INTERLEUKIN-1-BETA STIMULATES NITRITE PRODUCTION IN THE RAT OVARY - EVIDENCE FOR HETEROLOGOUS CELL-CELL INTERACTION AND FOR INSULIN-MEDIATED REGULATION OF THE INDUCIBLE ISOFORM OF NITRIC-OXIDE SYNTHASE

Recent studies suggest that endogenously generated nitric oxide (NO) may mediate the effects of cytokines in a variety of tissues. In an effort to determine whether NO generation mediates any of the intraovarian actions of interleukin-1 beta (IL-1 beta), we have looked for and characterized the accumulation of nitrite by IL-1 beta-treated, cultured whole ovarian dispersates. Application of IL-1 beta significantly enhanced basal nitrite release in a dose-, cell density- and time-dependent manner, the latter characterized by a lag time of about 20 h, suggestive of induction of NO synthase (NOS). Cellular NOS activity was also elevated by IL-1 beta. Sustained nitrite accumulation required continuous application of IL-1 beta. The maximally stimulating dose of IL-1 beta (50 ng/ml) produced a 10-fold increase in nitrite accumulation by 96 h of culture, an effect reduced 23% when cells were cultured in substrate (i.e., arginine)-free media. IL-1 beta-stimulated nitrite accumulation was reduced to control levels by the simultaneous application of an IL-1 beta receptor antagonist, thereby suggesting a specific receptor-mediated effect. Both the control and IL-1 beta-stimulated levels of nitrite accumulation were attenuated in a dose-dependent manner by inhibitors that favor the inducible form of NOS, In contrast, selective inhibitors of the constitutive form of NOS were significantly less potent. No inhibition was noted after application of an inactive stereoisomeric analogue. IL-1 beta-induced nitrite accumulation was shown to require cell-cell interaction between granulosa and theca-interstitial cells. IL-1 beta-induced nitrite accumulation was enhanced nearly 2-fold by the addition of insulin, although insulin alone had no effect. The synergy of IL-1 beta with a hormone (insulin) known to regulate ovarian function as well as the requirement for participation of both granulosa and theca-interstitial cells suggests a possible role for NO in mediating some of the pleiotropic actions of IL-1 beta in the ovary. These studies demonstrate an arginine-dependent, IL-1 beta-induced, receptor-mediated, insulin-enhanced stimulation of ovarian nitrite accumulation, presumably reflecting activation of the inducible isoform of NOS.