DIRECT EFFECT OF ENDOTHELIN-1 ON THE GRANULOSA-CELLS OF THE PORCINE OVARY

Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca2+ concentration ([Ca2+]i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using I-125-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0.59 nmol/l and the maximal binding capacity was 1.84 pmol/mg protein. Affinity-labelling of I-125-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1-100 nmol/l) induced rapid and transient increases in [Ca2+]i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[H-3]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ET(B)) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca2+]i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells.