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 Ca'+-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 X lop7 M ) and responsiveness to Ca2+ (force attained with 6.8 X Ca2+/force attained with 1.6 X lop6 M Ca2+), 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 X lop6 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 2 0.8 nmole/ min/mg actomyosin) nor the extent of phosphorylation (0.68 k 0.05 mole PO,/mole light chain) was altered at >5 X M Ca2+. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 pM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca2+ 36

Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.

“…Protein phosphatases reactive with MLC have been isolated from smooth muscle (22, 26, 29, 37-39, 45, reviewed 16, 40), as well as from cardiac (36,37,42) and skeletal muscle (14,15, reviewed in reference 16) and appear to be related to either type-1 (14,15,42, reviewed in reference 16) or type 2A enzymes (8,10,26,30). Both a polycation-modulated phosphatase holoenzyme (9) and the catalytic subunit of a skeletal protein phosphatase (41) have been found to decrease isometric tension of skinned smooth muscle and skeletal muscle fibers respectively. Both these protein phosphatase preparations are likely to be classed as type-2A phosphatases (16).…”

Dissociation between calcium influx blockage and smooth muscle relaxation by nifedipine in spontaneously hypertensive rats.

“…A decrease in calmodulin concentration, a phosphorylation of MLCK by AMP-dependent protein kinase, an increase in phosphatase activity, 25 or inhibitors that could interfere with the biological activity of calmodulin would result in a greater calcium requirement for a given level of myosin light chain phosphorylation and Figure 2) 19 report decreased calcium sensitivity in intact aortas of DOCA hypertensive rats precontracted with potassium chloride. However, they could not detect an altered sensitivity when the aortas were skinned.…”

“…29 These data are consistent, then, with the hypothesis that nifedipine produces smooth muscle relaxation in SHR through mechanisms other than calcium influx blockage, and also with the possibility that this pharmacological intervention may result in a diminished formation of the Ca 2+ -calmodulin-MLCK complex. This diminished formation could be the result of a binding of the drug to calmodulin, 30 a decrease in affinity of the Ca 2+ -calmodulin complex for the light chain kinase, an increase in phosphatase activity, 25 or a direct action of the drug on the MLCK. In connection with this, the binding of dihydropyridines to calmodulin has already been reported but at doses higher than 10" 10 M.…”

Vascular smooth muscle contractile elements. Cellular regulation.

“…Bovine aortic smooth muscle myosin light chain phosphatases have been identified as soluble enzymes that have biochemical characteristics of type 2A phosphatase. 41 A multisubunit form of the isolated catalytic subunit of aortic phosphatase 2A dephosphorylates myosin and causes relaxation of skinned fibers from porcine carotid artery 46 and uterine smooth muscle. 47 Gizzard smooth muscle contains four forms of myosin light chain phosphatase.…”