Ca(2+)- and protein kinase C-dependent stimulation of mitogen-activated protein kinase in detergent-skinned vascular smooth muscle.
Protein kinase C and mitogen-activated protein (MAP) kinase are expressed in all smooth muscle cells and believed to be important in several physiologically relevant properties of this muscle. Our goal was to determine if protein kinase C and MAP kinase are activated by a simple increase in cellular Ca(2+) and to determine if protein kinase C is an upstream activator of MAP kinase. These studies were performed in the Triton X-100 detergent-skinned preparation of the swine carotid artery, which allows control of the intracellular environment without influence from membrane or receptor-mediated modulation. The p42 and p44 isoforms of MAP kinase were activated in a concentration-dependent fashion by an increase in Ca2+. This was shown by in-the-gel kinase assay and direct measurement of MAP kinase phosphotransferase activity. Protein kinase C was also activated by an increase in Ca2+, as shown by a novel assay that measures total active protein kinase C in the tissue. Inhibition of protein kinase C activity completely abolished MAP kinase activity. Additionally, inhibition of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) also abolished MAP kinase activity. Using intact swine carotid arteries, we showed p42 and p44 MAP kinase to be activated by both histamine and phorbol dibutyrate, but only the p42 isoform was calcium-sensitive. Our results suggest that a Ca(2+)-dependent isoform of protein kinase C and CaM kinase II are upstream activators of MAP kinase in the swine carotid artery.
Katoch
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Department of Physiology, MCP Hahnemann University, Philadelphia, Pennsylvania 19129, USA.
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Moreland
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D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003902:Detergents; D004789:Enzyme Activation / Q000187:drug effects; D006632:Histamine / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D017830:Octoxynol; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology; D013552:Swine; D000096926:Benzenesulfonamides
D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003902:Detergents; D006632:Histamine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D017830:Octoxynol; D010703:Phorbol Esters; D011493:Protein Kinase C; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides