title: Intracellular mechanisms involved in copper-gonadotropin-releasing hormone (Cu-GnRH) complex-induced cAMP/PKA signaling in female rat anterior pituitary cells in vitro.
abstract: The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu(2+) ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine(2). A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes. To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1 μM antide, a GnRH antagonist; 0.1 μM cetrorelix, a GnRH receptor antagonist; 0.1 μM PACAP6-38, a PAC-1 receptor antagonist; 2 μM GF109203X, a protein kinase C inhibitor; 50 mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30 μM) and KT5720 (60 nM); factors affecting intracellular calcium activity: 2.5 mM EGTA; 2 μM thapsigargin; 5 μM A23187, a Ca(2+) ionophore; or 10 μg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1 μM Cu-GnRH for 0.5, 1, and 3 h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca(2+)-dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures. Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu-GnRH molecule including specific pattern of intracellular activity induced by complex in anterior pituitary cells in vitro.
authors.affiliation: Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jablonna n. Warsaw, Poland. Electronic address: a.gajewska@ifzz.pl.
authors.forename: Alina
authors.initials: A
authors.lastname: Gajewska
authors.affiliation: Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jablonna n. Warsaw, Poland.
authors.forename: Marlena
authors.initials: M
authors.lastname: Zielinska-Gorska
authors.affiliation: Neuroendocrinology Department, Medical Centre for Postgraduate Education, Marymoncka 99/103 st., 01-813 Warsaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Wolinska-Witort
authors.affiliation: Department of Animal Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A st. 10-719 Olsztyn, Poland.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Siawrys
authors.affiliation: Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jablonna n. Warsaw, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Baran
authors.affiliation: Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jablonna n. Warsaw, Poland.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Kotarba
authors.affiliation: Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jablonna n. Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Biernacka
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003300:Copper / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017208:Rats, Wistar; D011966:Receptors, LHRH / Q000037:antagonists & inhibitors / Q000378:metabolism; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D011966:Receptors, LHRH; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D005576:Colforsin; D007987:Gonadotropin-Releasing Hormone; D003300:Copper; D007986:Luteinizing Hormone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium
keywords: Cu-GnRH; Gonadoliberin; Intracellular calcium; PKC; Rat pituitary; cAMP