THE ROLE OF ENDOTHELIN-1 IN REGULATING HUMAN GRANULOSA-CELL PROLIFERATION AND STEROIDOGENESIS IN-VITRO

The effects of endothelin-1 (ET-1) on luteinized human granulosa cells (L-HGCs) have not been examined. It is well known that there are diff erences of actions of several autocrine/paracrine regulators between L -HGCs and GCs of other species, and therefore the present study was de signed to examine the effects of ET-11) on intracellular Ca2+ concentr ations ([Ca2+](i)) using the Ca2+-responsive fluorescent indicator Fur a-2, 2) on cell proliferation by the nonradioactive method using bromo deoxyuridine, and 3) on basal and gonadotropin-stimulated steroidogene sis, and to examine the expression of ET receptor messenger RNA (mRNA) using freshly isolated and cultured L-HGCs obtained from patients und ergoing in vitro fertilization. ET-1 increased [Ca2+](i) in L-HGCs in a dose-dependent manner between 1 and 1000 nmol/L. High concentrations (100-1000 nmol/L) of ET-1 produced a more rapid and transient increas e in [Ca2+](i) than that observed with low concentrations (1-10 nmol/L ) of ET-1. The increase in [Ca2+](i) elicited by ET-3 (1000 nmol/L) an d IRL-1620 (1000 nmol/L), a selective ET(B) receptor agonist, was 16% and 3% (us. ET-1, 100%), respectively. BQ-123 (1000 nmolL), an ET(A) r eceptor antagonist, inhibited the increase in [Ca2+](i) elicited by ET -1 (by 50% at 1000 nmol/L ET-1 and by > 90% at < 500 nmol/L ET-1). mRN As for the two known receptor subtypes (ET(A) and ET(B)) were also pre sent in L-HGCs; however, the expression of ET(A) receptor mRNA was muc h greater than that of ET(B) receptors. ET-1 stimulated cell prolifera tion in L-HGCs in a dose-dependent manner (1000 nmol/L, 210.5 i 13.1%; 100 nmol/L, 198 +/- 11%; 10 nmol/L, 146 +/- 18%; and 1 nmol/L, 103 +/ - 9%; vs. control, 100%). These stimulatory effects were completely bl ocked by BQ-123 (1000 nmol/L). ET-3 and IRL-1620 had no effects on cel l proliferation in L-HGCs. Significant stimulatory effects on cell pro liferation by the calcium ionophore, ionomycin (10-1000 nmol/L), were observed. ET-1, ET-3, and IRL-1620 attenuated basal progesterone secre tion in L-HGCs. These results suggest that ET(A) receptors predominant ly exist in L-HGCs and that ET-1 may stimulate cell proliferation of L -HGCs by increasing [Ca2+](i) via ET(A) receptors.