AT(1) calcium signaling in renal vascular smooth muscle cells

Experiments were conducted to gain insight into calcium signaling mechanism s triggered by angiotensin II (AngII) stimulation in vascular smooth muscle cells (SMC) freshly isolated from preglomerular vessels of normotensive Wi star Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Cytosolic calcium concentration ([Ca2+](i)) was measured using ratiometric Fura-2 flu orescence and a microscope-based photometer. Vascular SMC from preglomerula r vessels were isolated and dispersed using an iron oxide-sieving method co mbined with collagenase treatment. AngII produced rapid increases in [Ca2+] (i) that remained elevated for the duration of continued stimulation. The s ame pattern of time response was observed in WKY and in SHR. AngII elicited dose-dependent ;increases in [Ca2+](i) in groups of individual preglomerul ar arteriolar SMC from both strains. AngII (10(-10) M) induced an increase from baseline levels in WKY and SHR (37 +/- 9 and 32 +/- 13 nM: P < 0.05). In response to 10(-6) M AngII, steady-state responses were 165 +/- 30 and 1 70 +/- 35 nM (P < 0.01). The responses did not differ between strains (P > 0.4). The effects of AngII were inhibited by 88% by the AT(1) receptor bloc ker candesartan in renal SMC. In SMC pretreated with calcium-free medium, b aseline [Ca2+](i) fell by about 60 nM. Thereafter, AngII did not elicit any [Ca2+](i) response either in WKY or in SHR when calcium entry was prevente d. Also, after prestimulation by AngII, a calcium-free solution completely reversed the effects of AngII. This study shows that AngII acts through AT( 1) receptors to stimulate [Ca2+](i) by a predominant action on calcium entr y with no evidence for calcium mobilization. Other studies have demonstrate d that calcium entry in these SMC is mediated by voltage-gated, L-type entr y channels sensitive to dihydropyridine agents. No strain differences were noted between the actions of AngII on individual renal SMC from SHR and nor motensive control animals.