NEUROSTEROIDOGENESIS IN RAT RETINAS

Neurosteroids (steroids synthesized in the CNS) function by modulating neurotransmission. To establish an experimental model for investigati on of neurosteroid synthesis and regulation, independent of blood-born e steroids, we examined the steroidogenic activity of isolated rat ret inas. We identified progesterone, pregnenolone, dehydroepiandrosterone , desoxycorticosterone, 3 alpha,5 alpha-tetrahydrodesoxycorticosterone , 3 alpha-hydroxy-5 alpha-dihydroprogesterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone together with their esterified forms. As p regnenolone is the precursor of all steroids, its formation was studie d in detail as an index of a steroid-synthesizing tissue. Pregnenolone was identified further by gas chromatography coupled to mass spectrom etry, and its in vitro synthesis was inhibited by lovastatin, an inhib itor of mevalonolactone and cholesterol biosynthesis. We then examined pregnenolone synthesis in the presence of mevalonolactone as a precur sor of sterol formation together with lovastatin, which reduces endoge nous mevalonolactone synthesis, as well as with inhibitors of pregneno lone metabolism. The incorporation of mevalonolactone into pregnenolon e and its sulfate ester was time- and concentration-dependent and bloc ked by aminoglutethimide, a competitive inhibitor of cytochrome P450 s ide-chain cleavage (P450(scc)) enzyme. Immunocytochemical studies with a specific antibody to P450(scc) revealed a primary localization of t he enzyme at the retinal ganglion cell layer. A less pronounced immuno staining was also seen at cells of the inner nuclear layer. Compounds known to stimulate cyclic AMP content also stimulated pregnenolone for mation by rat retinas. These results demonstrate that rat retinas synt hesize steroids and, for the first time, they reveal the steroidogenic ability of neuronal cells. We propose rat retinas as an in vitro mode l system to study neurosteroidogenesis in the CNS.