NITRIC-OXIDE IS A MEDIATOR OF THE INHIBITORY EFFECT OF ACTIVATED MACROPHAGES ON PRODUCTION OF ANDROGEN BY THE LEYDIG-CELL OF THE MOUSE

We hypothesized that macrophage activation results in nitric oxide (NO ) production and that this NO acts directly on Leydig cells (LC) to al ter androgen synthesis. Both peritoneal macrophages and a murine macro phage cell line (RAW 264.7) were activated in vitro by sequential expo sure to interferon-gamma (50 U/ml) and then bacterial lipopolysacchari de (LPS; 100 ng/ml) for 24 h each. At various times after initiation o f activation, selected wells were harvested for identification of mess enger RNA for inducible NO synthase by RT-PCR. Amplicons of the predic ted 651-bp product were isolated, cloned, and sequenced to validate th e PCR procedure. Such amplicons first appeared between 2-4 h after exp osure to LPS, and staining increased in intensity for the rest of the study. Nitrite accumulation followed a similar time course. Similarly treated wells were washed after 24-h activation and cocultured with pu rified LC for a final 24-h incubation in the absence of interferon-gam ma and LPS. Basal and LH-stimulated production of androgen was estimat ed by RIA. In some experiments the NO synthase inhibitor N-omega-nitro -L-arginine methyl ester or the NO scavenger nyl)-4,4,5,5-tetramethyli midazoline-1-oxyl-3-oxide (C-PTIO) was added during activation and coc ulture. Coculture of LC with quiescent macrophages altered neither bas al nor LH-stimulated androgen production. Coculture with either type o f activated macrophage did not alter basal, but significantly reduced (by 50%) LH-stimulated, androgen production. N-omega-Nitro-L-arginine methyl ester and C-PTIO blocked the inhibitory effect. The NO donor S- nitroso-N-acetyl penicillamine at concentrations greater than 10(-5) M significantly inhibited LH-stimulated androgen production by purified LC (P < 0.01). The inhibitory effect of S-nitroso-N-acetyl penicillam ine was evident when exposure exceeded 4 h. Intermediates of steroidog enesis were added to elucidate the site of NO inhibition. The enzymati c inhibition occurred at least in part at 17 alpha-hydroxylase/C-17/20 lyase (P450c17). Enzyme inhibition was reversed by C-PTIO. Northern b lot analysis indicated that accumulation of messenger RNA for P450c17 was not significantly altered. Therefore, activation of macrophages re sults in decreased androgen production by cocultured LC. The inhibitio n is mediated in part by macrophage-derived NO acting directly on the LC via inhibition of at least one of the P450 steroidogenic enzymes.