Lipid hydroperoxide and cGMP are not involved in nitric oxide inhibition of steroidogenesis in bovine granulosa cells

The present study was performed to explore two of the possible signalling mechanisms through which nitric oxide (NO) inhibits steroidogenesis in bovine granulosa cells. Because cGMP is generally known to play a pivotal role in NO signal transduction, the first aim of the present study was to verify the presence of a functional NO-cGMP signalling pathway. Because non-cGMP-dependent pathways could be involved in the inhibition of steroidogenesis by NO, we examined the formation of lipid hydroperoxides (LPOs), possibly induced by NO. Using bovine granulosa cells collected from small (< 5 mm) and large (> 8 mm) follicles, the effectiveness of the NO donor s-nitroso-N-acetylpenicillamine (SNAP; 10(-3), 10(-4) and 10(-5) M) in stimulating cGMP production and the formation of LPOs was examined. The second aim of the present study was to determine whether the effects of NO on steroidogenesis could be mimicked by treatment of cells with a cGMP analogue (8-bromo-cGMP (8-Br-cGMP); 10(-3), 10(-4) and 10(-5) M) and whether these effects could be reversed by [H-1]-[1,2,3]oxadiaziolo[4,3a]quinoxaline- 1-one (ODQ; 10(-5) and 10(-4) M) an inhibitor of NO-sensitive soluble guanylate cyclase. The highest dose of SNAP used induced a significant (P<0.01) increase in cGMP levels, while other concentrations tested were ineffective. Neither concentration of ODQ used significantly inhibited basal cGMP output, while both concentrations counteracted the stimulatory effect of SNAP. Treatment of cells with 8-Br-cGMP and ODQ was ineffective in modifying steroidogenesis. Treatment with SNAP, at the three concentrations tested, had no significant effect on the level of LPOs. The present results suggest that NO inhibits steroidogenesis in bovine granulosa cells without involving cGMP and LPOs.