Rho kinase activation and ROS production contributes to the cooling enhanced contraction in cutaneous equine digital veins

Abstract

A decrease in environmental temperature can directly affect the contractility of cutaneous vasculature, mediated in part by α2-adrenoceptors. Most of the cellular mechanisms underlying the cooling-enhanced contractility to α2-adrenoceptor agonists have been reported in cutaneous arteries but little information is available on cutaneous veins. To investigate the cellular mechanisms associated with the cooling-enhanced contraction to UK-14304 (α2-adrenoceptor agonist), isolated equine digital veins (EDVs) were studied at 30°C and 22°C. The effects of inhibitors were studied on the contractile response to UK-14304 (0.1 μM). The cooling-enhanced responses were inhibited by Rho kinase inhibitors [maximum response to UK-14304 95.2 ± 8% of response to depolarizing Krebs solution (DKS) in control vessels cooled to 22°C, compared with 31.4 ± 6% in the presence of fasudil 1 μM and 75.8 ± 6% with Y-27632 0.1 μM] and the effects of these inhibitors were considerably less at 30°C (control response 56.4 ± 5% of DKS; 34.9 ± 6% with fasudil 1 μM and 50.6 ± 9% with Y-27632 0.1 μM). Furthermore, Western blotting showed that one of the downstream targets for Rho kinase activity, ezrin/radixin/moesin, was phosphorylated after cooling and reduced by fasudil (1 μM) only at 22°C. The activation of protein kinase C contributed to the contractile response, but predominantly at 30°C (maximum response 82.3 ± 9% of DKS for control; 57.7 ± 10% in the presence of chelerythrine 10 μM) with no significant effect at 22°C. The reduction of the response at 22°C by antioxidants, rotenone (14% reduction), and tempol (21% reduction) suggested the contribution of reactive oxygen species (ROS). No evidence was obtained to support the participation of tyrosine kinase. These data demonstrate that Rho kinase activation and the production of ROS contributes to the cooling-enhanced contraction in these cutaneous digital veins.