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

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% KCI. 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.