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%) 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 penicillamine was evident when exposure exceeded 4 h. Intermediates of steroidogenesis were added to elucidate the site of NO inhibition. The enzymatic inhibition occurred at least in part at 17alpha-hydroxylase/C(17/20) lyase (P450c17). Enzyme inhibition was reversed by C-PTIO. Northern blot analysis indicated that accumulation of messenger RNA for P450c17 was not significantly altered. Therefore, activation of macrophages results in decreased androgen production by cocultured LC. The inhibition is mediated in part by macrophage-derived NO acting directly on the LC via inhibition of at least one of the P450 steroidogenic enzymes.

An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

“…60,61 It is believed that NO inhibits the synthesis and release of testosterone from Leydig cells through the inhibition of steroidogenic enzymes, most likely P 450 scc, in a cGMP-independent manner. 6,62 Our current work adds newer perspective to the concept that NO derived from nNOS expressed in Leydig cells may be acting in an autocrine manner to regulate Leydig cell function. Specifically, NO produced from Tn-NOS-positive Leydig cells may act on these same cells as an intercellular and intracellular signaling molecule.…”

Nitric oxide synthase activity and progesterone release by isolated corpora lutea of rabbits in the early and mid-luteal phases of pseudopregnancy are modulated differently by prostaglandin E-2 and prostaglandin F-2alpha via adenylate cyclase and phospholipase C

“…In the past few years, NO has also emerged as a regulator of multiple functions within the reproductive system (Shukovski & Tsafriri 1995, Bonello et al 1996, Yamauchi et al 1997, and several findings suggest that it may have a significant role in the control of steroidogenesis in both ovary and testes (Adams et al 1992, Welch et al 1995, Olson et al 1996, Hesla et al 1997, Jablonka-Shariff & Olson 1997, Pomerantz & Pitelka 1998, Vega et al 1998.…”

Nitric Oxide and Cyclic Nucleotides: Their Roles in Junction Dynamics and Spermatogenesis

“…47 Other studies have demonstrated that the activated macrophage-produced NO is associated with a reduction in the testosterone producing activity in Leydig cells. 48 Notably, this inhibition is at least in part involved with the blockade of the P450 steroidogenic enzymes. 48 In short, the bi-directional relationship manifested by NOS/NO and the hormone/cytokine level is crucial in maintaining the physiological function of the testes.…”

“…48 Notably, this inhibition is at least in part involved with the blockade of the P450 steroidogenic enzymes. 48 In short, the bi-directional relationship manifested by NOS/NO and the hormone/cytokine level is crucial in maintaining the physiological function of the testes.…”