Histamine-induced Vasoconstriction Involves Phosphorylation of a Specific Inhibitor Protein for Myosin Phosphatase by Protein Kinase C α and δ Isoforms

Abstract

Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either Y27632 or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897-9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) α, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKCδ. Purified PKCδ was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKCα and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. Y27632 inhibited CPI-17 phosphorylation by purified PKCδ with IC50 of 0.6 μM (in the presence of 0.1 mM ATP) or 14 μM (2.0 mM ATP). Y27632 significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKCα/β inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKCα and especially PKCδ to phosphorylate CPI-17.