Characterization of G proteins involved in activation of nonselective cation channels and arachidonic acid release by norepinephrine/alpha1A-adrenergic receptors.
We demonstrated recently that norepinephrine activates Ca2+ -permeable nonselective cation channels (NSCCs) in Chinese hamster ovary cells stably expressing alpha1A-adrenergic receptors (CHO-alpha1A). Moreover, extracellular Ca2+ through NSCCs plays essential roles in norepinephrine-induced arachidonic acid release. The purpose of the present study was to identify the G proteins involved in the activation of NSCCs and arachidonic acid release by norepinephrine. For these purposes, we used U73122, an inhibitor of phospholipase C (PLC), and dominant negative mutants of G12 and G13 (G12G228A and G13G225A, respectively). U73122 failed to inhibit NSCCs activation by norepinephrine. The magnitudes of norepinephrine-induced extracellular Ca2+ influx in CHO-alpha1A microinjected with G13G225A were smaller than those in CHO-alpha1A. In contrast, the magnitudes of norepinephrine-induced extracellular Ca2+ influx in CHO-alpha1A microinjected with G12G228A were similar to those in CHO-alpha1A. In addition, neither a Rho-associated kinase (ROCK) inhibitor nor a phosphoinositide 3-kinase inhibitor affected norepinephrine-induced extracellular Ca2+ influx. G13G225A, but not G12G228A, also inhibited arachidonic acid release partially. These results demonstrate that 1) the Gq/PLC-pathway is not involved in NSCCs activation by norepinephrine, 2) G13 couples with CHO-alpha1A and plays important roles for norepinephrine-induced NSCCs activation, 3) neither ROCK- nor PI3K-dependent cascade is involved in NSCCs activation, and 4) G13 is involved in norepinephrine-induced arachidonic acid release in CHO-alpha1A.
Kawanabe
Yoshifumi
Y
Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan. ykawanabe@rics.bwh.harvard.edu
Hashimoto
Nobuo
N
Masaki
Tomoh
T
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000577:Amides / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D016466:CHO Cells; D015220:Calcium Channels / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D009638:Norepinephrine / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D014162:Transfection; D000077191:Wortmannin
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D000577:Amides; D000730:Androstadienes; D015220:Calcium Channels; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D004963:Estrenes; D010726:Phosphodiesterase Inhibitors; D011725:Pyridines; D011760:Pyrrolidinones; D018340:Receptors, Adrenergic, alpha-1; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C108830:Y 27632; D016718:Arachidonic Acid; D019204:GTP-Binding Proteins; D009638:Norepinephrine; D000077191:Wortmannin