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

1. Triton X-100-demembranated smooth muscle loses Ca2+-sensitizing responsi veness to protein kinase C (PKC) activators while intact and alpha-toxin-pe rmeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca2+ sensitization by PKC in the demembranated preparations . 2. Western blot analyses showed that the content of the PKC: alpha-isoform (PKC alpha) was markedly reduced and that the smooth muscle-specific protei n phosphatase-l inhibitor protein CPI-17 was not detectable, while the amou nt of calponin and actin still remained similar to those of intact strips. 3. Unphosphorylated recombinant CPI-17 alone induced a small but significan t contraction at constant Ca2+. Isoform-selective PKC inhibitors inhibited unphosphorylated but not prethiophosphorylated CPI-17-induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI-1 7. 4. Exogenously replenishing PKC alpha; alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphoryla tion at submaximal Ca2+. 5. PKC in the presence of CPI-17, but not the [T38A]-CPI mutant, markedly i nduced potentiation of both contraction and MLC phosphorylation. CPI-17 its elf was phosphorylated. 6. In in vitro experiments, CPI-17 was a much better substrate for PKC alph a: than calponin, caldesmon, MLC and myosin. 7. Our results indicate that PKC requires CPI-17 phosphorylation at Thr-38 but not calponin for reconstitution of the co.ntractile Ca2+ sensitization in the demembranated arterial smooth muscle.