Phosphatase-Mediated Modulation of Actin-Myosin Interaction in Bovine Aortic Actomyosin and Skinned Porcine Carotid Artery

Abstract. Since the Ca²⁺-regulatory mechanism for actin-myosin interaction in smooth muscle involves phosphorylation of the 20,000-Da myosin light chains, it was hypothesized that such interaction should be influenced by myosin phosphatase. Accordingly, we studied the effects of an aortic myosin light-chain phosphatase on Ca¹⁺-dependent actin-myosin interaction in detergent-skinned porcine carotid artery and bovine aortic native actomyosin. In skinned preparations, the aortic phosphatase (16 U/ml) markedly inhibited the rate of isometric contraction in low Ca²⁺ (6.8 × 10^-7 M) and responsiveness to Ca²⁺ (force attained with 6.8 × 10^-7 Ca²⁺/force attained with 1.6 × 10^-6 MCa²⁺), whereas relaxation was accelerated. Ca²⁺-dependent actomyosin ATPase activity and phosphorylation of the light chains were significantly and progressively depressed in the presence of increasing concentrations of phosphatase (0.1-0.9 U/ml). The concentration of Ca²⁺ (1.1 × 10^-6 M) required for half-maximal activation of either ATPase activity or light-chain phosphorylation increased by 70% in the presence of 0.1 U phosphatase/ml. Neither the maximal rate of Ca²⁺-sensitive ATP hydrolysis (39 ± 0.8 nmole/ min/mg actomyosin) nor the extent of phosphorylation (0.68 ± 0.05 mole PO₄/mole light chain) was altered at >5 × 10^-6 M Ca²⁺. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 μM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca²⁺ (10^-8to 1.25 × 10^-5 M). However, significant phosphorylation was present (20-25% of maximum) in the absence of Ca²⁺-dependent ATPase activity and only 15% of the maximal rate of ATP hydrolysis was expressed until phosphorylation attained 50% of its maximal value. These findings are consistent with the ordered model of myosin phosphorylation suggested by A. Persechini and D. J. Hartshorne (Science (Washington, DC), 213:1383-285, 1961] (36). They also suggest that myosin phosphatase may participate in modulating actin-myosin interactions in vascular smooth muscle.