Nitric oxide synthase expression and nitric oxide/cyclic GMP pathway in swine granulosa cells

The present investigation was undertaken to verify if the two nitric oxide synthase isoforms, eNOS and iNOS, are present in swine granulosa cells and whether the enzyme soluble guanylate cyclase is functionally active in the same cells and can account for NO effects. Using western blotting, the pres ence of endothelial NO synthase was demonstrated in freshly collected cells ; on the contrary, iNOS expression was not observed in the same cells eithe r before or after culture with the inflammatory cytokine hTNF-alpha. The tr eatment with a strong NO donor (S-Nitroso-L-acetyl penicillamine, SNAP) det ermined an increase of cGMP levels in culture media, which was attenuated b y the combined treatment with an inhibitor of NO-sensitive soluble guanylat e cyclase, 1H-[1,2,3]oxadiaziolo [4,3a]quinoxaline -1-one (ODQ). The cGMP a nalog, 8 bromo-cGMP, mimicked the strong inhibitory effect exerted by SNAP on estradiol 17 beta and progesterone production, while ODQ did not modify steroids concentrations in culture media. These observations demonstrate th e presence of a follicular NO-generating system, which in swine granulosa c ells seems to include only the endothelial NOS isoform. Furthermore, the ni tric oxide/cyclic GMP system seems to be functionally active in these cells , since cGMP appears to mediate NO action, even if it cannot account comple tely for NO inhibitory effect on steroidogenesis. (C) 2001 Elsevier Science Inc. All rights reserved.