Effects of nitric oxide and cGMP on inhibin A and inhibin subunit mRNA levels from cultured rat granulosa cells

Abstract

Objective To determine the effects of nitric oxide (NO) and cGMP on inhibin A and inhibin subunit mRNA levels from cultured rat granulosa cells. Design Basic research study. Setting University research laboratory. Animal(s) Primary cell culture of granulosa cells obtained from estrogen-treated, immature Sprague-Dawley female rats. Intervention(s) Functionally immature rat granulosa cells were incubated for 48 hours with media alone; FSH; forskolin; the NO generator DETA/NO; an inhibitor of soluble guanylyl cyclase (ODQ); and/or a cell-permeable cGMP analog. Main outcome measure(s) Media concentrations of inhibin A were measured by solid-phase immunosorbent assay. Inhibin α and βA subunit mRNA levels were determined by Northern and slot blot analyses. Result(s) Whereas FSH caused a 20-fold increase in inhibin A levels compared with untreated granulosa cells, the NO generator DETA/NO significantly inhibited FSH-stimulated inhibin A concentrations. Similarly, cotreatment with FSH plus dibutyryl cGMP significantly attenuated inhibin A concentrations, compared with those in cells treated with FSH alone. Incubation with forskolin (FSK) stimulated inhibin A levels sevenfold, whereas cotreatment with FSK plus DETA/NO or FSK plus dibutyryl cGMP effectively decreased inhibin A concentrations. The effects of NO on inhibin A levels were not prevented by cotreatment with an inhibitor of soluble guanylyl cyclase. In addition, there was no influence of DETA/NO or dibutyryl cGMP on inhibin subunit mRNA levels. Conclusion(s) These findings indicate that NO and cGMP can attenuate inhibin A concentrations through actions at one or more post-FSH receptor sites. These influences may reflect inhibition of inhibin A secretion, rather than gene expression and protein synthesis. In addition, NO decreases inhibin A concentrations through both cGMP-dependent and -independent pathways. These results suggest local roles for NO and cGMP in the regulation of granulosa cell function.