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 m echanisms through which nitric oxide (NO) inhibits steroidogenesis in bovin e granulosa cells. Because cGMP is generally known to play a pivotal role i n 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-d ependent pathways could be involved in the inhibition of steroidogenesis by NO, we examined the formation of lipid hydroperoxides (LPOs), possibly ind uced 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-ace tylpenicillamine (SNAP; 10(-3), 10(-4) and 10(-5) M) in stimulating cGMP pr oduction and the formation of LPOs was examined. The second aim of the pres ent study was to determine whether the effects of NO on steroidogenesis cou ld 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 r eversed by [H-1]-[1,2,3]oxadiaziolo[4,3a]quinoxaline- 1-one (ODQ; 10(-5) an d 10(-4) M) an inhibitor of NO-sensitive soluble guanylate cyclase. The hig hest dose of SNAP used induced a significant (P<0.01) increase in cGMP leve ls, while other concentrations tested were ineffective. Neither concentrati on of ODQ used significantly inhibited basal cGMP output, while both concen trations counteracted the stimulatory effect of SNAP. Treatment of cells wi th 8-Br-cGMP and ODQ was ineffective in modifying steroidogenesis. Treatmen t with SNAP, at the three concentrations tested, had no significant effect on the level of LPOs. The present results suggest that NO inhibits steroido genesis in bovine granulosa cells without involving cGMP and LPOs.