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The aim of the present study was to evaluate the effects of pituitary adenylate cyclase activating polypeptide (PACAP) on testosterone production in isolated adult rat Leydig cells and its possible mechanisms of action. PACAP-38 stimulated testosterone secretion in a dose-dependent manner with a minimal and a maximal efficacious dose of 1.0 nM and 100 nM, respectively. PACAP-27 was without effect on testosterone secretion at any dose tested. Similarly, vasoactive intestinal peptide did not stimulate steroidogenesis nor interfere with PACAP-38 activity, as well as preincubation of Leydig cells with the vasoactive intestinal peptide-antagonist [Lys(1), Pro(2,5), Arg(3,4), Tyr(6)]-vasoactive intestinal peptide. Removal of extracellular Ca2+ did not inhibit the stimulatory effects of PACAP-38 on Leydig cell testosterone production. Neither PACAP-38 nor PACAP-27 modified intracellular free Ca2+ and cAMP levels at any dose tested thus excluding a role for Ca2+ and cAMP in the stimulatory effects of PACAP. PACAP-38 was able to induce a plasma membrane depolarization that was dependent on an influx of Na+ from the extracellular medium as confirmed by the monitoring of intracellular Na+ with the Na+-sensitive fluorescent dye sodium benzofuran isophtalate. When Na+ was removed from the extracellular medium, PACAP-38 did not stimulate testosterone production, demonstrating that Na+ influx through the plasma membrane is strictly related to the stimulatory effects of this peptide. In addition, preincubation of Leydig cells in the presence of pertussis-toxin (500 ng/ml for 5 h) significantly reduced PACAP-38-stimulated effects both on plasma membrane depolarization and testosterone secretion. These results demonstrate that PACAP-38 stimulates testosterone secretion in isolated adult rat Leydig cells through the interaction with a novel PACAP receptor subtype coupled to a pertussis toxin sensitive G protein whose activation induces a Na+-dependent depolarization of the plasma membrane and testosterone production.

Pituitary Adenylate Cyclase Activating Polypeptide Stimulates Rat Leydig Cell Steroidogenesis Through a Novel Transduction Pathway