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

Abstract

1 Using P-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 M and maximum effects were obtained at 1O LM. MLC was mainly monophosphorylated, rather than diphosphorylated, in the presence of Ca2+. ED50 value for Ca2+ was 0.54 iM 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 fM CL-A produced a contraction, the maximum amplitude of which was similar to that evoked by 10 lIM Ca2+. CL-A (0.03-3 tM) 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 JAM and maximum effects were obtained at 3 gM. In the presence of CL-A, MLC was not only monophosphorylated but also diphosphorylated. ED50 values for CL-A were 0.39 JAM for tension, 0.44 JAM for the monophosphorylated form of MLC and 0.54 JuM 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+. 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 JM Ca2+ failed to produce contraction. Under these conditions, the amplitude of the contraction induced by 311M 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+-calmodulinindependent (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.

Rho‐kinase mediates diphosphorylation of myosin regulatory light chain in cultured uterine, but not vascular smooth muscle cells

“…In contrast, myocytes with tonic contractile patterns, such as hVM require prolonged contractions with relatively small changes in amplitude over time. Such speculation is supported by reports showing that pRLC corresponds linearly with tension development in hVM [27] and that ppRLC in hVM appears to be less important than pRLC [28][29][30]. These pathways were elucidated through the use of combinations of physiological agonists and pharmacological inhibitors.…”

Zipper-interacting Protein Kinase Induces Ca2+-free Smooth Muscle Contraction via Myosin Light Chain Phosphorylation

“…It has been known that smooth muscle contracts even in the absence of Ca 2ϩ if protein phosphatase inhibitors such as okadaic acid (16,(42)(43)(44), calyculin A (45,46), tautomycin (16,47), or microcystin (16 -19) are added. Recently, Weber et al (19) reported that microcystin induced Ca 2ϩ -free contractions of Triton X-100-skinned rat caudal arterial smooth muscle and that during the Ca 2ϩ -free contraction, myosin regulatory light chain was phosphorylated at both Ser 19 and Thr 18 .…”

Importance of inositol (1,4,5)‐trisphosphate, intracellular Ca2+ release and myofilament Ca2+ sensitization in 5‐hydroxytryptamine‐evoked contraction of rabbit mesenteric artery

“…H-7 can inhibit cyclic nucleotide-dependent protein kinases in addition to protein kinase C (Hidaka et al, 1984), although the possible role of these enzymes in responses to 5-HT will require further study (see Sumner et al, 1992). It has also been suggested that H-7 directly inhibits myosin light chain kinase (Suzuki & Itoh, 1993), but the concentration of H-7 used in the present study did not inhibit contraction to Ca2+, so that the 5-HT-induced increase in Ca2+ sensitivity may well involve protein kinase C. Previous studies on the rabbit mesenteric artery have also shown that H-7 and another protein kinase C inhibitor, staurosporine can inhibit noradrenaline, endothelin and GTP-7-S-induced contractions at fixed [Ca2+]j, and that the protein kinase C activator phorbol 12,13-dibutyrate increased the Ca2+-sensitivity of the contractile filaments (Nishimura et al, 1991).…”