MECHANISM OF ACTION OF PEPTIDE HORMONES IN STEROIDOGENIC CELLS

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Cytochrome P450-17alpha (CYP17), the key enzyme for the synthesis of androgens and glucocorticoids, catalyses two consecutive oxidation reactions to produce 17alpha-hydroxylated (hydroxylase) intermediate and androstene products (17-20 lyase). Within the conserved domain (343 to 363), charged amino acids contribute to lyase and hydroxylase activities. Arg 346 is essential for lyase activity and Arg 363 is of importance for hydroxylase activity. Genomic analysis revealed that this domain is contained in exon 6. Substitution of Arg 361 and Glu 358 within the conserved domain with alanine abolished all hydroxylase and lyase activities. These mutants showed a pattern of premature degradation in transfection studies. Although the mutant protein levels were close to wild type at early times, the mutant protein was inactive at these time periods. Pulse chase studies revealed marked decreases in half-life from 10 hours to 2 hours. Additional substitutions show that a negative charge and the extended length of the side chain of Glu 358 is of importance for the hydroxylase and lyase activities. Our studies indicate that Glu 358 and Arg 361 may contribute to the folding of the steroid binding domain of CYP17 and that their substitution results in misfolding and degradation. In the Leydig cells, serotonin is an acute stimulus of CRF secretion and its action is exerted via high-affinity low capacity 5HT2 receptors that are located in the plasma membrane. In recent studies we defined the regulation of serotonin secretion in cultured Leydig cells. Adult Leydig cells secrete considerable quantities of serotonin. The release was acutely stimulated by human chorionic gonadotropin and by cyclic adenosine monophosphate, indicating its major role in gonadotropin-induced serotonin release. hCG stimulation did not require extracellular calcium. However, ionomycin was a potent stimulus of serotonin release, indicating that increases in cytoplasmic calcium could also induce amine release. The serotonin content of Leydig cells decreased during stimulation of serotonin release. Immunocytochemical studies revealed specific staining of serotonin in the interstitial cells of the adult rat testis. Testicular serotonin has local antireproductive actions and may serve as the trigger of an autocrine microcircuit that buffers the gonadotropin stimulus. Intratesticular increases of serotonin concentration could occur in conditions known to increase testicular (varicocele) and central (stress) serotonin, and may contribute to the disruption of testicular function observed in these states.