Effects of okadaic acid on isometric tension and myosin phosphorylation of chemically skinned guinea-pig taenia coli

Abstract

1. In guinea-pig taenia coli skinned with Triton X-100, the marine sponge toxin okadaic acid (OA; 0.1-10 microM) produced a dose-dependent enhancement of isometric tension in the presence of low concentrations (0.1-1 microM) of Ca2+. 2. The Ca2+-tension relation of the skinned taenia showed a high co-operativity (Hill coefficient, h = 5) in the presence of 0.2 microM-calmodulin. The concentration of Ca2+ required to obtain half-maximal tension (ED50) was 1.8 microM. OA (5 microM) reduced the co-operativity (h = 2.3) and increased the Ca2+ sensitivity (ED50 = 0.92 microM-Ca2+). OA further increased the tension produced with 30 microM-Ca2+, while it failed to produce any mechanical effect in Ca2+-free solution. When the calmodulin concentration was increased the Ca2+ sensitivity increased as well, but the co-operativity was not affected both in the absence and in the presence of OA. 3. The level of myosin phosphorylation was analysed by two-dimensional gel electrophoresis. OA produced an increase in phosphorylated light chains and a concomitant decrease in unphosphorylated light chains. The effect was completely reversed when OA was washed out. 4. In solutions containing more than 1 microM-Ca2+, a third protein band appeared on the gels next to the bands of light chains. OA markedly increased the third band which disappeared when OA and Ca2+ were simultaneously removed. 5. OA reversibly slowed down both relaxation and dephosphorylation induced by Ca2+ removal following activation with 30 microM-Ca2+. Complete relaxation did not occur in the presence of more than 1 microM-OA. The concentration of OA required to produce a 50% reduction (ID50) of the relaxation rate was 78 nM. 6. The phosphatase activity in the taenia extract was inhibited by OA (1-10 microM) in a dose-dependent manner. The inhibition was well described as a mixed noncompetitive inhibition, and the dose-inhibition relation was shifted to the right when the concentration of substrate (phosphorylated light chains) was increased. The lower and upper limits of the change of ID50 produced by changing the substrate concentration were estimated to be 10 and 165 nM-OA, respectively. 7. These results strongly suggest that the tension enhancement and the slow-down of relaxation are both causally related to inhibition of myosin phosphatase activity by OA.