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 differences of actions of several autocrine/paracrine regulators between L-HGCs and GCs of other species, and therefore the present study was designed to examine the effects of ET-1 1) on intracellular Ca2+ concentrations ([Ca2+]i) using the Ca(2+)-responsive fluorescent indicator Fura-2, 2) on cell proliferation by the nonradioactive method using bromodeoxyuridine, and 3) on basal and gonadotropin-stimulated steroidogenesis, and to examine the expression of ET receptor messenger RNA (mRNA) using freshly isolated and cultured L-HGCs obtained from patients undergoing 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 increase 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) and IRL-1620 (1000 nmol/L), a selective ETB receptor agonist, was 16% and 3% (vs. ET-1, 100%), respectively. BQ-123 (1000 nmol/L), an ETA receptor 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). mRNAs for the two known receptor subtypes (ETA and ETB) were also present in L-HGCs; however, the expression of ETA receptor mRNA was much greater than that of ETB receptors. ET-1 stimulated cell proliferation in L-HGCs in a dose-dependent manner (1000 nmol/L, 210.5 +/- 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 blocked by BQ-123 (1000 nmol/L). ET-3 and IRL-1620 had no effects on cell proliferation in L-HGCs. Significant stimulatory effects on cell proliferation by the calcium ionophore, ionomycin (10-1000 nmol/L), were observed. ET-1, ET-3, and IRL-1620 attenuated basal progesterone secretion in L-HGCs. These results suggest that ETA receptor predominantly exist in L-HGCs and that ET-1 may stimulate cell proliferation of L-HGCs by increasing [Ca2+]i via ETA receptors.
Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk 23507, USA.
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S
Kamada
P F
PF
Blackmore
T
T
Kubota
S
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Oehninger
Y
Y
Asada
K
K
Gordon
G D
GD
Hodgen
T
T
Aso
D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000494:pharmacology / Q000502:physiology*; D005260:Female; D006062:Gonadotropins / Q000494:pharmacology; D006107:Granulosa Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D017466:Receptors, Endothelin / Q000235:genetics; D013256:Steroids / Q000096:biosynthesis*
D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D006062:Gonadotropins; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D012333:RNA, Messenger; D017466:Receptors, Endothelin; D013256:Steroids; C074308:sovateltide; C072247:cyclo(Trp-Asp-Pro-Val-Leu); D002118:Calcium