Norepinephrine stimulates mitogen-activated protein kinase activity in GT1-1 gonadotropin-releasing hormone neuronal cell lines.
The GT1-1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1-1 cells with 75 mM KCl, 10 microM BayK 8644, or 1 microM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1-1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.
Sawada
T
T
Department of Obstetrics and Gynecology, Osaka University Medical School, Suita, Japan.
Ohmichi
M
M
Koike
K
K
Kanda
Y
Y
Kimura
A
A
Masuhara
K
K
Ikegami
H
H
Inoue
M
M
Miyake
A
A
Murata
Y
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology; D009638:Norepinephrine / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D007987:Gonadotropin-Releasing Hormone; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D002118:Calcium; D009638:Norepinephrine