title: Protein kinase C mediation of Ca(2+)-independent contractions of vascular smooth muscle.
abstract: Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca(2+)-independent isoenzyme of PKC. Of the two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, identified in ferret aorta, PKC epsilon is more likely to mediate the contractile response because (i) PKC epsilon, but not PKC zeta, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKC epsilon translocates from the sarcoplasm to the sarcolemma, whereas PKC zeta, translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKC epsilon, but not PKC zeta, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKC epsilon is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of alpha, beta, and gamma isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKC epsilon but is a very poor substrate of PKC zeta. A signal transduction pathway is proposed to explain Ca(2+)-independent contraction 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 the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (approximately 0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKC epsilon is a component of contractile 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+.
authors.affiliation: Smooth Muscle Research Group, Faculty of Medicine, University of Calgary, AB, Canada. walsh@acs.ucalgary.ca
authors.forename: M P
authors.initials: MP
authors.lastname: Walsh
authors.forename: A
authors.initials: A
authors.lastname: Horowitz
authors.forename: O
authors.initials: O
authors.lastname: Clément-Chomienne
authors.forename: J E
authors.initials: JE
authors.lastname: Andrea
authors.forename: B G
authors.initials: BG
authors.lastname: Allen
authors.forename: K G
authors.initials: KG
authors.lastname: Morgan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D000096985:Calponins
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D007527:Isoenzymes; D008840:Microfilament Proteins; D011493:Protein Kinase C; D000096985:Calponins