Kamada S, Blackmore P F, Kubota T, Oehninger S, Asada Y, Gordon K, Hodgen G D, Aso T
Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk 23507, USA.
J Clin Endocrinol Metab. 1995 Dec;80(12):3708-14. doi: 10.1210/jcem.80.12.8530623.
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.
内皮素 -1（ET -1）对人黄素化颗粒细胞（L -HGCs）的作用尚未得到研究。众所周知，L -HGCs与其他物种的颗粒细胞（GCs）在几种自分泌/旁分泌调节因子的作用方面存在差异，因此本研究旨在探讨ET -1的作用：1）使用Ca(2 +)响应荧光指示剂Fura -2检测其对细胞内Ca2 +浓度（[Ca2 +]i）的影响；2）使用溴脱氧尿苷通过非放射性方法检测其对细胞增殖的影响；3）检测其对基础及促性腺激素刺激的类固醇生成的影响，并使用从接受体外受精的患者中新鲜分离和培养的L -HGCs检测ET受体信使核糖核酸（mRNA）的表达。ET -1在1至1000 nmol/L之间以剂量依赖性方式增加L -HGCs中的[Ca2 +]i。高浓度（100 -1000 nmol/L）的ET -1比低浓度（1 -10 nmol/L）的ET -1能使[Ca2 +]i产生更快速和短暂的增加。ET -3（1000 nmol/L）和选择性ETB受体激动剂IRL -1620（1000 nmol/L）引起的[Ca2 +]i增加分别为16%和3%（相对于ET -1为100%）。ETA受体拮抗剂BQ -123（1000 nmol/L）抑制了ET -1引起的[Ca2 +]i增加（在1000 nmol/L ET -1时抑制50%，在<500 nmol/L ET -1时抑制>90%）。两种已知受体亚型（ETA和ETB）的mRNA也存在于L -HGCs中；然而，ETA受体mRNA的表达远高于ETB受体。ET -1以剂量依赖性方式刺激L -HGCs的细胞增殖（1000 nmol/L时为210.5±13.1%；100 nmol/L时为198±11%；10 nmol/L时为146±18%；1 nmol/L时为103±9%；相对于对照为100%）。这些刺激作用被BQ -123（1000 nmol/L）完全阻断。ET -3和IRL -1620对L -HGCs的细胞增殖无影响。观察到钙离子载体离子霉素（10 -1000 nmol/L）对细胞增殖有显著刺激作用。ET -1、ET -3和IRL -1620减弱了L -HGCs中的基础孕酮分泌。这些结果表明，ETA受体主要存在于L -HGCs中，并且ET -1可能通过ETA受体增加[Ca2 +]i来刺激L -HGCs的细胞增殖。
