This study examined contribution of Na(+)-dependent processes to the regulation of free cytosolic calcium (Ca2+i) in cultured vascular smooth muscle cells (VSMC) using fura-2. Removal of Na+ from superfusate (replacement with choline) resulted in an increment of Ca2+i that was greatly augmented by pretreatment with ouabain. Under both conditions, Ca2+i increase was followed by partial recovery to a new steady state that was still significantly higher than that seen before removal of external Na+ (Na+o). In ouabain-pretreated cells lowering of Na+o caused progressive increases in Ca2+i. Addition of NiCl2, a Na(+)-Ca2+ exchange inhibitor, completely blocked the increase in Ca2+i produced by removal of Na+o, indicating that the Na(+)-Ca2+ antiporter was responsible for observed Ca2+i changes. Ca2+i increase produced by reduction of Na+o was also seen after depletion of inositol trisphosphate-sensitive Ca2+ stores with repeated pulses of angiotensin II or after blockade of sarcoplasmatic reticulum Ca2+ release with TMB-8 but was not observed in the absence of external Ca2+. These observations indicate that the source of Ca2+i increase in response to changes in the transmembrane Na+ gradient is largely external, and potentiation of the Ca2+i surge by ouabain suggests Ca2+ influx via the Na(+)-Ca2+ exchanger operating in the reverse mode. The relative contribution of a Na(+)-dependent and -independent component of Ca2+i recovery was investigated by superfusing cells with ionomycin in a Na(+)-free medium and later adding Na+ to the medium. This Ca2+ ionophore increased Ca2+i to a peak, and this was followed by a rapid but partial recovery to a new steady state. Readdition of varying amounts of Na+ to the superfusate, in the continued presence of ionomycin, resulted in concentration-related decline in Ca2+i, thereby uncovering a substantial contribution of a Na(+)-dependent mechanism of Ca2+i regulation. Decline of Ca2+i produced by readdition of Na+ was blocked by addition of NiCl2 to the superfusate. Our findings thereby provide evidence for Ca2+i regulation in VSMC via a Na(+)-dependent mechanism, consistent with a Na(+)-Ca2+ exchanger, which acts as a Ca2+ efflux mechanism when Ca2+i is elevated. Na(+)-Ca2+ exchanger acts as a Ca2+ influx mechanism when intracellular Na+ is elevated by prior exposure to ouabain.

Hypertension is a well-known side effect of ciclosporin A (CsA). In the present study the mechanisms of vasoconstriction in renal vessels were examined in the isolated perfused rat kidney. Kidneys were perfused with constant flow at a temperature of 37 °C with Tyrode’s solution equilibrated with 95% O₂/5% CO₂. CsA was dissolved in ethanol. 500 and 2000 ng/ml increased resistance of renal vessels by 0.97 ± 0.55 × 10⁵ and 2.29 ± 1.33 × 10⁵ dyn s cm^-5, respectively (mean values ± SD, n = 12). The vasoconstriction developed gradually over 4 min. The vasopressor effect of CsA was not changed by saralasin (10^-6 M), nifedipine (10^-6 M) and ketanserin (10^-6 M), but was completely blocked by phentolamine and prazosin (each 10^-6M). CsA-induced vasoconstriction was not prevented by perfusion with Ca²⁺-free solution containing 2 mmol EGTA. Similarly, pretreatment with reserpine to deplete sympathetic nerve endings from catecholamines did not affect CsA-induced vasoconstriction. The findings suggest that CsA-induced vasoconstriction is mediated by stimulation of α₁-receptors. Ca²⁺ influx does not play a role for CsA-induced vasoconstriction. Prolonged perfusion of rat kidneys with the vehicle cremophor EL elicits an irreversible increase in perfusion pressure.

The effects of captopril on the response of cytosolic free Ca 2+ concentration in cultured vascular smooth muscle cells of aortas from Wistar-Kyoto and spontaneously hypertensive rats to angiotensin II (Ang II) and bradykinin were studied using fura 2. Incubation with captopril for longer than 10 minutes caused a decreased response of cytosolic free Ca 2+ to Ang II and bradykinin. Maximal effects of captopril were observed after a 40-minute incubation. The inhibitory effect of captopril was abolished in Ca 2+ -free medium, suggesting that captopril acts by blocking Ca 2+ influx. Similar effects were observed with enalaprilat. Isometric contraction of aortic strips induced by Ang II in normotensive rats was reduced from 6.5±2.5 to 1.8±0.6 mN by a 40-minute incubation with 1 /tmol/L captopril (P=.O16). Enalaprilat similarly decreased the Ang II-induced contraction. Besides the inhibition of the angiotensin converting enzyme, direct effects of Ang II converting enzyme inhibitors on vascular contraction and Ca 2+ influx in vascular smooth muscle cells may be of therapeutic relevance. (Hypertension. 1993;22:806-811.) KEY WORDS • calcium channel • captopril • angiotensin converting enzyme inhibitors • muscle, smooth, vascular A ngiotensin converting enzyme (ACE) inhibitors / \ were introduced in antihypertensive therapy in A. A . 1977.' The main mechanism of action is lowering the angiotensin II (Ang II) production in both plasma and tissues containing renin and the ACE. The long-term antihypertensive action of ACE inhibitors is not strictly correlated with inhibition of circulating ACE.2 -3 Although the inhibition of tissue ACE 4 and possibly the accumulation of bradykinin by inhibition of kininase II 5 may help to explain the antihypertensive effects, other non-ACE-dependent mechanisms cannot be excluded. Among those, a postjunctional blockade of a-adrenergic vasoconstriction, 6 an inhibition of norepinephrine release from sympathetic neurons, 7 and an inhibition of Na + ,K + -ATPase 8 have been demonstrated. However, these effects were found with concentrations of an ACE inhibitor far exceeding those effective in blocking ACE. Furthermore, long-term ACE inhibition in spontaneously hypertensive rats (SHR) was found to affect Ca 2+ handling in vascular smooth muscle cells (VSMCs).9 Because this was observed after intact animals had been treated with an ACE inhibitor, the question remained open as to whether ACE inhibitors can affect Ca 2+ handling in VSMCs directly. To examine this question, we studied the effect of ACE inhibitors on agonist-induced Ca 2+ transients in cultured VSMCs and on isometric contraction of aortic strips. Methods Cell CultureAll experiments were carried out using rat thoracic aortas isolated from 6-month-old male SHR (weight, 300 to 400 g; systolic blood pressure, 195 ±4 mm Hg, mean±SEM) of the Munster strain (Medizinische Universitats-PolikJinik, Munster, Germany) as previously described 10 and age-matched male normotensive Wistar-Kyoto (WKY) rats (systolic blood pressure, 116±3 mm Hg ). V...

To evaluate the influence of the sarcoplasmic Ca(2+)-ATPase, isometric vasoconstrictions of aortic strips from spontaneously hypertensive rats from the Münster strain (SHR) and normotensive Wistar-Kyoto rats (WKY) were measured after inhibition of Ca(2+)-ATPase by thapsigargin. Inhibition of Ca(2+)-ATPase by thapsigargin caused a biphasic contractile response of the aorta in both SHR and WKY (maximum increase of tension: 1.7 +/- 0.3 x 10(-3) Newton and 2.1 +/- 0.3 x 10(-3) Newton, respectively; mean +/- SE). The second peak of the contractile response was abolished in the absence of external calcium or by inhibition of transplasmamembrane calcium influx by nifedipine, indicating that the second peak occurs as a consequence of calcium influx from the extracellular space. The initial peak of the contractile response after thapsigargin administration was abolished in the presence of an intracellular calcium antagonist, 8-(diethylamino-)-octyl-3,4,5-trimethoxybenzoate (TMB-8), indicating that the initial response was due to calcium release from intracellular stores. Measurements using the fluorescent dye fura2 showed that thapsigargin increased the cytosolic free calcium concentration ([Ca2+]i) in SHR by 72.6 +/- 7.3 nmol/l (n = 34) and in WKY by 53.3 +/- 6.6 nmol/l (n = 39), showing no significant differences between the two strains. The inhibition of Ca(2+)-ATPase increases [Ca2+]i and causes vasoconstriction. The vasoconstriction produced by thapsigargin is not significantly different between SHR and WKY.

1. The effect of insulin on cytosolic free Ca2+ concentration was measured using fura-2 in vascular smooth muscle cells of normotensive and spontaneously hypertensive rats. 2. In both strains, insulin increased cytosolic free Ca2+ concentration in a concentration range between 10(-6) and 10(-3) units/ml. The maximum increase in cytosolic free Ca2+ concentration was observed with 10(-5) units/ml insulin (107 +/- 25 and 82 +/- 27 nmol/l in spontaneously hypertensive rats and normotensive rats, respectively). 3. The effect of insulin was dependent on extracellular Ca2+ and was enhanced by stimulation of protein kinase C. 4. Thus insulin appears to induce a Ca2+ influx in vascular smooth muscle cells only over a certain range of concentrations. No significant difference in the response to insulin of cells from normotensive and hypertensive rats was observed.