title: Activation of intracellular pathways with forskolin and phorbol ester increases LHRH mRNA level in the rat hypothalamus superfused in vitro.
abstract: Two intracellular signal transduction mechanisms such as cAMP-protein kinase a and phosphatidylinositol (PI) turnover-protein kinase c are known to be dually involved in the regulation of luteinizing hormone-releasing hormone (LHRH) release. However, it is not yet evident that the activation of two intracellular pathways affects the LHRH gene expression. The present study aims, therefore, to determine whether the activation of two intracellular pathways affects changes in LHRH mRNA. To this end, we took advantage of an in vitro superfusion system, where rat hypothalamic tissues were superfused with media containing forskolin (FKN) and/or phorbol-12-myristate-13-acetate (PMA). Superfusates were collected at 10-min intervals and LHRH release was determined by radioimmunoassay. After a 2-h superfusion period, the post-superfusion hypothalami were recovered and poly (A) RNA fractions were isolated. Alterations in LHRH mRNA in response to FKN and/or PMA were determined by an RNA-blot hybridization assay using a 32P-end-labeled LHRH oligonucleotide (29-mer) probe. In vitro perfusion of hypothalamic fragments with PMA and/or FKN stimulated LHRH release as well as LHRH mRNA. The combined infusion of FKN and PMA did not produce an additive effect on the LHRH mRNA levels, but it was effective in synergistically increasing LHRH secretion in vitro. These data clearly demonstrate that the biosynthetic machinery of LHRH is influenced by activation of two intracellular pathways, both cAMP-protein kinase a and phosphatidyl-inositol turnover-protein kinase c, indicating the transsynaptic regulation of hypothalamic LHRH gene expression.
authors.affiliation: Department of Zoology, College of Natural Sciences, Seoul National University, South Korea.
authors.forename: B J
authors.initials: BJ
authors.lastname: Lee
authors.forename: K
authors.initials: K
authors.lastname: Kim
authors.forename: W K
authors.initials: WK
authors.lastname: Cho
mesh_terms: D000818:Animals; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics*; D007031:Hypothalamus / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis / Q000187:drug effects / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D005576:Colforsin; D007987:Gonadotropin-Releasing Hormone; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate