Ca2+- 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 i n all smooth muscle cells and believed to be important in several physiolog ically relevant properties of this muscle. Our goal was to determine if pro tein kinase C and MAP kinase are activated by a simple increase in cellular Ca2+ 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 intrac ellular environment without influence from membrane or receptor-mediated mo dulation. The p42 and p44 isoforms of MAP kinase were activated in a concen tration-dependent fashion by an increase in Ca2+. This was shown by in-the- gel kinase assay and direct measurement of MAP kinase phosphotransferase ac tivity. Protein kinase C was also activated by an increase in Ca2+, as show n by a novel assay that measures total active protein kinase C in the tissu e. Inhibition of protein kinase C activity completely abolished MAP kinase activity. Additionally, inhibition of Ca2+/calmodulin-dependent protein kin ase II (CaM kinase II) also abolished MAP kinase activity. Using intact swi ne carotid arteries, we showed p42 and p44 MAP kinase to be activated by bo th histamine and phorbol dibutyrate, but only the p42 isoform was calcium-s ensitive. Our results suggest that a Ca2+-dependent isoform of protein kina se C and CaM kinase II are upstream activators of MAP kinase in the swine c arotid artery. (C) 1999 Wiley-Liss, Inc.