Effects of calyculin A on tension and myosin phosphorylation in skinned smooth muscle of the rabbit mesenteric artery
Authors: Suzuki Akito , Itoh Takeo 
Published: July 2012

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+.
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.
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+.
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.
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.
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.
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.