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

Abstract

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) (EC 50 values against CCh and Tg of 8.473.3 (n ¼ 6) and 6.172.1 (n ¼ 7) mm, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mm external potassium (6977% (n ¼ 4) inhibition using 10 mm Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light-chain phosphatase, calyculin-A.3 In b-escin-permeabilised preparations, both CCh and Tg produced significant increases in tone over-and-above that produced by a combination of calcium (1 mm) and GTP (100 mm). These responses to CCh and Tg were inhibited by Y27632 (10 mm). 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.

Comparative pharmacological analysis of Rho-kinase inhibitors and identification of molecular components of Ca2+ sensitization in the rat lower urinary tract

“…In agreement with our results, recent reports demonstrated that KClinduced contraction involves activation of Rho-kinase in rat tail artery [41] and canine tracheal smooth muscle [42]. Y-27632 has also been shown to inhibit non-receptor-mediated contractions in mouse anococcygeus muscle [43] and to block Ca 2+ entry in rat arteries [44]. Interestingly, KCl-induced contractions of trigone were not significantly affected by either Rho-kinase inhibitor, providing evidence that the Rho-kinase-mediated Ca 2+ sensitization mechanism is not particularly involved in KCl depolarization-coupled contractile responses of this tissue.…”

Rho/Rho Kinase and Phosphoinositide 3-Kinase Are Parallel Pathways in the Development of Spontaneous Arterial Tone in Deoxycorticosterone Acetate-Salt Hypertension

“…Nobe and Paul (2001) show that, although in a transient phase of contraction Y27632 reduced force and intracellular calcium, treatment during the sustained phase of contraction indicates that inhibition of MYPT can alter force without changing calcium. In contrast, Rho/Rho-kinase is involved with voltage-dependent calcium channels in regulating tone of the renal afferent arteriole (Nakamura et al, 2003) and may have a role in mediating intracellular calcium signaling (Ayman et al, 2003). Also, Rho/Rho-kinase contributes to calcium mobilization induced by agonist stimulation in cultured cells (Chong et al, 1994).…”

Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

“…48 In mouse anococcygeus smooth muscle, Ca 2+ sensitization induced by phenylephrine, norepinephrine, and CCh was markedly antagonized by ROCK inhibitors, and the contractile responses to KCl-induced depolarization or excitatory electrical field stimulation were also highly sensitive to ROCK inhibitors. 87,88 The phosphorylation of MYPT1, CPI-17, and MLC in various GI smooth muscles in response to contractile agonists has been investigated by a number of laboratories. These studies show that, in general, exogenously added contractile agonists elicit variable increases in MLC S19, CPI-17 T38, and MYPT1 T694 and T852 phosphorylation depending on the agonist used and the smooth muscle employed.…”