Nitric oxide is a mediator of the inhibitory effect of activated macrophages on production of androgen by the Leydig cell of the mouse
Abstract
We hypothesized that macrophage activation results in nitric oxide (NO) production and that this NO acts directly on Leydig cells (LC) to alter androgen synthesis. Both peritoneal macrophages and a murine macrophage cell line (RAW 264.7) were activated in vitro by sequential exposure to interferon-gamma (50 U/ml) and then bacterial lipopolysaccharide (LPS; 100 ng/ml) for 24 h each. At various times after initiation of activation, selected wells were harvested for identification of messenger RNA for inducible NO synthase by RT-PCR. Amplicons of the predicted 651-bp product were isolated, cloned, and sequenced to validate the PCR procedure. Such amplicons first appeared between 2-4 h after exposure 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 purified LC for a final 24-h incubation in the absence of interferon-gamma and LPS. Basal and LH-stimulated production of androgen was estimated by RIA. In some experiments the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO) was added during activation and coculture. Coculture of LC with quiescent macrophages altered neither basal nor LH-stimulated androgen production. Coculture with either type of activated macrophage did not alter basal, but significantly reduced (by 50%)…
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