Pretreatment with phorbol ester and an LHRH asgonist 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-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).

Propranolol stimulates testicular interstitial fluid formation and testosterone secretion in rats

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.

Modulatory effects of adrenergic agonists on testosterone secretion from rat dispersed testicular cells or Percoll-purified Leydig cells

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