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

Triton X-100-demembranated smooth muscle loses Ca2+-sensitizing responsiveness to protein kinase C (PKC) activators while intact and α-toxin-permeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca2+ sensitization by PKC in the demembranated preparations. Western blot analyses showed that the content of the PKC α-isoform (PKCα) was markedly reduced and that the smooth muscle-specific protein phosphatase-1 inhibitor protein CPI-17 was not detectable, while the amount of calponin and actin still remained similar to those of intact strips. Unphosphorylated recombinant CPI-17 alone induced a small but significant contraction at constant Ca2+. Isoform-selective PKC inhibitors inhibited unphosphorylated but not pre-thiophosphorylated CPI-17-induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI-17. Exogenously replenishing PKCα alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca2+. PKC in the presence of CPI-17, but not the [T38A]-CPI mutant, markedly induced potentiation of both contraction and MLC phosphorylation. CPI-17 itself was phosphorylated. In in vitro experiments, CPI-17 was a much better substrate for PKCα than calponin, caldesmon, MLC and myosin. Our results indicate that PKC requires CPI-17 phosphorylation at Thr-38 but not calponin for reconstitution of the contractile Ca2+ sensitization in the demembranated arterial smooth muscle. The primary mechanism of smooth muscle contraction is reversible phosphorylation of myosin light chain (MLC) at Ser-19 (Kamm & Stull, 1985; Hartshorne, 1987). This phosphorylation was initially assumed to be a result of regulation of Ca2+/calmodulin-dependent MLC kinase by cytoplasmic Ca2+ only, since activity of phosphoprotein phosphatase toward MLC was presumed to be constant. It is now well established that physiological phosphorylation of MLC in smooth muscle is caused by not only an increase in the phosphorylating enzyme activity but also a decrease in the dephosphorylating enzyme activity through G protein(s) (Kitazawa et al. 1991b; Kubota et al. 1992). Regulation of the MLC phosphatase is one of the current topics in smooth muscle research as well as an important field in general cell biology (Somlyo & Somlyo, 1994; Hartshorne et al. 1998). During stimulation of smooth muscle contraction, MLC phosphatase activity is reduced to increase the contractile Ca2+ sensitivity (Ca2+ sensitization) and this is possibly mediated by multiple second messengers/signalling pathways: the rho A-rho kinase (Kimura et al. 1996), protein kinase C (PKC; Masuo et al. 1994) and arachidonic acid pathways (Gong et al. 1992). In contrast cyclic GMP, a vasodilator second messenger, has been demonstrated to increase MLC phosphatase activity and reduce the contractile Ca2+ sensitivity (Ca2+ desensitization) during vascular smooth muscle relaxation (Wu et al. 1996; Lee et al. 1997). During stimulation in smooth muscle, protein kinase and phosphatase targeting the same protein MLC work co-operatively to effect a response rather than working to achieve homeostasis by counterbalancing each other. Yet the precise mechanism of each signalling pathway regulating MLC phosphatase remains unclear. The activation of PKC by diacylglycerol (DAG) analogues and phorbol esters in intact and permeabilized arterial smooth muscle instructs the MLC phosphatase to decrease its activity but has no effect on MLC kinase activity (Itoh et al. 1993; Masuo et al. 1994). The resultant gain in MLC phosphorylation at Ser-19 thus increases the Ca2+ sensitivity of smooth muscle contraction. The precise mechanism for the modulation of MLC phosphatase by PKC remains unknown, although in vitro evidence controverts the direct phosphorylation of MLC phosphatase by PKC (Hartshorne et al. 1998). CPI-17, a novel smooth muscle-specific regulatory protein, has been demonstrated to be phosphorylated by PKC, thereby inhibiting in vitro and in situ MLC phosphatase and increasing the Ca2+ sensitivity of both MLC phosphorylation and contraction (Eto et al. 1995, 1997; Li et al. 1998; Senba et al. 1999). This protein is postulated to be a mediator between PKC activation and inhibition of MLC phosphatase. There are several reports, however, showing that phorbol esters and agonists evoked a contraction of intact smooth muscle without an associated increase in MLC phosphorylation under Ca2+-free conditions (see review by Singer, 1996). It has been suggested that PKC activation may relieve the MLC phosphorylation-independent inhibition of contraction by affecting the thin filament-associated proteins caldesmon and/or calponin (see reviews by Allen & Walsh, 1994). These proteins can be phosphorylated in vitro by several protein kinases including PKC, but the evidence for in situ phosphorylation and its significance is conflicting (see review by Horowitz et al. 1996). 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 Ca2+ (Inagaki et al. 1987; Sutton & Haeberle, 1990; Parente et al. 1992). This is in contrast to the Ca2+-sensitizing effect of PKC activators in intact and mildly (α-toxin- or β-escin-) permeabilized smooth muscles (Masuo et al. 1994). In this study, we first confirmed the previous results, that our Triton X-100-demembranated arterial strips lose contractile responsiveness to PKC activators, and then examined what constituents remain in the preparations using Western immunoblot analysis. Finally, we attempted to reconstitute the Ca2+-sensitizing response of the demembranated strips to active PKC.