Effects of the constitutively active proteolytic fragment of protein kinase C on the contractile properties of demembranated smooth muscle fibres

Abstract

The role of protein kinase C (PKC) in regulating the contractile state of smooth muscle was investigated using the constitutively active catalytic fragment of PKC (PKM) with skinned (demembranated) chicken gizzard fibres. PKM attenuated a submaximal contraction in gizzard smooth muscle skinned fibres, but not in rabbit cardiac skinned fibres. PKM-mediated relaxation of submaximal contractions of smooth muscle was accompanied by a reduction in the rate of ATP hydrolysis in the fibre and by phosphorylation of the 20 kDa light chain of gizzard myosin at the PKC sites (serine-1, serine-2 and threonine-9). In addition, several other endogenous proteins were phosphorylated by PKM. However, the inhibitory effects on tension and ATPase are consistent with the biochemical effects of PKC-catalysed phosphorylation of myosin, i.e. reduction of the actin-activated MgATPase activity of myosin prephosphorylated at serine-19 by myosin light chain kinase. Pretreatment of skinned fibres with PKM and ATP gamma S in the absence of Ca2+ had no inhibitory effect on the subsequent submaximal Ca(2+)-activation of force. Consistent with this observation, PKC was not able to utilize ATP gamma S as a substrate, confirming that the observed effects were the result of PKM-catalysed protein phosphorylation. We suggest that PKC may have two distinct effects on smooth muscle contraction: translocation of PKC to the sarcolemma on stimulation results in phosphorylation of a protein(s) other than myosin and a slow, sustained contraction; in some circumstances PKC may undergo proteolysis to PKM resulting in myosin phosphorylation at PKC-specific sites, a reduction in ATPase activity and relaxation of the muscle.

Calcium-dependent mechanisms of regulation of smooth muscle contraction

“…Furthermore, PKM treatment of skinned fibers submaximally contracted at pCa 5.6 induced relaxation. On the other hand, maximal contractions occurring at pCa 3.8 were unaffected by PKM addition or pretreatment (Parente et al 1990(Parente et al , 1991. These results, which are consistent with the conclusions of other skinned fiber studies (Inagaki et al 1987) and the in vitro effect of PKC phosphorylation of myosin that reduces the actin-activated M~~+ ATPase activity (Nishikawa et al 1983), indicate that, at least at submaximal [ca2+], PKC-catalyzed phosphorylation of myosin can affect contraction.…”

Reconstitution of protein kinase C‐induced contractile Ca2+ sensitization in Triton X‐100‐demembranated rabbit arterial smooth muscle

“…Several reports have shown that direct application of PKC enzyme in heavily permeabilized smooth muscles, which still retained the regulatory/contractile apparatus including MLC kinase and phosphatase, failed to cause an increase in contractile force levels at submaximal levels of Ca 2+ (Inagaki et al 1987; Sutton & Haeberle, 1990; Parente et al 1992). This is in contrast to the Ca 2+ ‐sensitizing effect of PKC activators in intact and mildly (α‐toxin‐ or β‐escin‐) permeabilized smooth muscles (Masuo et al 1994).…”

Phorbol Ester-Induced Contractions of Swine Carotid Artery Are Supported by Slowly Cycling Crossbridges Which Are Not Dependent on Calcium or Myosin Light Chain Phosphorylation

“…Sev eral investigators have suggested that PKC-catalyzed MLC phosphorylation is not involved in the regulation of smooth muscle contraction [11][12][13], In fact, if phosphory lation at the PKC sites plays any role in crossbridge regu lation it is to inhibit activity [35,36], In addition, phos phorylation of the MLC kinase during phorbol esterinduced contractions of intact tissue does not appear to be of physiological importance [37], As such, the MLC kinase/phosphatase system and the thick filament do not appear to be responsible.…”