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Involvement of Nitric Oxide Synthase in the Mechanism of Histamine-Induced Inhibition of Leydig Cell Steroidogenesis via Histamine Receptor Subtypes in Sprague-Dawley Rats¹

Laboratory of Molecular Endocrinology and Signal Transduction,³ Institute of Biology and Experimental Medicine–Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), CP 1428, Buenos Aires, Argentina Physical Chemistry–Programa de Radicales Libres, School of Pharmacy and Biochemistry,⁴ Department of Human Biochemistry, School of Medicine,⁵ and Department of Biological Chemistry, School of Sciences,⁶ University of Buenos Aires (UBA), CP 1428, Buenos Aires, Argentina

ABSTRACT

This study was conducted to shed light on the so far unexplored intracellular mechanisms underlying negative modulation of Leydig cell steroidogenesis by histamine (HA). Using the MA-10 cell line and highly purified rat Leydig cells as experimental models, we examined the effect of the amine on biochemical steps known to be modulated by HA or involved in LH/hCG action. In agreement with previous findings, HA at 10 µM showed a potent inhibitory effect on hCG-stimulated steroid synthesis, regardless of the gonadotropin concentration used. Moreover, HA decreased not only LH/hCG-induced cAMP production but also steroid synthesis stimulated by the permeable cAMP analog dibutyryl cAMP (db-cAMP). Considering the post-cAMP sites of HA action, it is shown herein that HA markedly inhibited db-cAMP-stimulated steroidogenic acute regulatory (STAR) protein expression, as well as steps catalyzed by P450-dependent enzymes, mainly the conversion of cholesterol to pregnenolone by cholesterol side-chain cleavage enzyme (CYP11A). The antisteroidogenic action of HA was blocked by addition of the phospholipase C (PLC) inhibitor U73122, and HA significantly augmented inositol triphosphate (IP₃) production, suggesting a major role for the PLC/IP₃ pathway in HA-induced inhibition of Leydig cell function. Finally, HA increased nitric oxide synthase (NOS) activity, and the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) markedly attenuated the effect of the amine on steroid synthesis. On the basis of our findings, HA antagonizes the gonadotropin action in Leydig cells at steps before and after cAMP formation. NOS activation is the main intracellular mechanism by which HA exerts its antisteroidogenic effects.

histamine, Leydig cells, nitric oxide, testis, testosterone

¹Supported by Agencia Nacional de Promoción Científica y Tecnológica grant PICT 2005 5-38281, by CONICET grant PIP 5525, by Grant Carrillo-Oñativia to C.M. and O.P.P., and by UBA grant X814 to O.P.P.

Correspondence: ²FAX: 54 011 4786 2564; e-mail: mondillo{at}dna.uba.ar