title: Divergent roles of protein kinase C in luteinizing hormone biosynthesis versus release in rat anterior pituitary cells.
abstract: We previously demonstrated that protein kinase C (PKC) activators, i.e. L-alpha-1,2-dioctanoyl glycerol (C8) and phorbol 12-myristate 13-acetate (PMA), mimic the stimulatory effects of GnRH on both LH glycosylation and release. To further evaluate the roles of PKC, we determined: 1) the interaction between PKC activator and GnRH; and 2) the effects of depleting cellular PKC with a high dose of PMA on LH glycosylation vs. release. Anterior pituitaries excised from ovariectomized rats were enzymatically dispersed and cultured. In series 1 experiments, day 3 monolayer cells were incubated in the presence of radiolabeled precursors and GnRH (0, 1, or 100 nM), with or without C8 (200 microM). In series 2 experiments, day 2 cells were pretreated with either PMA (1 microM) or vehicle (0.08% dimethyl sulfoxide) for 24 h and then incubated with diluent, GnRH (1 nM), or PMA (20 nM), and radiolabeled precursors for 4 h. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine ([14C]A) and [3H]glucosamine ([3H]GA), respectively, into LH. Immunoreactive LH (IRLH) was measured by RIA. In series 1 experiments, C8 increased basal release of IRLH, potentiated IRLH release stimulated by 1 nM GnRH, but not by 100 nM GnRH. C8 elevated total [3H]GA-LH but had no additive effects with GnRH. In series 2 experiments, PMA pretreatment inhibited subsequent PMA-stimulated IRLH release. However, PMA pretreatment did not affect GnRH-induced IRLH release even though PMA pretreatment decreased cellular IRLH content. In comparison, PMA pretreatment reduced both GnRH- and PMA-stimulated total [3H]GA-LH. PMA pretreatment had no effects on total [14C]A-LH in the presence of GnRH or PMA, but reduced the basal level. In summary, PKC activators had no additive effects on either IRLH release or LH glycosylation stimulated by a maximal dose of GnRH. However, PMA pretreatment decreased GnRH-induced LH glycosylation without depressing LH release. These results suggest differential roles of PKC in the actions of GnRH on LH glycosylation vs. LH release.
authors.affiliation: Institute of Physiology, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
authors.forename: T C
authors.initials: TC
authors.lastname: Liu
authors.forename: H F
authors.initials: HF
authors.lastname: Pu
authors.forename: G L
authors.initials: GL
authors.lastname: Jackson
mesh_terms: D000409:Alanine / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005944:Glucosamine / Q000378:metabolism; D006031:Glycosylation; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D007986:Luteinizing Hormone / Q000096:biosynthesis* / Q000378:metabolism*; D010052:Ovariectomy; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium
chemical_list: D002250:Carbon Radioisotopes; D014316:Tritium; D007987:Gonadotropin-Releasing Hormone; D007986:Luteinizing Hormone; D011493:Protein Kinase C; D005944:Glucosamine; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine