Characterization of the endothelin receptor in primary cultures of human aortic smooth muscle cells.

We characterized the endothelin receptor subtypes in primary cultures of human aortic smooth muscle cells (HASMCs) by binding studies. [125I]-Endothelin (ET)-1 saturation experiments showed the existence of a homogeneous population of binding sites with the high affinity (KD value) of 97 +/- 37 pM and maximum number of binding sites (Bmax) of 54 +/- 10 fmol/mg protein. However, almost no specific [125I]-ET-3 binding was observed. Inhibition of [125I]-ET-1 binding in the HASMCs membrane by nonlabeled compounds showed the following order of effectiveness: ET-1 = ET-2 = FR139317 >> ET-3. These results suggest that the endothelin receptor of HASMCs is of the ETA type. We also studied the effect of ET-1 on the cytosolic [Ca2+]i in HASMCs loaded with fura-2/AM. In 1.3 mM Ca2+, ET-1 produced a dose-dependent, biphasic increase in signal with a maximal effect at 10 nM. At this concentration, ET-1 produced a transient increase in [Ca2+]i that reached a peak at 1 min, which was followed by a slow but sustained increase in [Ca2+]i. This second phase was attenuated in Ca(2+)-deficient medium. Furthermore, ET-1 increased inositol 1,4,5-triphosphate in a time- and dose-dependent manner. These results suggest that the endothelin receptors of HASMCs are of the ETA type, which couple with Ca2+ channels.