Endothelin receptor subtypes in mesenteric vascular smooth muscle cells of spontaneously hypertensive rats

Calcium responses to the endothelin B receptor (ETB) selective agonist IRL-1620 (IRL) and to endothelin 1 (ET-1) in the absence and presence of the endothelin A receptor (ETA) antagonist BQ-123 were determined in primary cultured unpassaged vascular smooth muscle cells derived from mesenteric resistance vessels of 3-, 9-, and 17-week-old spontaneously hypertensive rats (SHR), Wistar rats, and Wistar-Kyoto (WKY) rats. Intracellular free calcium concentration ([Ca2+]i) was measured microphotometrically and by digital imaging using fura 2 methodology. ET receptor affinity and density were determined in membrane preparations from the mesenteric vascular bed from these rats. Binding studies were performed using [125I]ET-1 in the presence of increasing concentrations of ET-1, BQ-123, or IRL. Basal [Ca2+]i was significantly greater (p < 0.01) in adult SHR compared with age-matched normotensive groups. IRL (10(-7) mol/L) and ET-1 (10(-9) mol/L) increased [Ca2+]i in cells from all strains of all age groups. Responses were significantly lower (p < 0.05) in vascular smooth muscle cells from 9- and 17-week SHR compared with cells from age-matched Wistar and WKY rats. Concentration-response curves for IRL were blunted in cells from 17-week SHR versus cells from WKY rats. The selective ETA receptor antagonist BQ-123 (10(-7)-10(-5) mol/L) had no effect on IRL-induced [Ca2+]i but significantly reduced ET-1-induced [Ca2+]i by 135 +/- 4, 80 +/- 6, and 91 +/- 4 nmol/L in cells from 17-week SHR, WKY, and Wistar groups, respectively. The selective ETB receptor antagonist BQ-788 (10(-6) mol/L) significantly reduced ET-1-induced [Ca2+]i responses in 17-week rats. The Bmax for ET-1 binding was significantly lower in mesenteric artery membrane preparations from 17-week SHR (627 +/- 163 fmol/mg protein) compared with WKY and Wistar rats (1190 +/- 43 and 1059 +/- 55 fmol/mg protein, respectively). These data demonstrate the presence of both ETA and ETB receptors in rat mesenteric vascular smooth muscle cells. In adult SHR, reduced responses of vascular smooth muscle cells to IRL, which represents ETB-mediated effects, and to ET-1, may be partly due, as demonstrated in the binding studies, to lower ET receptor density than in normotensive rats