Receptor-independent activation of Rho-kinase-mediated calcium sensitisation in smooth muscle

1 The aim of this work was to determine whether Rho-kinase-mediated calcium sensitisation contributes to contractions of the mouse anococcygeus smooth muscle and, if so, whether the process was activated by receptor-dependent or receptor-independent mechanisms. 2 The Rho-kinase inhibitor Y27632 produced concentration-dependent decreases in tone raised by either the muscarinic receptor agonist carbachol (CCh), or the sarco-endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Tg) (EC50 values against CCh and Tg of 8.4 +/- 3.3 (n = 6) and 6.1 +/- 2.1 (n = 7) muM, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mM external potassium (69 +/- 7% (n = 4) inhibition using 10 muM Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light-chain phosphatase, calyculin-A. 3 In beta-escin-permeabilised preparations, both CCh and Tg produced significant increases in tone over-and-above that produced by a combination of calcium (1 muM) and GTP (100 muM). These responses to CCh and Tg were inhibited by Y27632 (10 muM). 4 Western blot analysis of fractionated tissue samples probed for RhoA immunoreactivity, indicated that both CCh and Tg were able to induce translocation of RhoA from the cytosol to the membrane. 5 These findings suggest that Rho-kinase-mediated calcium sensitisation is activated by both receptor-dependent and receptor-independent mechanisms in the mouse anococcygeus.