The effects of cyclopiazonic acid on intracellular Ca2+ in aortic smooth muscle cells from DOCA-hypertensive rats

Braz J Med Biol Res, February 1997, Volume 30(2) 257-267

The effects of cyclopiazonic acid on intracellular Ca ²⁺ in aortic smooth muscle cells from DOCA-hypertensive rats

R.C.A. Tostes^1,3, D.W. Wilde², L.M. Bendhack⁴ and R.C. Webb¹

¹Departments of Physiology and ²Anesthesiology, University of Michigan, Ann Arbor, MI 48109-0622, USA

³Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto and ⁴Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-900 Ribeirão Preto, SP, Brasil

^{Correspondence and Footnotes}

Abstract

We tested the hypothesis that cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase, increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) in aortic myocytes and that the increase in [Ca²⁺]_i is higher in aortic cells from deoxycorticosterone acetate (DOCA)-hypertensive rats. Male Sprague-Dawley rats, 250-300 g, underwent uninephrectomy, received a silastic implant containing DOCA (200 mg/kg) and had free access to water supplemented with 1.0% NaCl and 0.2% KCl. Control rats were also uninephrectomized, received normal tap water, but no implant. Intracellular Ca²⁺ measurements were performed in aortic myocytes isolated from normotensive (systolic blood pressure = 120 ± 3 mmHg; body weight = 478 ± 7 g, N = 7) and DOCA-hypertensive rats (195 ± 10 mmHg; 358 ± 16 g, N = 7). The effects of CPA on resting [Ca²⁺]_i and on caffeine-induced increase in [Ca²⁺]_i after [Ca²⁺]_i depletion and reloading were compared in aortic cells from DOCA and normotensive rats. The phasic increase in [Ca²⁺]_i induced by 20 mM caffeine in Ca²⁺-free buffer was significantly higher in DOCA aortic cells (329 ± 36 nM, N = 5) compared to that in normotensive cells (249 ± 16 nM, N = 7, P<0.05). CPA (3 µM) inhibited caffeine-induced increases in [Ca²⁺]_i in both groups. When the cells were placed in normal buffer (1.6 mM Ca²⁺, loading period), after treatment with Ca²⁺-free buffer (depletion period), an increase in [Ca²⁺]_i was observed in DOCA aortic cells (45 ± 11 nM, N = 5) while no changes were observed in normotensive cells. CPA (3 µM) potentiated the increase in [Ca²⁺]_i (122 ± 30 nM, N = 5) observed in DOCA cells during the loading period while only a modest increase in [Ca²⁺]_i (23 ± 10 nM, N = 5) was observed in normotensive cells. CPA-induced increase in [Ca²⁺]_i did not occur in the absence of extracellular Ca²⁺ or in the presence of nifedipine. These data show that CPA induces Ca²⁺ influx in aorta from both normotensive and DOCA-hypertensive rats. However, the increase in [Ca²⁺]_i is higher in DOCA aortic cells possibly due to an impairment in the mechanisms that control [Ca²⁺]_i. The large increase in [Ca²⁺]_i in response to caffeine in DOCA cells probably reflects a greater storage of Ca²⁺ in the SR.

Key words: vascular smooth muscle, intracellular calcium mobilization, caffeine, cyclopiazonic acid, sarcoplasmic reticulum, deoxycorticosterone (DOCA) hypertension

Introduction

Cyclopiazonic acid (CPA), a mycotoxin from Aspergillus and Penicillium, is a selective inhibitor of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase and is widely used as a pharmacological tool for investigating the role and turnover of intracellular Ca²⁺. CPA has been shown to inhibit the SR Ca²⁺-ATPase in skeletal (1), smooth (2,3) and cardiac muscle (4). The specific inhibition of the SR Ca²⁺-ATPase by CPA may be related to the inhibition of conformational changes associated with ATP hydrolysis and Ca²⁺ transport (1). By inhibiting the ATP-driven Ca²⁺ sequestration, CPA reduces the storage capacity of the SR for Ca²⁺ and inhibits contractions that depend on intracellular Ca²⁺ release (5-7).

CPA has been reported to increase intracellular Ca²⁺ concentration ([Ca²⁺]_i) in endothelial cells from bovine pulmonary arteries (8), but has no effect on membrane currents of single myocardial cells (4). In aorta from deoxycorticosterone acetate (DOCA)-hypertensive rats, CPA induced contractions in the presence of extracellular Ca²⁺, but not in Ca²⁺-free buffer, suggesting that CPA activates Ca²⁺ influx (7). CPA-induced contractions were not observed in aorta from normotensive rats.

In the present study we tested the hypothesis that CPA increases [Ca²⁺]_i in rat aortic smooth muscle cells and that the CPA-induced increase in [Ca²⁺]_iis higher in aortic cells from DOCA-hypertensive rats. Intracellular Ca²⁺ measurements were performed in aortic smooth muscle cells isolated from normotensive and DOCA-hypertensive rats and the effects of CPA on resting [Ca²⁺]_i and on the increase in [Ca²⁺]_istimulated by 20 mM caffeine were evaluated. The effects of CPA and caffeine on [Ca²⁺]_iwere compared to the effects of CPA and caffeine on vessel tone according to a protocol obtained under similar experimental conditions.

Material and Methods

DOCA-salt induced hypertension

Adult male Sprague-Dawley rats weighing 250-300 g (Charles River Laboratories, Inc., Portage, MI) underwent uninephrectomy (small flank incision, left side) under sodium pentobarbital anesthesia (50 mg/kg, ip). In the same surgery, a silastic implant containing DOCA (200 mg/kg) was implanted subcutaneously behind the skull. Control (normotensive) rats were also uninephrectomized, but received no implant. After surgery, DOCA-treated rats received water supplemented with 1.0% NaCl and 0.2% KCl and normotensive rats received normal tap water. All animals were fed standard laboratory rat chow and had free access to both food and water. The rats were housed individually in a room kept at constant temperature (24^oC) with a day-night cycle interval of 12 h each. Systolic blood pressure (SBP) was determined in conscious restrained rats by the standard tail cuff method (pneumatic transducer), before and after surgery. The experimental protocols performed in this study were in accordance with the State of Michigan guidelines on animal care as well as the standards and policies of the Unit for Laboratory Animal Medicine, University of Michigan.

Intracellular Ca ²⁺ measurements

Four to six weeks after surgery, rats were anesthetized with sodium pentobarbital (60-80 mg/kg, ip) and killed by pneumothorax. The thoracic aorta was removed, placed in physiological salt solution (PSS) and cleaned of fat and connective tissue. The PSS contained 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂PO₄, 1.17 mM MgSO₄.7H₂O, 14.9 mM NaHCO₃, 26 µM CaNa₂-EDTA, 1.6 mM CaCl₂.H₂O and 5.5 mM dextrose. Ca²⁺-free buffer contained 1 mM ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and no Ca²⁺ was added.

Vascular smooth muscle cells (VSMC) were isolated from aortas of normotensive and DOCA-hypertensive rats according to the method of Furspan and Webb (9). Briefly, the thoracic aorta was rapidly removed, placed in 0.1 mM Ca²⁺ Hanks' solution at 22^oC and cleaned of fat and connective tissue. The composition of Hanks' solution was 140 mM NaCl, 5.4 mM KCl, 0.44 mM KH₂PO₄, 0.42 mM NaH₂PO₄, 4.17 mM NaHCO₃, 26 µM CaNa₂-EDTA, 0.10 mM CaCl₂.H₂O, 5.0 mM HEPES and 5.5 mM dextrose, pH 7.35. Vessels were cut into 1-mm segments, and placed into 5 ml of 0.1 mM Ca²⁺ Hanks' solution containing 10 mg/ml bovine serum albumin (type I), 1 mg/ml soybean trypsin inhibitor, 5 mM taurine, 0.4 mg/ml dithiothreitol, 0.6 mg/ml type I collagenase, and 0.6 mg/ml papain (all from Sigma, St. Louis, MO). Vessels were incubated at 37^oC with gentle shaking for 40 min. After incubation, vessel fragments were removed with a pipette and placed in 15-ml plastic capped tubes and resuspended in 0.1 mM Ca²⁺ Hanks' solution containing albumin, trypsin inhibitor and taurine as described above. Fragments were washed three times in this solution to remove enzymes and then incubated with 5 µM Fura-2 acetoxy methylester (Fura-2/AM) for 45 min at room temperature. After loading with Fura-2/AM, single cells were released from vessel fragments by gentle pipette agitation. Isolated VSMC in suspension were transferred to a recording chamber on the stage of a Leitz Diavert inverted fluorescence microscope equipped with quartz optics. The recording chamber was maintained at 37^oC while being superfused at 3 ml/min with PSS equilibrated with 95% O₂and 5% CO₂. All VSMC were allowed to rest for 30 min for complete de-esterification of Fura-2 from its acetoxy methylester form to the Ca²⁺-sensitive Fura-2 pentapotassium salt. Single VSMC with relaxed fusiform morphology were isolated in the window of the photomultiplier (PMT). Fluorescence Fura-2 emission was excited alternately with 340-nm and 380-nm bandpass (± 5 nm) filters mounted in a computer-controlled filter wheel (Lambda-10, Sutter Instruments, Novato, CA). Emitted light was filtered with a 500-530 bandpass filter. Fluorescence emissions were captured with the PMT, recorded and processed on a Mac II fx computer using data acquisition/analysis software (LabView 2, Version 2.2.1). The computer also controlled the filter wheel alternation up to a maximum rate of 90 ms/cycle. Isolated VSMC exhibited no detectable autofluorescence. Maximum and minimum [Ca²⁺]_i were determined at the end of each experimental protocol by treating the cells with PSS containing 1.6 mM Ca²⁺ plus 1 µM ionomycin and 15 mM EGTA (0 mM Ca²⁺), respectively. The ratios of 340/380 nm were then used to calculate[Ca²⁺]_i according to the equation of Grynkiewicz et al. (10):

where: F = fluorescence ratio at 340/380 nm; F_mx= the fluorescence ratio in saturating Ca²⁺ (ionomycin) solution; F_mn = the fluorescence ratio in Ca²⁺-free (EGTA) solution, and Sf₂/Sb₂ = the ratio of 380-nm signals in ionomycin and EGTA solutions. It is important to note that external calibration methods using an assumed dissociation constant (K_d) for Fura-2/AM provide only a reasonable estimate of actual [Ca²⁺]_i. Therefore, we have calculated the K_d (297 nM) for Fura-2 in our system. Calibration curves were obtained for most of the cells (basal ratio values as well as agonist-induced changes in the ratio were constant in all cells) and average values were calculated. [Ca²⁺]_i was determined for all cells using the derived calibration curve. The experimental protocol was performed in various aortic cells from the same rat and the average values were then determined. "N" represents the number of rats used in each protocol.

Experimental protocol

Caffeine-induced increases in [Ca²⁺]_i and changes in resting [Ca²⁺]_iwere evaluated in single aortic smooth muscle cells as described in Figure 1. Briefly, aortic cells were stimulated with 3 µM 5-hydroxytryptamine (5-HT) (1 min), and rinsed in Ca²⁺-free buffer for 10 min to deplete intracellular Ca²⁺ stores (depletion of intracellular Ca²⁺ stores was demonstrated by the lack of changes in [Ca²⁺]_i upon subsequent exposure to 5-HT or caffeine). After Ca²⁺ depletion, intracellular Ca²⁺ stores were loaded by placing the cells in PSS containing 1.6 mM Ca²⁺ for 10 min (loading period). The bathing medium was then replaced with Ca²⁺-free buffer and the increase in [Ca²⁺]_i in response to 20 mM caffeine was induced after a 2-min exposure to Ca²⁺-free PSS. The magnitude of this last response was taken as a measure of the SR Ca²⁺ content (for additional details, see Refs. 7,11,12). The experiment was performed in the presence of 3 µM CPA or vehicle (DMSO). Experiments previously performed in our laboratory (7) have shown that 3 µM CPA inhibits Ca²⁺ uptake into the SR in the aorta from both DOCA and normotensive rats [IC₅₀ (-log M) = 5.52 ± 0.23 normotensive vs 5.30 ± 0.05 DOCA, N = 6].

The same protocol was used in aortas from normotensive and DOCA rats for contraction measurements to correlate force and [Ca²⁺]_i. Aortic strips were stimulated with 3 µM 5-HT and, when maximal contraction was achieved, vessels were rinsed in Ca²⁺-free buffer for 10 min and subsequently in PSS containing 1.6 mM Ca²⁺ for an additional 10 min. Arteries were stimulated with 20 mM caffeine after a 1-min exposure to Ca²⁺-free buffer. The protocol was performed in the presence of 3 µM CPA or vehicle (DMSO). The magnitude of the last response to caffeine was taken as a measure of the functional capacity of the SR to release Ca²⁺.

Figure 1
- [Ca²⁺]_i of rat aortic smooth muscle cells. Aortic myocytes from DOCA-hypertensive and normotensive rats were stimulated with 3 µM 5-HT for 1 min and the protocol described in Material and Methods was performed. Briefly, following stimulation with 5-HT, aortic cells were rinsed in Ca²⁺-free buffer for 10 min to deplete intracellular Ca²⁺ stores. After Ca²⁺ depletion, intracellular Ca²⁺ stores were loaded by placing the cells in PSS containing 1.6 mM Ca²⁺ for 10 min. After a 2-min exposure to Ca²⁺-free PSS the cells were stimulated with 20 mM caffeine. Tracings A (normotensive) and B (DOCA) illustrate control (no CPA) responses in aortic cells using DMSO as vehicle. Changes in [Ca²⁺]_i during the Ca²⁺-loading period and in response to 20 mM caffeine were higher in aortic cells from DOCA-hypertensive rats compared to normotensive cells. Tracings C (normotensive) and D (DOCA) illustrate the effects of CPA. CPA (3 µM) potentiated the increase in [Ca²⁺]_i during the loading period both in DOCA and normotensive aortic cells. However, the increase in [Ca²⁺]_i during the Ca²⁺-loading period was higher in aortic cells from DOCA-hypertensive rats compared to normotensive cells. CPA also attenuated phasic responses to 20 mM caffeine in DOCA-hypertensive and in normotensive aortic cells. Tracings are representative of 5-7 experiments and values indicated in the figure were obtained from one of these experiments.

Statistical analysis

Data are reported as means ± SEM. The Student t-test was used to compare observations between DOCA-hypertensive and normotensive groups and differences related to treatment. The Bonferroni correction was used during multiple testing procedures. Values of P<0.05 were considered to be statistically significant.

Drugs

All drugs were prepared daily and kept on ice during the course of the experiments. 5-Hydroxytryptamine (5-HT) hydrochloride, caffeine, cyclopiazonic acid (CPA) and deoxycorticosterone acetate (DOCA) were purchased from Sigma Chemical Co., St. Louis, MO. Ionomycin (Calbiochem, La Jolla, CA), CPA and Fura-2/AM (Molecular Probes, Eugene, OR) were prepared as stock solutions and dissolved in dimethyl sulfoxide (DMSO, Fisher Chemical, Fair Lawn, NJ). The final concentration of DMSO in the bath chamber did not exceed 0.1% v/v. The DOCA implant was prepared as 1:2 parts DOCA/Silastic (Dow Chemical Co., Midland, MI) .

Results

Systolic blood pressure values were significantly higher (P<0.05) in the DOCA group (195 ± 10 mmHg; N = 7) than in the normotensive group (120 ± 3 mmHg; N = 7), 4-6 weeks after surgery. Mean body weight was 358 ± 16 g for DOCA rats (N = 7) and 478 ± 7 g for normotensive rats (N = 7; P<0.05).

Effects of CPA on increases in [Ca ²⁺ ] _i stimulated by caffeine

Changes in [Ca²⁺]_iare indicated by alterations in the fluorescence ratio at 340 and 380 nm (340/380 ratio). Basal [Ca²⁺]_ivalues were significantly higher in DOCA aortic cells compared to normotensive cells (86.4 ± 14.4 nM, N = 5 vs 43.0 ± 6.7 nM, N = 5, respectively; P<0.05). Since the resting [Ca²⁺]_i in aortic cells differed between normotensive and DOCA rats, 5-HT- and caffeine-induced increases in [Ca²⁺]_i as well as changes in resting [Ca²⁺]_i in aortic smooth muscle cells from normotensive and DOCA rats were reported as the differential increase in [Ca²⁺]_i, i.e., peak [Ca²⁺]_i minus resting [Ca²⁺]_i. Figure 1 illustrates the protocol employed in this study. The cells were stimulated with 3 µM 5-HT and a phasic increase in [Ca²⁺]_i was observed. Ca²⁺-free buffer containing 3 µM CPA or vehicle was then introduced into the recording chamber and the cells were allowed to equilibrate for 10 min. After this period, 1.6 mM Ca²⁺ buffer containing CPA or vehicle was placed in the chamber and the cells were allowed to equilibrate for an additional 10 min. Ca²⁺-free buffer was then reintroduced into the bath chamber and allowed to equilibrate for 2 min before 20 mM caffeine was introduced. The differential peak increase in [Ca²⁺]_iinduced by caffeine in Ca²⁺-free buffer was taken as a measure of Ca²⁺ released from intracellular stores. No differences were observed in 3 µM 5-HT-induced increase in [Ca²⁺]_i between normotensive and DOCA aortic cells. The increase in [Ca²⁺]_i observed with 5-HT was 445 ± 49 nM for cells from DOCA rats, N = 5, and 350 ± 89 nM for cells from normotensive rats, N = 7. As can be observed in the representative traces in Figure 1, the increase in [Ca²⁺]_iobserved in response to 20 mM caffeine was higher in aortic cells from DOCA rats (Figure 1B, 329 ± 36 nM, N = 5, P<0.05) compared to normotensive aortic cells (Figure 1A, 249 ± 16 nM, N = 7). Incubation with 3 µM CPA significantly reduced (P<0.05) the subsequent increase in [Ca²⁺]_i stimulated by 20 mM caffeine both in normotensive (Figure 1C) and DOCA aortic cells (Figure 1D). This concentration of CPA was utilized because the effects of CPA, indicated by IC₅₀ (-log M) values, on caffeine-induced phasic contractions did not differ between DOCA (5.95 ± 0.10, N = 6) and normotensive (5.76 ± 0.25, N = 6) aorta and this concentration produces significant inhibition of caffeine-induced contractions (7). The increase in [Ca²⁺]_i in response to caffeine in the presence of 3 µM CPA was DOCA = 141 ± 29 nM (N = 5) vs normotensive = 102 ± 14 nM (N = 5).

Effects of CPA on resting [Ca ²⁺ ] _i

During the loading period, no changes in [Ca²⁺]_i were observed in normotensive aortic cells (Figure 1A). However, in DOCA cells, a small increase in [Ca²⁺]_i (45 ± 11 nM, N = 5; P<0.05; Figure 1B) was observed when Ca²⁺ was reintroduced into the chamber bath. When the same protocol was used in the presence of 3 µM CPA, both normotensive (Figure 1C) and DOCA-hypertensive rat aortic cells (Figure 1D) exhibited increases in [Ca²⁺]_i during the loading period. However, the increase was higher in DOCA aortic cells compared to normotensive cells (122 ± 30 nM, N = 5 vs 23 ± 10 nM, N = 5, respectively; P<0.05). No changes in [Ca²⁺]_i during the loading period were observed in aortic cells from DOCA rats when 3 µM CPA and 1 µM of the voltage-activated L-type Ca²⁺ channel antagonist nifedipine were introduced simultaneously into the chamber bath (Figure 2D). These results suggest that an increased extracellular Ca²⁺ influx mediates this response in aortas from hypertensive rats.

Figure 3 summarizes the alterations in force and [Ca²⁺]_i exhibited by aortic strips and isolated smooth muscle cells, respectively, from DOCA and normotensive rats for the protocol employed in this study. Aortic strips and isolated cells were stimulated with 3 µM 5-HT. Ca²⁺-free buffer containing 3 µM CPA or vehicle was then introduced into the recording chamber for 10 min to deplete intracellular Ca²⁺ stores. During the loading period, 1.6 mM Ca²⁺ buffer containing CPA or vehicle was placed in the chamber for an additional 10 min. Ca²⁺-free buffer was then reintroduced into the bath before 20 mM caffeine was added. The bars represent the changes in tone and [Ca²⁺]_i observed after 3 µM 5-HT stimulation, during the loading period, and in response to 20 mM caffeine. No differences in 5-HT-induced contraction or increase in [Ca²⁺]_i were observed between the DOCA and normotensive groups. Changes in tone or [Ca²⁺]_i during the loading period under control conditions (vehicle) were observed only in DOCA preparations. In the presence of 3 µM CPA, aortic strips and isolated cells from both DOCA and normotensive rats presented changes in tone and [Ca²⁺]_i during the loading period, but the changes were higher in DOCA preparations. The contractile responses and increases in [Ca²⁺]_i stimulated by caffeine were higher in the DOCA group compared to those in the normotensive group. CPA inhibited caffeine-induced contractions and increases in [Ca²⁺]_i in both groups.

Figure 2
- Effects of nifedipine on CPA-induced contraction and the increase in [Ca²⁺]_i in DOCA aortic smooth muscle cells. The same protocol as described in the legend to Figure 1 was performed in aortas and isolated aortic myocytes from DOCA-hypertensive rats in the presence of 3 µM CPA (A and B) or CPA plus 1 µM nifedipine (C and D). Nifedipine inhibited both contractile responses and the increase in [Ca²⁺]_i during the Ca²⁺-loading period in DOCA aortic cells. Tracings are representative of 6 experiments and values indicated in the figure were obtained from one of these experiments.

Figure 3
- Changes in tone (A) and [Ca²⁺]_i (B) in DOCA-hypertensive and normotensive aortic smooth muscle cells. Alterations in force observed in DOCA aortas in the presence of vehicle or CPA, during the protocol previously described (Figures 1 and 2), are comparable to the alterations in [Ca²⁺]_i. The bars indicate mean ± SEM for changes in force (mg, N = 10-12) and [Ca²⁺]_i (nM, N = 5-7). DOCA, DOCA-hypertensive; control, normotensive; CPA, cyclopiazonic acid (3 µM). *P<0.05 compared to the normotensive group; ⁺P<0.05 compared to control conditions (vehicle).

Discussion

The present study was performed to determine the effects of CPA, a specific and reversible inhibitor of the SR Ca²⁺-ATPase, on [Ca²⁺]_i of aortic cells isolated from normotensive and DOCA-hypertensive rats. We have tested the hypothesis that CPA increases [Ca²⁺]_i in aortic smooth muscle cells and that the increase in [Ca²⁺]_i is higher in cells from DOCA-hypertensive rats. Changes in [Ca²⁺]_i stimulated by caffeine as well as the effects of CPA on caffeine-induced responses were also evaluated in both types of vascular smooth muscle cells. Cyclopiazonic acid is an indole tetramic acid metabolite derived from tryptophan which is produced by fungi such as Aspergillus and Penicillium. The specific inhibition of the SR Ca²⁺-ATPase by CPA may be related to the inhibition of conformational changes associated with ATP hydrolysis and Ca²⁺ transport (1). However, unlike thapsigargin, a tumor-promoting sesquiterpene lactone which binds irreversibly to the SR Ca²⁺-ATPase, CPA does not block the enzyme in the E₁ or E₂ state (1,13,14).

These agents promote depletion of intracellular Ca²⁺ stores by inhibiting Ca²⁺ uptake into the SR and, consequently, they inhibit responses to drugs that release Ca²⁺ from intracellular stores (5-7). CPA reduces or abolishes contractions induced by caffeine in guinea pig ileum (3), in rat skeletal muscle and mesenteric arteries (5,6) and in aortas from normotensive and DOCA-hypertensive rats (7). In the present study, CPA also decreased the caffeine-induced phasic increase in [Ca²⁺]_i (Figure 1C,D) in rat aortic myocytes, consistent with an inhibitory action of CPA on Ca²⁺ uptake into the SR. The CPA-induced inhibition of the caffeine-induced increase in [Ca²⁺]_i is not related to the inhibition of cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase or the increase in cAMP, since no inhibition of caffeine-induced responses was observed in the presence of vehicle.

The caffeine-induced differential peak increase in [Ca²⁺]_i in Ca²⁺-free buffer, a reflection of Ca²⁺ released from the SR, was higher in DOCA aortic cells compared to normotensive cells (Figure 1A,B). We have also observed that phasic contractile responses to caffeine in Ca²⁺-free buffer were enhanced in aorta from DOCA rats (7). Taken together, these observations support our previous suggestion of an expansion of intracellular Ca²⁺ stores in vessels from DOCA-hypertensive rats (7).

The basal [Ca²⁺]_i was higher in DOCA aortic smooth muscle cells compared to normotensive cells. Elevated intracellular Ca²⁺ levels have been reported in aortic smooth muscle cells from spontaneously hypertensive rats (SHR) compared to normotensive Wistar Kyoto (WKY) rats (15,16) and have been associated with increased vascular reactivity in hypertension (17-21). Cytochemical studies on aortic smooth muscle cells from DOCA rats have demonstrated increased cytoplasmic Ca²⁺ and elevated Ca²⁺ in the sarcoplasmic area (22,23), a fact that also supports our observations.

An increase in [Ca²⁺]_iduring the loading period was only observed in aortic cells from DOCA-hypertensive rats. Chen et al. (24) have proposed that the SR regulates [Ca²⁺]_i and acts as a superficial buffer barrier by taking up Ca²⁺ that enters the smooth muscle cell. In this model, the SR may determine the threshold [Ca²⁺]_i that induces contraction and may regulate the steady-state [Ca²⁺]_i. A dysfunction in the SR Ca²⁺ uptake would decrease the ability of the smooth muscle cells to control [Ca²⁺]_i and would result in increased [Ca²⁺]_i. We have previously suggested that the increase of [Ca²⁺]_i in DOCA aorta, indicated by spontaneous contractions, is not due to an impairment of Ca²⁺ uptake into the SR, but to increased cell membrane permeability to Ca²⁺ (7). Since Ca²⁺ uptake into the superficial SR is mediated mainly by Ca²⁺-ATPase, which is a saturable active transporter, it could be bypassed by a high rate of Ca²⁺ entry. Ca²⁺ entering at slower rates could be effectively removed from the cytoplasm before activation of the contractile proteins. Since the spontaneous increase in [Ca²⁺]_i in DOCA aortic cells is only observed in the presence of external Ca²⁺, a higher Ca²⁺ influx may occur in these cells.

CPA-induced increase in [Ca²⁺]_i was observed in both aortic smooth muscle cells from DOCA and normotensive rats. Baró and Eisner (25) have shown that thapsigargin increases [Ca²⁺]_i in smooth muscle cells isolated from the rat mesenteric artery by inducing Ca²⁺ release from the norepinephrine- and caffeine-sensitive stores. In our studies, the [Ca²⁺]_i increase was not due to Ca²⁺ release from intracellular stores since it was also observed after depletion and during loading of the intracellular Ca²⁺ stores. The modulatory effects of the SR on Ca²⁺ entry through the plasma membrane have been proposed in the capacitative model (26,27), which suggests that depletion of intracellular Ca²⁺ stores activates extracellular Ca²⁺ entry, possibly in order to refill the intracellular Ca²⁺ stores. In lymphocytes (28) and in Xenopus oocytes (29), the emptying of intracellular Ca²⁺ stores activates Ca²⁺ influx through the release of an intracellular messenger. CPA can indirectly promote intracellular Ca²⁺ store depletion since it inhibits Ca²⁺ uptake into the SR. The empty stores may trigger the influx of external Ca²⁺ and, in this sense, may explain the different effects of CPA which have been reported. In endothelial cells, CPA induces external Ca²⁺ influx through non-selective cation channels probably by a continuous leak of Ca²⁺ from the endoplasmic reticulum (8). However, in myocardial cells no changes were observed in membrane currents in response to CPA (4). In the present study, the observation that the CPA-induced increase in [Ca²⁺]_i as well as the CPA-induced changes in tone did not occur in Ca²⁺-free buffer and were blocked by nifedipine supports the idea that CPA activates Ca²⁺ influx. Similarly, in skeletal muscle arterioles, it has been shown that the CPA-induced increase in tone is inhibited by nifedipine and Ca²⁺-free PSS (6). The mechanisms by which CPA increases [Ca²⁺]_i are unknown, but there are suggestions that CPA inhibits Ca²⁺-dependent K⁺ channels (30) and increases Ca²⁺ influx through voltage-operating Ca²⁺ channels (31).

An increased transmembrane Ca²⁺ flux as well as altered activity of Ca²⁺ channels have been reported in vessels from hypertensive animals (32-36). Furthermore, Soltis and Field (37) reported an increased reactivity to KCl and to norepinephrine in femoral arteries from DOCA rats, which was related to increased sensitivity to extracellular Ca²⁺, supporting the suggestion of an augmented membrane permeability to Ca²⁺. While an increased membrane permeability to Ca²⁺ in DOCA aortic cells can account for the alterations observed, alternative mechanisms must be considered. For example, reduced Ca²⁺ extrusion through plasma membrane Ca²⁺-ATPase, and increased sensitivity of the contractile proteins to Ca²⁺ have been reported in the literature (19,20). Interactions between Na⁺ and [Ca²⁺]_i should also be taken into consideration. Changes in the transmembrane Na⁺ gradient may alter, for example, the activity of the Na⁺-Ca²⁺ exchanger, which couples Ca²⁺ efflux to Na⁺ influx, an important mechanism for Ca²⁺ extrusion following a transient rise in [Ca²⁺]_i (38). In fact, an increased intracellular Na⁺ content has been shown in vascular smooth muscle from DOCA-hypertensive rats (39,40). The increased intracellular Na⁺ content may alter the Na⁺-Ca²⁺ exchange system, with a consequent increase in [Ca²⁺]_i.

The data presented here support the hypothesis that CPA increases [Ca²⁺]_i in vascular smooth muscle from DOCA-hypertensive and normotensive rats and that the increase in [Ca²⁺]_i is higher in DOCA aortic smooth muscle cells. The alterations in Ca²⁺ handling observed in DOCA aorta do not involve an inadequate SR buffering ability, as previously suggested, but are possibly related to an increased membrane permeability to Ca²⁺. The augmented intracellular Ca²⁺ levels in DOCA aorta may be the consequence of increased Ca²⁺ influx and may be responsible for the greater contractile responses to caffeine observed in this hypertensive model.

References

1. Seidler NS, Jona I, Vegh M & Martonosi A (1989). Cyclopiazonic acid is a specific inhibitor of the Ca²⁺-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry, 264: 17816-17823.

2. Deng HW & Kwan CY (1991). Cyclopiazonic acid (CPA) is a sarcoplasmic reticulum Ca²⁺ pump inhibitor of rat aortic smooth muscle. A functional study. Acta Pharmacologica Sinica, 12: 53-58.

3. Uyama Y, Yamazumi Y & Watanabe M (1992). Effects of cyclopiazonic acid, a novel Ca²⁺-ATPase inhibitor, on contractile responses in skinned ileal smooth muscle. British Journal of Pharmacology, 106: 208-214.

4. Takahashi S, Kato Y, Adachi M, Agata N, Tanaka H & Shigenobu K (1995). Effects of cyclopiazonic acid on rat myocardium: Inhibition of calcium uptake into the sarcoplasmic reticulum. Journal of Pharmacology and Experimental Therapeutics, 272: 1095-1100.

5. Shima H & Blaustein M (1992). Modulation of evoked contractions in rat arteries by ryanodine, thapsigargin, and cyclopiazonic acid. Circulation Research, 70: 968-977.

6. Watanabe J, Karibe A, Horiguchi S, Keitoku M, Satoh S, Takishima T & Shirato K (1993). Modification of myogenic intrinsic tone and [Ca²⁺]_i of rat isolated arterioles by ryanodine and cyclopiazonic acid. Circulation Research, 73: 465-472.

7. Tostes RCA, Traub O, Bendhack LM & Webb RC (1995). Sarcoplasmic reticulum Ca²⁺ uptake is not decreased in aorta from DOCA-hypertensive rats: functional assessment with cyclopiazonic acid. Canadian Journal of Physiology and Pharmacology, 73: 1536-1545.

8. Pasyk E, Inazu M & Daniel EE (1985). CPA enhances Ca²⁺ entry in cultured bovine pulmonary arterial endothelial cells in an IP₃-independent manner. American Journal of Physiology, 268: H138-H146.

9. Furspan PB & Webb RC (1990). Potassium channels and vascular reactivity in genetically hypertensive rats. Hypertension, 15: 687-691.

10. Grynkiewicz G, Poenie M & Tsien RY (1985). A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry, 260: 3440-3450.

11. Karaki H, Kubota H & Urakawa N (1979). Mobilization of stored calcium for phasic contraction induced by norepinephrine in rabbit aorta. European Journal of Pharmacology, 56: 237-245.

12. Perry PA & Webb RC (1991). Agonist-sensitive calcium stores in arteries from steroid hypertensive rats. Hypertension, 17: 603-611.

13. Lytton J, Westlin M & Hanley MR (1991). Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca²⁺-ATPase family of calcium pumps. Journal of Biological Chemistry, 266: 17067-17071.

14. Witcome M, Michelangeli F, Lee AG & East M (1991). The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E₂ form of the Ca²⁺Mg²⁺-ATPase. FEBS Letters, 304: 109-113.

15. Sugiyama T, Yoshizumi M, Takaku F & Yazaki Y (1990). Abnormal calcium handling in vascular smooth muscle cells of spontaneously hypertensive rats. Journal of Hypertension, 8: 369-375.

16. Papageorgiou P & Morgan KG (1991). Intracellular free-Ca²⁺ is elevated in hypertrophic aortic muscle from hypertensive rats. American Journal of Physiology, 260: H507-H517.

17. Bohr DF & Webb RC (1984). Vascular smooth muscle function and its changes in hypertension. American Journal of Medicine, 77: 3-16.

18. Kwan CI (1985). Dysfunction of calcium handling by smooth muscle in hypertension. Canadian Journal of Physiology and Pharmacology, 63: 366-374.

19. Mulvany MJ & Nyborg N (1980). Increased sensitivity of mesenteric resistance vessels in young and adult spontaneously hypertensive rats. British Journal of Pharmacology, 71: 585-596.

20. Mecca TE & Webb RC (1984). Vascular responses to serotonin in steroid hypertensive rats. Hypertension, 6: 887-892.

21. Longhurst PA, Rice PJ, Taylor DA & Fleming WW (1988). Sensitivity of caudal arteries and the mesenteric vascular bed to norepinephrine in Doca-salt hypertension. Hypertension, 12: 133-142.

22. Aguas AP & Nickerson PA (1983). Increased Ca²⁺ in the sarcoplasm of aortic smooth muscle cells from rats made hypertensive with DOC. A quantitative ultrastructural and cytochemical study. Journal of Submicroscopy and Cytology, 15: 425-431.

23. Nickerson PA & Yang F (1988). Calcium distribution in aortic smooth muscle cells of deoxycorticosterone-hypertensive rats. A quantitative cytochemical study. Journal of Submicroscopy in Cytology and Pathology, 20: 317-324.

24. Chen Q, Cannell M & van Breemen C (1992). The superficial buffer barrier in vascular smooth muscle. Canadian Journal of Physiology and Pharmacology, 70: 509-514.

25. Baró I & Eisner DA (1992). The effects of thapsigargin on [Ca²⁺]_i in isolated rat mesenteric artery vascular smooth muscle cells. Pflügers Archiv, 420: 115-117.

26. Putney Jr JW (1986). A model for receptor-regulated Ca²⁺ entry. Cell Calcium, 7: 1-12.

27. Putney Jr JW (1990). Capacitative calcium entry revisited. Cell Calcium, 11: 611-624.

28. Randriamampita C & Tsien RY (1993). Emptying of intracellular Ca²⁺ stores releases a novel small messenger that stimulates Ca²⁺ influx. Nature, 364: 809-814.

29. Parekh AB, Terlau H & Stuhmer W (1993). Depletion of InsP₃ stores activates a Ca²⁺ and K⁺ current by means of a phosphatase and a diffusible messenger. Nature, 364: 814-817.

30. Suzuki M, Muraki K, Imaizumi Y & Watanabe M (1992). Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca²⁺ pump, reduces Ca²⁺-dependent K⁺ currents in guinea-pig smooth muscle cells. British Journal of Pharmacology, 107: 134-140.

31. Uyama Y, Imaizumi Y & Watanabe M (1993). Cyclopiazonic acid, an inhibitor of Ca²⁺-ATPase in the sarcoplasmic reticulum, increases excitability in ileal smooth muscle. British Journal of Pharmacology, 110: 565-572.

32. Aoki K & Asano M (1986). Effect of Bay K 8644 and nifedipine on femoral arteries of spontaneously hypertensive rats. British Journal of Pharmacology, 88: 221-230.

33. Hermsmeyer K, Sturek M & Marvin W (1989). Vascular muscle Ca²⁺ channel modulation in hypertension. Journal of Cardiovascular Pharmacology, 14 (Suppl 6): 45-48.

34. Noon JP, Rice PJ & Baldessarini RJ (1978). Calcium leak as a cause of high resting tension in vascular smooth muscle from spontaneously hypertensive rats. Proceedings of the National Academy of Sciences, USA, 75: 1605-1607.

35. Wilde DW, Furspan PB & Szocik JF (1994). Calcium current in smooth muscle cells from normotensive and genetically hypertensive rats. Hypertension, 24: 739-746.

36. Ohya Y, Abe I, Fujii K, Takata Y & Fujishima M (1993). Voltage-dependent Ca²⁺ channels in resistance arteries from spontaneously hypertensive rats. Circulation Research, 73: 1090-1099.

37. Soltis EE & Field FP (1986). Extracellular calcium and altered vascular responsiveness in the deoxycorticosterone acetate-salt rat. Hypertension, 8: 526-532.

38. Van Breemen C, Aaronson P & Loutzenhiser R (1979). Sodium-calcium interactions in mammalian smooth muscle. Pharmacological Reviews, 30: 167-208.

39. Friedman SM, McIndoe RA & Tanaka M (1988). The relation of cellular sodium to the onset of hypertension induced by DOCA-saline in the rat. Journal of Hypertension, 6: 63-69.

40. Krep H, Price DA, Soszynski P, Tao QF, Graves SW & Hollenberg NK (1995). Volume sensitive hypertension and the digoxin-like factor. American Journal of Hypertension, 8: 921-927.

Acknowledgments

The authors thank Ms. Mary Lloyd for excellent technical assistance.

Address for correspondence: R.C.A. Tostes, Laboratório de Hipertensão, Departamento de Farmacologia, Instituto de Ciências Biomédicas I, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900 São Paulo, SP, Brasil. Fax: [phone]. E-mail: [e-mail]

Research supported by the National Institutes of Health (No. HL18575; R.C. Webb), the American Heart Association-MI (No. 38G5945; D.W. Wilde) and by CNPq (R.C.A. Tostes). Publication supported by FAPESP. Received July 3, 1996. Accepted November 14, 1996.

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  20 Oct 1998
• Date of issue
  Feb 1997

History

• Accepted
  14 Nov 1996
• Received
  03 July 1996