PROTEIN-KINASE-C MEDIATION OF CA2-INDEPENDENT CONTRACTIONS OF VASCULAR SMOOTH-MUSCLE()

Tumour-promoting phorbol esters induce slow, sustained contractions of Vascular smooth muscle, suggesting that protein kinase C (PKC) may pl ay a role in the regulation of smooth muscle contractility. In some ca ses, e.g., ferret aortic smooth muscle, phorbol eater induced contract ions occur without a change in [Ca2+](i) or myosin phosphorylation. Di rect evidence for the involvement of PKC came from the use of single s aponin-permeabilized ferret aortic cells. A constitutively active cata lytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a p eptide inhibitor of PKC. Contractions of similar magnitude occurred ev en when the [Ca2+] was reduced to close to zero, implicating a Ca2+-in dependent isoenzyme of PKC. Of the two Ca2+-independent PKC isoenzymes , epsilon and zeta, identified in ferret aorta, PKC epsilon is more li kely to mediate the contractile response because (i) PKC epsilon, but not PKC zeta, is responsive to phorbol eaters; (ii) upon stimulation w ith phenylephrine, PKC epsilon translocates from the sarcoplasm to the sarcolemma, whereas PKC zeta translocates from a perinuclear localiza tion to the interior of the nucleus; and (iii) when added to permeabil ized single cells of the ferret aorta at pCa 9, PKC epsilon, but not P KC zeta, induced a contractile response similar to that induced by phe nylephrine. A possible substrate of PKC epsilon is the smooth muscle s pecific, thin filament associated protein, calponin. Calponin is phosp horylated in intact smooth muscle strips in response to carbachol, end othelin-1, phorbol eaters, or okadaic acid. Phosphorylation of calponi n in vitro by PKC (a mixture of alpha, beta, and gamma isoenzymes) dra matically reduces its affinity for F-actin and alleviates its inhibiti on of the cross-bridge cycling rate. Calponin is phosphorylated in vit ro by PKC epsilon but is a very poor substrate of PKC zeta. A signal t ransduction pathway is proposed to explain Ca2+-independent contractio n of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+ transient. The consequent activation of PKC epsilon would result in calponin phosphorylation, its release from th e thin filaments, and alleviation of inhibition of cross-bridge cyclin g. Slow, sustained contraction then results from a slow rate of cross- bridge cycling because of the basal level of myosin light chain phosph orylation (approximate to 0.1 mol P-i/mol light chain). We also sugges t that signal transduction through PKC epsilon is a component of contr actile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to alpha(1)-adrenergic agonists than to K+.