Thiophosphorylation-induced Ca2+ sensitization of guinea-pig ileum contractility is not mediated by Rho-associated kinase

1. Incubation of beta -escin-permeabilized guinea-pig longitudinal ileal sm ooth muscle with ATP gammaX under conditions that do not lead to thiophosph orylation of regulatory light chains of myosin (r-MLC) increased subsequent Ca2+ sensitivity of force and r-MLC phosphorylation. In this study we test ed whether this is due to activation of the Rho and/or Rho-associated kinas e (ROK) as it is the case in agonist-induced Ca2+ sensitization. 2. The increase in Ca2+ sensitivity induced by pretreatment with ATP gammaX at pCa > 8 with the myosin light chain kinase ((MLCK) inhibitor ML-9 in ri gor solution was associated with S-35 incorporation into the regulatory sub unit of myosin light chain phosphatase (MLCP), MYPT1, and several other hig h molecular mass proteins. No thiophosphorylation of r-MLC, MLCK, caldesmon , calponin and CPI-17 was detected. 3. While the relatively specific inhibitor of ROK, Y 27632, inhibited the c arbachol-induced increase in Ca2+ sensitivity with an IC50 of 1.4 muM, the ATP gammaS-induced increase in Ca2+ sensitivity and thiophosphorylation of MYPT1 was not inhibited. Inhibiton of Rho by exoenzyme C3 also had no effec t. 4. Only staurosporine (2 muM), but not the PKC inhibitor peptide 19-31, nor genistein nor PD 98059, inhibited the ATP gammaS-induced Ca2+ sensitizatio n of force, r-MLC phosphorylation, and the S-35 incorporation into MYPT1. 5. The staurosporine-sensitive kinase(s) appeared to be tightly associated with the contractile apparatus because treatment of Triton-skinned preparat ions with ATP gammaS also induced a staurosporine-sensitive increase in Ca2 + sensitivity of contraction. Since there was very little immunoreactivity with antibodies to p(21)-associated kinase (PAK) in Triton-skinned preparat ions, the staurosporine-sensitive kinase most probably is not PAK. 6. GTP gammaS had an additive effect on ATP gammaS-induced sensitization at saturating concentrations of ATP gammaS. The additional effect of GTP gamm aS was inhibited by Y 27632. 7. We conclude that treatment with ATP gammaS under ATP-free conditions, un masks a staurosporine-sensitive kinase which induces a large increase in Ca 2+ sensitivity that is most likely to be due to thiophosphorylation of MYPT 1. The kinase is distinct from ROK. The physiological significance of this kinase, which is tightly associated with the contractile apparatus, is not known at present.