Effects of calyculin A on tension and myosin phosphorylation in skinned smooth muscle of the rabbit mesenteric artery

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Abstract
1 Using β-escin and ionomycin-treated skinned smooth muscle strips of the rabbit mesenteric artery, the effects of calyculin A (CL-A, an inhibitor of type 1 and 2A phosphatases) on mechanical activities, phosphorylation of myosin light chain (MLC) and the relationship between the two were studied in Ca2+-free solution containing 4 mm EGTA and these effects were compared with those evoked by Ca2+. 2 The threshold concentration of Ca2+ required to increase either tension or MLC-phosphorylation was 0.1 μm and maximum effects were obtained at 10 μm. MLC was mainly monophosphorylated, rather than diphosphorylated, in the presence of Ca2+. ED50 value for Ca2+ was 0.54 μm for either tension or MLC-phosphorylation. The relationship between tension and MLC-phosphorylation is linear in the pCa range 7–5.5. 3 In Ca2+-free solution (containing either 20 mm EGTA or 4 mm EGTA with or without 4 mm BAPTA), 3 μm CL-A produced a contraction, the maximum amplitude of which was similar to that evoked by 10 μm Ca2+. CL-A (0.03–3 μm) concentration-dependently increased both tension and MLC-phosphorylation in Ca2+-free solution containing 4 mm EGTA. The threshold concentration of CL-A required for the increase in either tension or MLC-phosphorylation was 0.03 μm and maximum effects were obtained at 3 μm. In the presence of CL-A, MLC was not only monophosphorylated but also diphosphorylated. ED50 values for CL-A were 0.39 μm for tension, 0.44 μm for the monophosphorylated form of MLC and 0.54 μm for all phosphorylated (mono + di) forms. The relationship between tension and the monophosphorylated form of MLC was linear over the concentration range studied and was similar to that for Ca2+. 4 H-7 (3 μm, an inhibitor of protein kinase C) inhibited neither the tension nor phosphorylation of MLC induced by 10 μm Ca2+ or 3 μm CL-A. At a high concentration (30 μm), H-7 slightly inhibited both the tension and phosphorylation of MLC induced by either stimulant without a change in the tension-MLC-phosphorylation relationship. KN-62, an inhibitor of Ca2+-calmodulin-dependent protein kinase II, did not modify either the tension or the phosphorylation of MLC induced by 10 μm Ca2+ or 3 μm CL-A. CK-II, another inhibitor of Ca2+-calmodulin-dependent protein kinase II, did not inhibit the contraction induced by 3 μm CL-A. 5 SM-1 (0.03–0.3 mm) and ML-9 (0.1 and 0.3 mm), inhibitors of MLC-kinase, each lowered the resting level of MLC-phosphorylation in Ca2+-free solution and also inhibited both the tension and MLC-phosphorylation induced by 10 μm Ca2+ or 3 μm CL-A, in a concentration-dependent manner. Neither SM-1 nor ML-9 modified the relationship between tension and either monophosphorylated or all phosphorylated (mono + di) forms of MLC in the presence of Ca2+ or CL-A. 6 In a solution containing MgITP (the substrate for myosin ATPase but not for MLC-kinase) with no MgATP, 10 μm Ca2+ failed to produce contraction. Under these conditions, the amplitude of the contraction induced by 3 μm CL-A was greatly diminished in comparison with that induced in the presence of MgATP. 7 The present results suggest that in smooth muscle cells of the rabbit mesenteric artery, CL-A in Ca2+-free solution, produces a maximum contraction through an indirect activation of Ca2+-calmodulin-independent (constitutively active) MLC-kinase via its inhibitory action on MLC-phosphatases. Based on this evidence, it is hypothesized that, in these cells, a constitutively active MLC-kinase may be present, though its action may be concealed by that of endogenous MLC-phosphatase.