α1-adrenoceptor-mediated phosphorylation of myosin in rat-tail arterial smooth muscle

Abstract

The mechanism of α1-adrenoceptor-mediated contraction was investigated in helical strips of the rat-tail artery. Muscle strips with the endothelium removed contracted in response to the α1-adrenoceptor agonist cirazoline, with half-maximal contraction at 0.23 μM. The contractile response to a submaximal concentration of cirazoline (0.3 μM) was biphasic, with a rapid phasic component peaking at approx. 30 s, followed by sustained tonic contraction. Phosphorylation of the 20 kDa light chain of myosin (LC20) in response to 0.3 μM cirazoline was also biphasic and closely matched the time-course of contraction. Resting LC20 phosphorylation levels were 0.22 ± 0.06 mol of P(i)/mol of LC20 (n = 3) and reached a maximum of 0.58 ± 0.08 mol of P(i)/mol of LC20 (n = 3). Phosphopeptide mapping and phosphoamino acid analysis revealed that LC20 phosphorylation occurred exclusively at serine-19. The sustained phase of contraction was eliminated by removal of extracellular Ca2+ and the phasic response was eliminated by depletion of endogenous Ca2+ stores. Both phases of the contractile response were restored by readdition of Ca2+ to the bathing medium. LC20 phosphorylation and both phases of the contractile response to 0.3 μM cirazoline were inhibited by the myosin light-chain kinase inhibitor ML-9 (30 μM). Resting LC20 phosphorylation, however, was unaffected by ML-9. Finally, both phasic and tonic responses to 0.3 μM cirazoline were partially inhibited by chloroethylclonidine (50 μM), suggesting the involvement of both α(1A) and α(1B) adrenoceptors in these contractile responses.