Pretreatment with phorbol ester and an LHRH agonist reduces testosterone production and protein kinase C activity in rat Leydig cells challenged with PDBu and LHRH.
We have investigated the role of protein kinase C (PK-C) in luteinizing hormone-releasing hormone (LHRH)-induced testosterone secretion from purified rat Leydig cells (70-80-day old Sprague-Dawley rats) by pretreating the cells in vitro with 200 mM phorbol 12,13-dibutyrate (PDBu) (a known procedure to down-modulate this enzyme in most cell types) and 1 muM [D-Ala6,Des-Gly10]-LHRH ethylamide, an LHRH agonist (LHRH-A). Following pretreatment we measured PK-C activity and secretion of testosterone in response to subsequent challenges with the PK-C activator PDBu (20-2000 nM) and with LHRH (0.001-1.0 muM) and the Ca(2+)-mobilizing secretagogue A23187 (0.1-100 microM) in the same cell preparation. PDBu and LHRH-A pretreatments caused a reduction in testosterone secretion in response to subsequent exposure to PDBu or LHRH. Both pretreatments decreased PK-C activity in crude and purified extracts of the same cells. The magnitude of reduction of the secretory response was greater than that of enzyme activity for both PDBu and LHRH-A pretreatment (68.9% reduction of testosterone secretion vs 54.7% reduction of PK-C activity in PDBu-pretreated cells and 78.6% reduction of testosterone production vs 36.6% reduction of PK-C activity in LHRH-A-pretreated cells). The effect of phorbol ester pretreatment on PDBu- or LHRH-stimulated testosterone secretion and PK-C activity was specific (no measurable effect with 4 alpha-PDBu, an inactive phorbol ester). While PDBu and LHRH-A pretreatment reduced Leydig cell responsiveness to PDBu or LHRH, the secretion of testosterone in response to the Ca2+ -mobilizing secretagogue A23187 was similar in PDBu- and LHRH-A-pretreated and in control (non-pretreated) cells. We conclude that down-modulation of protein kinase C by prolonged exposure of Leydig cells to phorbol esters or LHRH-A results in decreased PK-C activity and testosterone secretion. These results provide the first evidence that pretreatment with LHRH-A, which does not enter the cell, can affect the steroidogenesis and PK-C activity responses to PDBu (the intracellular ligand of PK-C).
Wanderley
M I
MI
Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. miw@npd.ufe.br
Negro-Vilar
A
A
D000818:Animals; D007987:Gonadotropin-Releasing Hormone / Q000008:administration & dosage* / Q000819:agonists* / Q000494:pharmacology; D066298:In Vitro Techniques; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000008:administration & dosage* / Q000494:pharmacology; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013739:Testosterone / Q000096:biosynthesis*
D010703:Phorbol Esters; D007987:Gonadotropin-Releasing Hormone; D015240:Phorbol 12,13-Dibutyrate; D013739:Testosterone; D011493:Protein Kinase C