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

Incubation of β-escin-permeabilized guinea-pig longitudinal ileal smooth muscle with ATPγS under conditions that do not lead to thiophosphorylation of regulatory light chains of myosin (r-MLC) increased subsequent Ca 2+ sensitivity of force and r-MLC phosphorylation. In this study we tested whether this is due to activation of the Rho and/or Rho-associated kinase (ROK) as it is the case in agonist-induced Ca 2+ sensitization. The increase in Ca 2+ sensitivity induced by pretreatment with ATPγS at pCa > 8 with the myosin light chain kinase (MLCK) inhibitor ML-9 in rigor solution was associated with 35 S incorporation into the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1, and several other high molecular mass proteins. No thiophosphorylation of r-MLC, MLCK, caldesmon, calponin and CPI-17 was detected. While the relatively specific inhibitor of ROK, Y 27632, inhibited the carbachol-induced increase in Ca 2+ sensitivity with an IC 50 of 1.4 μ m , the ATPγS-induced increase in Ca 2+ sensitivity and thiophosphorylation of MYPT1 was not inhibited. Inhibiton of Rho by exoenzyme C3 also had no effect. Only staurosporine (2 μ m ), but not the PKC inhibitor peptide 19–31, nor genistein nor PD 98059, inhibited the ATPγS-induced Ca 2+ sensitization of force, r-MLC phosphorylation, and the 35 S incorporation into MYPT1. The staurosporine-sensitive kinase(s) appeared to be tightly associated with the contractile apparatus because treatment of Triton-skinned preparations with ATPγS also induced a staurosporine-sensitive increase in Ca 2+ sensitivity of contraction. Since there was very little immunoreactivity with antibodies to p 21 -associated kinase (PAK) in Triton-skinned preparations, the staurosporine-sensitive kinase most probably is not PAK. GTPγS had an additive effect on ATPγS-induced sensitization at saturating concentrations of ATPγS. The additional effect of GTPγS was inhibited by Y 27632. We conclude that treatment with ATPγS under ATP-free conditions, unmasks a staurosporine-sensitive kinase which induces a large increase in Ca 2+ sensitivity that is most likely to be due to thiophosphorylation of MYPT1. 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.