CALCIUM-INDEPENDENT PHOSPHORYLATION OF SMOOTH-MUSCLE MYOSIN LIGHT CHAIN BY OKADAIC ACID ISOLATED FROM BLACK SPONGE (HALICHONDRIA-OKADAI)

  • 1 December 1987
    • journal article
    • research article
    • Vol. 243  (3) , 1167-1173
Abstract
Previously, we have shown that okadaic acid (OA), isolated from black sponge (Halichondria okadai) causes contraction even in the absence of Ca++ in the saponin-permealized taenia isolated from guinea pig cecum. In the present study, mechanism of action of OA was examined using native actomyosin extracted from chicken gizzard smooth muscle. In the absence of Ca++, OA (0.1-1 .mu.M) induced superprecipitation and increased the Mg++-adenosine triphosphatase activity. The OA-induced superprecipitation was enhanced by Ca++ at a concentration (> 0.1 .mu.M) which did not activate the calmodulin-dependent myosin light chain (MLC) kinase. The effect of OA was not affected by the calmodulin inhibitor, trifluoperazine, at a concentration (100 .mu.M) needed to inhibit the Ca++-induced response, but was inhibited markedly by the nonselective kinase inhibitors, amiloride (1 mM) and K-252a (5 .mu.M). The OA-induced superprecipitation in the absence of Ca++ was accompanied by phosphorylation of the 20 K dalton MLC, which also was enhanced by low concentration of Ca++ (> 0.1 .mu.M). OA did not change the phosphatase activity which dephosphorylates the phosphorylated MLC. An activator of Ca++- and phospholipid-dependent protein kinase, 12-O-tetradecanoylphorbol 13-acetate (1 .mu.M) did not modulate superprecipitation or phosphorylation of MLC in the presence and absence of OA. Furthermore, inhibitors of Ca++ and phospholipid-dependent protein kinase, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (400 .mu.M) and polymyxin B (100 .mu.g/ml), affected neither superprecipitation nor phosphorylation of MLC induced by OA. With a reconstituted system containing purified myosin and MLC kinase, OA induced only slight phosphorylation of MLC. These results suggest that the activation of contractile elements by OA is due to the phosphorylation of MLC. This effect may be attributable only partially to the direct activation by OA of MLC kinase, but not Ca++ and phospholipid-dependent protein kinase.