Characterization of .ALPHA.T3-1 Cells Stably Transfected with Luteininzing Hormone .BETA.-Subunit Complementary Deoxyribonucleic Acid

YONEHARA TOSHIE, YAMADA YOKO, KANASAKI HARUHIKO, YAMAMOTO HIDEYUKI, FUKUNAGA KOHJI, MIYAZAKI KOHJI, MIYAMOTO EISHICHI

doi:10.1507/endocrj.50.341

Luteinizing hormone (LH) consists of α- and β-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 αT3-1 cells with rat LHβ-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 α-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 μM 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 μM 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 LHβ-subunit synthesis, and that LH2 cells are a useful model for the study of LH secretion induced by several secretagogues.

Characterization of .ALPHA.T3-1 Cells Stably Transfected with Luteininzing Hormone .BETA.-Subunit Complementary Deoxyribonucleic Acid

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