Characterization of alphaT3-1 cells stably transfected with luteininzing hormone beta-subunit complementary deoxyribonucleic acid.
Luteinizing hormone (LH) consists of alpha- and beta-subunits, and synthesis and secretion of LH are regulated by gonadotropin-releasing hormone (GnRH). In order to examine the molecular mechanisms by which GnRH regulates LH secretion, we transfected alphaT3-1 cells with rat LHbeta-subunit cDNA under the control of a constitutive promoter and established a stable cell line of LH2 cells which secreted LH in response to GnRH. Pulsatile and continuous GnRH pretreatments increased gene expression of the alpha-subunit and synthesis of LH, and enhanced the LH secretion by brief treatments with GnRH and 56 mM KCl. The LH secretions were partially blocked by elimination of extracellular Ca2+. GnRH-induced LH secretion was completely inhibited by calphostin C (a protein kinase C inhibitor) and 1 microM wortmannin. In contrast to the GnRH induction, high K+-induced LH secretion was inhibited by KN93, a Ca2+/calmodulin-dependent protein kinase II inhibitor, as well as by 1 microM wortmannin. We also confirmed that activation of cAMP-pathway induced LH secretion, but activation of mitogen-activated protein (MAP) kinase pathway was not involved in LH secretion. These results suggest that GnRH directly regulates LH secretion as well as LHbeta-subunit synthesis, and that LH2 cells are a useful model for the study of LH secretion induced by several secretagogues.
Yonehara
Toshie
T
Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan.
Yamada
Yoko
Y
Kanasaki
Haruhiko
H
Yamamoto
Hideyuki
H
Fukunaga
Kohji
K
Miyazaki
Kohji
K
Miyamoto
Eishichi
E
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000502:physiology*; D015151:Immunoblotting; D037101:Luteinizing Hormone, beta Subunit / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D009479:Neuropeptides / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C495403:Adcyap1 protein, rat; D018076:DNA, Complementary; D037101:Luteinizing Hormone, beta Subunit; D009281:Naphthalenes; D009479:Neuropeptides; D010455:Peptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; C065738:MLCK peptide; D007987:Gonadotropin-Releasing Hormone; D000242:Cyclic AMP; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C058819:calphostin C