NITRIC-OXIDE - A MODULATOR FOR THE EPIDERMAL GROWTH-FACTOR RECEPTOR EXPRESSION IN DEVELOPING OVARIAN GRANULOSA-CELLS

The present study was designed to assess the involvement of nitric oxi de (NO) in the regulation of the epidermal growth factor (EGF) recepto r during development of rat granulosa cells, which were prepared by pu ncturing ovaries of diethylstilbestrol-treated rats. The immature cell s were cultured for 48 h with follicle-stimulating hormone (FSH) to be transformed into mature cells. A marked accumulation of guanosine 3', 5'-cyclic monophosphate (cGMP) was observed during development. The ac cumulation of cGMP, but not of adenosine 3',5'-cyclic monophosphate (c AMP), was suppressed by specific inhibitors of NO synthase, L-N-G-mono methyl-L-arginine (L-NMMA) and L-N-G-nitro-L-arginine, and a selective inhibitor of the inducible NO synthase, aminoguanidine. The L-NMMA-in duced suppression was partially reversed by addition of L-arginine to cultures but not D-arginine, indicating that NO formation is inhibited by competing with analogues of L-arginine for NO synthase. Treatment with oxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl- 3-oxide, an an tagonist of NO, caused suppression in the increase of EGF binding site s, whereas exposure of the cells to sodium nitroprusside (SNP), an NO donor, caused elevation in EGF binding sites, with increasing extra- a nd intracellular cGMP levels. Analysis of the EGF receptor by affinity labeling with I-125-labeled EGF revealed that the intensity of the cr oss-linked receptor molecule with a molecular mass of 180 kDa was incr eased by exposure to SNP. The facilitatory effect of SNP on the EGF re ceptor was observed when the cAMP-dependent pathway was fully activate d by FSH. However, the NO effect may be mediated by a cGMP-independent pathway, as 8-bromo-cGMP did not mimic the action of SNP. These resul ts indicate that the L-arginine-NO system may contribute to the regula tion of EGF receptor expression in developing granulosa cells stimulat ed by FSH.