 2). Similar results were obtained with vinblastine in either strain (WKY: amplitude, 48 ± 6% of control values; slope, 53 ± 5%; total Ca2+i, 49 ± 6%; n = 143, P <=  0.0001 for each; SHR: amplitude, 62 ± 4%; slope, 51 ± 4%; total Ca2+i, 68 ± 5%; n = 152, P <=  0.0001 for each).

In the nominal absence of external Ca2+, ANG II-induced Ca2+i mobilization from internal stores was significantly reduced by nocodazole in both strains (Table 2). Similar results were obtained with vinblastine (results not shown). Experiments were also performed with Taxol, which hyperpolymerizes microtubules (38). Pretreatment with Taxol (9.4 µmol/l, incubation for 60 min) significantly reduced ANG II-induced Ca2+i mobilization in both WKY rats (amplitude, 45 ± 2% of control values; slope, 45 ± 2%; n = 120, P <=  0.0001 for each) and SHR (amplitude, 61 ± 3%; slope, 53 ± 2%; n = 33, P <=  0.01 for each).

Ca2+ influx elicited by ANG II was not significantly altered after pretreatment with nocodazole (n = 307, P = NS), vinblastine (n = 143, P = NS), or Taxol (n = 37, P = NS) in both WKY rats and SHR (results not shown).

Effect of disorganizing the actin and microtubule networks on thapsigargin-induced Ca2+ influx. The addition of thapsigargin (3 µmol/l) in the absence of external Ca2+ induced a transient Ca2+i increase in both strains that occurred at a slower rate than that observed with ANG II. Reintroduction of Ca2+ in the medium induced a Ca2+ influx of a magnitude similar to that observed with ANG II. Incubation of VSMC with thapsigargin for 5 min abolished the response to subsequent infusion of ANG II in the two strains (results not shown), as previously reported (9). Furthermore, thapsigargin completely emptied intracellular Ca2+ stores, because ionomycin addition did not elicit any increase in cell Ca2+ (results not shown).

In cells from SHR pretreated with cytochalasin D for 30 min, the Ca2+i transient and the capacitative Ca2+ entry induced by thapsigargin were not significantly different from those observed in control cells (results not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of disorganizing actin or microtubule networks on ANG II-induced Ca2+i increase was assessed in VSMC from SHR and WKY rats. This was achieved with the use of specific agents at concentrations that disorganize the networks without affecting cell shape. Cytoskeletal disruption was found to induce a marked modification in Ca2+ transport processes without affecting steady-state Ca2+i levels or Ca2+i storage capacity. The main result of this study is that when cells pretreated with 2 µmol/l cytochalasin D are exposed to ANG II, the agonist action on Ca2+i is markedly reduced in SHR but not in WKY rats. In VSMC, the ANG II-induced Ca2+i increase results from both Ca2+ release from intracellular stores and Ca2+ influx across the membrane via Ca2+ channels. The effect of cytochalasin D in the SHR could be explained by a reduced mobilization of Ca2+i from internal stores and a decreased Ca2+ influx. This effect is reversible, suggesting that hampering the organization of the actin network has no deleterious effect on VSMC. The lack of effect on Ca2+i release mechanisms observed in WKY rats was not dose dependent, suggesting an insensitivity of Ca2+ signaling pathways to actin depolymerization and to cell shape changes that occurred at high concentrations of cytochalasin D (5-20 µmol/l). This suggests that the signaling pathway linking AT1-receptor activation to Ca2+ storage pools is different in SHR compared with WKY rats.

Several mechanisms could be responsible for these effects in SHR. Cytoskeletal disruption could result in a failure of ANG II to increase IP3 levels. In this regard the integrity of the network was reported to be required for biosynthesis of polyphosphoinositides and activation of phospholipase C by ANG II in adrenocortical cells (14). In contrast, Pedrosa-Ribeiro et al. (33) found no evidence for alteration in the formation or action of IP3 after cytoskeletal disorganization in NIH 3T3 cells. They suggested that cytoskeletal disruption altered the spatial relationship between phospholipase C and IP3 receptors, impairing phospholipase C-dependent Ca2+ signaling. On the other hand, Kraus-Friedmann (21) proposed that the interaction of IP3 with its receptor could induce a conformational change in the cytoskeleton, sensed by the ryanodine binding Ca2+ channel and resulting in its opening. An alternate mechanism could be that cytoskeletal disruption could alter the activation of IP3 receptors in the endoplasmic reticulum, thus impairing Ca2+ release from storage pools (15, 45). The present data do not allow us to differentiate between these mechanisms.

Agonist-stimulated release of Ca2+ from the intracellular stores is accompanied by repletion of the store by Ca2+ influx from the extracellular space (48). In aortic VSMC from SHR and WKY, the major Ca2+-entry mechanisms induced by ANG II were reported to be voltage independent (7, 30, 46). The present study confirms these results, because nifedipine was without effect on Ca2+ influx.

The role of Na+/Ca2+ exchanger in Ca2+ influx, although well documented in several cell preparations, remains a subject of controversy in VSMC (27). In this study, the participation of this exchanger in ANG II-induced Ca2+ influx is negligible. Alternately, Na+ loading may not be sufficient to elicit a significant influx via the exchanger, or Ca2+ influx may be too small to be detected by fura 2 (27, 41).

Our results showed that ANG II-induced Ca2+ influx was inhibited by the disorganization of actin filaments in SHR, whereas no effect could be observed in WKY rats. In contrast, neither the release nor the entry of Ca2+ in response to Ca2+-pool depletion induced by thapsigargin was significantly affected by microfilament disruption in either strain. In VSMC, Ca2+ influx is known to be elicited by either IP3 or an increase in cell Ca2+. Studies using agents such as thapsigargin, which inhibits the Ca2+-ATPase, have demonstrated that Ca2+-store depletion provides a full and sufficient signal for activation of capacitative Ca2+ entry (3, 19, 34). This study shows that, in SHR, the actin network does not play an obligatory role in the activation of capacitative Ca2+ entry but that its integrity is necessary to elicit Ca2+ influx on the activation of AT1 receptors. This is in agreement with the study from Pedrosa-Ribeiro et al. (33), who have shown a physical interaction between IP3 receptors and capacitative Ca2+ entry that involved the actin cytoskeleton. In contrast, these results argue against the hypothesis that Ca2+-pool depletion promotes an insertion of vesicles containing capacitative Ca2+ channels, because exocytosis requires an intact cytoskeleton.

Another main result of this study is that tubulin depolymerization induced a reduction in ANG II-induced Ca2+ increase in cells from both SHR and WKY rats, linked to a reduced mobilization of Ca2+i from internal stores. Surprisingly, hyperpolarization of the microtubule network with Taxol produced the same results. This strongly suggests that the link of AT1-receptor activation to Ca2+-transport processes depends on the dynamic state of the microtubule system. This is in accordance with previous results showing that microtubules respond to various stimuli by changes in their dynamic arrangement and that this is necessary to several cell functions (26, 43, 44).

It would be premature to establish a link between these results obtained in cultured cells and the alteration of VSMC properties observed in hypertension. The involvement of cytoskeleton in contractile response depends on the type of smooth muscle cell. Thus disruption of the actin or microtubule network has been found to attenuate the contraction of rat aortic or uterine smooth muscle cells (11, 25, 51). In contrast, disruption of the microtubule network was shown to increase the contractile response of pulmonary (39) or mesenteric beds (24). Unfortunately, there is, as yet, no information relating hypertension to the involvement of the cytoskeleton in contractile processes.

In conclusion, the present results provide novel insights into the regulation of ANG II-induced Ca2+ signaling by cytoskeletal elements. The data clearly establish that an intact actin network is required to release Ca2+i from storage pools and to activate Ca2+ influx in SHR, suggesting that actin elements play a major role in linking IP3 to its receptor. In contrast, no evidence linking actin to an ANG II effect could be observed in normotensive WKY rats. On the other hand, a functionally intact microtubule system is a necessary prerequisite for ANG II-induced Ca2+i release from internal stores in both strains.


    ACKNOWLEDGEMENTS

We thank Dr. Evelyne Coudrier for support and for useful, stimulating discussion and Drs. Pascal Ferré and Randy Thomas for reading the manuscript.


    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: G. Dagher, INSERM U465, Faculté Broussais-Hotel Dieu, 15 Rue de l'École de Médecine, 75006 Paris, France (E-mail: dagher{at}ccr.jussieu.fr).

Received 31 July 1998; accepted in final form 23 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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