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 responsiveness to protein kinase C (PKC) activators while intact and alpha-toxin-permeabilized 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 (PKCalpha) 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. 3. 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. 4. Exogenously replenishing PKCalpha alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca2+. 5. 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. 6. In in vitro experiments, CPI-17 was a much better substrate for PKCalpha 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 contractile Ca2+ sensitization in the demembranated arterial smooth muscle.
Kitazawa
T
T
Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20007, USA. tkitaz01@gusun.georgetown.edu
Takizawa
N
N
Ikebe
M
M
Eto
M
M
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D020536:Enzyme Activators / Q000494:pharmacology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism / Q000502:physiology; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D017830:Octoxynol / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D020536:Enzyme Activators; D007527:Isoenzymes; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D017830:Octoxynol; D011493:Protein Kinase C; D002118:Calcium