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

Gabriele Pfitzer, Dagmar Sonntag-Bensch and Dragana Brkic-Koric

1. Incubation of -escin-permeabilized guinea-pig longitudinal ileal smooth muscle with ATPS under conditions that do not lead to thiophosphorylation of regulatory light chains of myosin (r-MLC) increased subsequent Ca²⁺ 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 Ca²⁺ sensitization.
2. The increase in Ca²⁺ sensitivity induced by pretreatment with ATPS at pCa > 8 with the myosin light chain kinase (MLCK) inhibitor ML-9 in rigor solution was associated with ³⁵S 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.
3. While the relatively specific inhibitor of ROK, Y 27632, inhibited the carbachol-induced increase in Ca²⁺ sensitivity with an IC₅₀ of 1.4 µM, the ATPS-induced increase in Ca²⁺ sensitivity and thiophosphorylation of MYPT1 was not inhibited. Inhibiton of Rho by exoenzyme C3 also had no effect.
4. Only staurosporine (2 µM), but not the PKC inhibitor peptide 19-31, nor genistein nor PD 98059, inhibited the ATPS-induced Ca²⁺ sensitization of force, r-MLC phosphorylation, and the ³⁵S incorporation into MYPT1.
5. The staurosporine-sensitive kinase(s) appeared to be tightly associated with the contractile apparatus because treatment of Triton-skinned preparations with ATPS also induced a staurosporine-sensitive increase in Ca²⁺ sensitivity of contraction. Since there was very little immunoreactivity with antibodies to p²¹-associated kinase (PAK) in Triton-skinned preparations, the staurosporine-sensitive kinase most probably is not PAK.
6. GTPS had an additive effect on ATPS-induced sensitization at saturating concentrations of ATPS. The additional effect of GTPS was inhibited by Y 27632.
7. We conclude that treatment with ATPS under ATP-free conditions, unmasks a staurosporine-sensitive kinase which induces a large increase in Ca²⁺ 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.

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