Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms

Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosp horylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specifi c protein called CPI-17 (for protein kinase C-potentiated inhibitory protei n for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphory lated and converted to a potent inhibitor for myosin phosphatase. Phosphory lation of CPI-17 is diminished by pretreatment with either Y27632 or GF1092 03x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woo dsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897-9900). H ere we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extrac ts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) alph a, and the second kinase was purified to homogeneity as a 45-kDa protein, a nd identified by sequencing as PKC delta. Purified PKC delta was 3-fold mor e reactive with CPI-17 compared with myelin basic protein, whereas purified PKC alpha and recombinant RhoA-activated kinases (Rho-associated coiled-co il forming protein Ser/Thr kinase and protein kinase N) showed equal activi ty with CPI-17 and myelin basic protein. Y27632 inhibited CPI-17 phosphoryl ation by purified PKC delta with IC50 of 0.6 mum (in the presence of 0.1 mm ATP) or 14 mum (2.0 mm ATP). Y27632 significantly suppressed CPI-17 phosph orylation in smooth muscle cells, and the contraction of permeabilized rabb it femoral artery induced by stimulation with phorbol ester. GF109203x inhi bited phorbol ester-induced contraction of rabbit femoral artery by 80%, wh ereas a PKC alpha/beta inhibitor, Go6976, reduced contraction by 47%. The r esults imply that histamine stimulation elicits contraction of vascular smo oth muscle through activation of PKCa and especially PKC delta to phosphory late CPI-17.