The hypotheses to be tested by the experiments in this proposal are: 1. Acute and chronic ethanol (EtOH) exposure in adult male rats, acting at the pituitary level, is disruptive to luteinizing hormone (LH) synthesis and secretion at multiple steps. Pretranslationally EtOH perturbs intracellular transmembrane signalling and diminishes LHbeta mRNA synthesis and stability; post-translationally EtOH alters LH glycosylation and secretion. 2. Ethanol exposure does not modify luteinizing hormone releasing hormone (LHRH) gene transcription or secretion in adult male rats. The experiments are logical extensions of our previous studies demonstrating that acute EtOH exposure profoundly reduces serum LH and steady state LHbeta mRNA (but not the common alpha subunit mRNA), without affecting LHRH mRNA content or secretion. The pituitary experiments will examine the effects of a single injection of I.P. EtOH compared with I.P. saline and chronic (3 and 14 days) EtOH administration by gastric cannula on key events in pituitary LH synthesis and secretion. First we will examine EtOH's effects on the membrane translocation and activity of protein kinase C since this is a key intracellular transduction pathway in the LHRH-LH cascade. This will be done both after in vivo EtOH administration on whole pituitary and after in vitro EtOH exposure to an enriched gonadotrope cell population. Secondly, since we have shown that EtOH markedly reduces steady-state LHbeta mRNA, we will determine EtOH's impact on LHbeta mRNA synthesis (by nuclear run-on assays) and stability. Thirdly, since our previous work suggested that EtOH blocked LH secretion as well as synthesis, we will investigate its effects on LH trafficking through cytoplasm (by subcellular distribution analysis) and on LH glycosylation, important to secretion. The hypothalamic experiments will address chronic EtOH effects only since our previous work has failed to show any acute EtOH effects on hypothalamic LHRH physiology. The chronic administration of EtOH (3 and 14 days of EtOH or control diet by gastric cannula) on LHRH mRNA (by quantitative PCR), LHRH content, and LHRH secretion will be determined. In complimentary studies, the effect of chronic in vitro EtOH exposure on LHRH mRNA, peptide content, and secretion from the LHRH clonal cell line, GT1, will be assessed. The hypotheses are plausible in light of EtOH's effects on cell systems other than pituitary and hypothalamus, and can be accomplished since all the techniques are already operative in our laboratory. The experiments proposed meld the techniques of classical endocrine physiology (RIA's, cell culture) with those of modern molecular biology (Northern and Western blots, PCR analysis and nuclear run-on assays).
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