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

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 Ca 2+ ‐free solution containing 4 m m EGTA and these effects were compared with those evoked by Ca 2+ . 2 The threshold concentration of Ca 2+ required to increase either tension or MLC‐phosphorylation was 0.1 μ m and maximum effects were obtained at 10 μ m . MLC was mainly monophosphorylated, rather than diphosphorylated, in the presence of Ca 2+ . ED 50 value for Ca 2+ was 0.54 μ m for either tension or MLC‐phosphorylation. The relationship between tension and MLC‐phosphorylation is linear in the pCa range 7–5.5. 3 In Ca 2+ ‐free solution (containing either 20 m m EGTA or 4 m m EGTA with or without 4 m m BAPTA), 3 μ m CL‐A produced a contraction, the maximum amplitude of which was similar to that evoked by 10 μ m Ca 2+ . CL‐A (0.03–3 μ m ) concentration‐dependently increased both tension and MLC‐phosphorylation in Ca 2+ ‐free solution containing 4 m m EGTA. The threshold concentration of CL‐A required for the increase in either tension or MLC‐phosphorylation was 0.03 μ m and maximum effects were obtained at 3 μ m . In the presence of CL‐A, MLC was not only monophosphorylated but also diphosphorylated. ED 50 values for CL‐A were 0.39 μ m for tension, 0.44 μ m for the monophosphorylated form of MLC and 0.54 μ m 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 Ca 2+ . 4 H‐7 (3 μ m , an inhibitor of protein kinase C) inhibited neither the tension nor phosphorylation of MLC induced by 10 μ m Ca 2+ or 3 μ m CL‐A. At a high concentration (30 μ m ), 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 Ca 2+ ‐calmodulin‐dependent protein kinase II, did not modify either the tension or the phosphorylation of MLC induced by 10 μ m Ca 2+ or 3 μ m CL‐A. CK‐II, another inhibitor of Ca 2+ ‐calmodulin‐dependent protein kinase II, did not inhibit the contraction induced by 3 μ m CL‐A. 5 SM‐1 (0.03–0.3 m m ) and ML‐9 (0.1 and 0.3 m m ), inhibitors of MLC‐kinase, each lowered the resting level of MLC‐phosphorylation in Ca 2+ ‐free solution and also inhibited both the tension and MLC‐phosphorylation induced by 10 μ m Ca 2+ or 3 μ m 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 Ca 2+ or CL‐A. 6 In a solution containing MgITP (the substrate for myosin ATPase but not for MLC‐kinase) with no MgATP, 10 μ m Ca 2+ failed to produce contraction. Under these conditions, the amplitude of the contraction induced by 3 μ m 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 Ca 2+ ‐free solution, produces a maximum contraction through an indirect activation of Ca 2+ ‐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.