ABSTRACT
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-Glyl0]-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 Ca2+ mobilizing secretagogue A23187 (0.1-1OO muM) 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 per cent reduction of testosterone secretion vs 54.7 per cent reduction of PK-C activity in PDBu-pretreated cells and 78.6 per cent reduction of testosterone production vs 36.6 per cent 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).
Subject(s)
Rats , Male , Animals , Gonadotropin-Releasing Hormone/administration & dosage , Gonadotropin-Releasing Hormone/agonists , In Vitro Techniques , Leydig Cells/drug effects , Phorbol 12,13-Dibutyrate/pharmacology , Phorbol Esters/administration & dosage , Protein Kinase C/metabolism , Testosterone/biosynthesis , Gonadotropin-Releasing Hormone/pharmacology , Phorbol Esters/pharmacology , Protein Kinase C/drug effects , Rats, Sprague-DawleyABSTRACT
We investigated the effect of intratesticularly injected propranolol on testicular interstitial fluid (TIF) formation and on testosterone levels in the TIF of intact adult male Wistar rats (4-9 rats per group). D1-propranolol at doses of 0.6, 1.2 or 6.0 mg/kg was injected into the left (L) testis whereas the right (R) testis (control testis) received vehicle. d1-propranolol (6.0 mg/kg) caused a significant increase in both TIF volume (329 per cent) and TIF levels of testosterone (257 per cent) in the L testis but not in the R (control) testis 3 h post-injection. In rats treated simultaneously with human chorionic gonadotropin (hCG, 5 IU/rat, sc) the same dose of propranolol (6.0 mg/kg) significantly increased the stimulatory effect of hCG on testosterone secretion by 1.8-fold, but hCG did not modify the stimulatory effect of propranolol on TIF volume. These results demonstrate a direct stimulatory effect of propranolol on TIF volume and testosterone secretion, both under basal and hCG-stimulated conditions.
Subject(s)
Rats , Animals , Male , Chorionic Gonadotropin/pharmacology , Leydig Cells/drug effects , Propranolol/pharmacology , Testosterone/metabolism , Extracellular Space/drug effects , Rats, WistarABSTRACT
Freshly dispersed testicular interstitial cells as well as Percoll-purified Leydig cells were studied in vitro in order to evaluate the effect of adrenergic agonists on testosterone (T) secretion. Epinephrine and phenylephrine did not change the rate of T release under basal conditions in freshly dispersed interstitial cells, but enhanced it during human chorionic gonadotropin (hCG) stimulation. Norepinephrine and clonidine had no effect on T secretion. In contrast, in Percoll-purified Leydig cells epinephrine increased T release both under basal and hCG-stimulated conditions. These data demonstrate that neurotransmitters may participate in T secretion from isolated Leydig cells