Systemic administration of alcohol to adult rats inhibits leydig cell activity: Time course of effect and role of nitric oxide.
BACKGROUND
Alcohol has been shown to interfere with testosterone (T) release from Leydig cells. However, the mechanisms responsible for this phenomenon, which may include decreased activity of the luteinizing hormone-releasing hormone (LHRH)-LH axis, as well as a direct influence of the drug on the testes, are not fully understood. In this work, we investigated the influence of alcohol, administered intragastrically (i.g.) or delivered via vapors, on Leydig cell activity and T release. Leydig cell function was studied by measuring changes in the levels of the steroidogenic proteins steroidogenic acute regulatory (StAR), the peripheral-type benzodiazepine receptor (PBR), and the cytochrome P450 side-chain cleavage enzyme (P450scc). Testosterone release was studied under basal conditions or in response to human chorionic gonadotropin (hCG). Finally, to identify potential factors mediating the influence of alcohol, we measured the testicular variant of the neuronal nitric oxide (NO) synthase (NOS), TnNOS, in semipurified Leydig cells.


METHODS
Adult male Sprague-Dawley rats were either injected with alcohol i.g. once or exposed to alcohol vapors (4 h/d) for 1 or 5 days. Controls received the vehicle (i.g. model) or were kept in boxes through which no vapors were circulated. Following these treatments, one series of experiments was devoted to investigate Leydig cell responsiveness by measuring plasma T levels before or after the intravenous injection of hCG (1 U/kg). In another series of experiments, we used semipurified Leydig cell preparations to measure StAR, PBR, P450scc, and TnNOS by Western blot analysis.


RESULTS
In the i.g. model, the T response to hCG was blunted for 12 hours following alcohol injection, but showed a rebound at 48 hours. Levels of StAR protein and of PBR, but not of P450scc, were significantly decreased within 10 minutes of drug administration. While StAR then remained depressed for 24 hours, PBR values were variable over this time course. By 48 hours, StAR, PBR, and P450scc levels had increased above control values. Both StAR and PBR levels showed correlations with plasma T levels. In the alcohol vapor models, both regimens of the drug also significantly depressed StAR and PBR protein concentrations, blunted the T response to hCG, and did not alter P450scc. Finally, we observed that alcohol delivered i.g. or via vapors up-regulated TnNOS levels in Leydig cells, but that blockade of NO formation failed to restore a normal T response to hCG.


CONCLUSIONS
Collectively, these results suggest that (a) the ability of Leydig cells to release T does not show a simple correlation with changes in StAR, PBR, and P450scc levels; (b) the time course of the alcohol-induced changes were protein-specific; and (c) despite the ability of alcohol to stimulate TnNOS expression, NO does not appear to mediate the inhibitory influence of this drug on testicular steroidogenesis in the models that we studied.
Herman
Melissa
M
Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037, USA.
Kang
Sang Soo
SS
Lee
Soon
S
James
Peter
P
Rivier
Catherine
C
D000280:Administration, Inhalation; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002469:Cell Separation; D002492:Central Nervous System Depressants / Q000008:administration & dosage / Q000097:blood / Q000494:pharmacology*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000008:administration & dosage / Q000097:blood / Q000494:pharmacology*; D007202:Indicators and Reagents; D007275:Injections, Intravenous; D007985:Leydig Cells / Q000187:drug effects*; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D013997:Time Factors; D000098682:Steroidogenic Acute Regulatory Protein
D002492:Central Nervous System Depressants; D006063:Chorionic Gonadotropin; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D010750:Phosphoproteins; D011963:Receptors, GABA-A; D000098682:Steroidogenic Acute Regulatory Protein; D009569:Nitric Oxide; D000431:Ethanol; D013739:Testosterone; D052248:Nitric Oxide Synthase Type I; D002786:Cholesterol Side-Chain Cleavage Enzyme; D019331:NG-Nitroarginine Methyl Ester