The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.

Smooth muscle contraction depends on the state of myosin phosphorylation and hence on the balance of myosin light chain kinase and phosphatase activity. Effects of okadaic acid isolated from black sponge on both enzyme activities and contractility were studied in chemically skinned fibers from guinea pig taenia coli. The toxin strongly inhibits myosin phosphatase and enhances tension development.

SUMMARY1. In guinea-pig taenia coli skinned with Triton X-100, the marine sponge toxin okadaic acid (OA; 0-1-10 ,UM) produced a dose-dependent enhancement of isometric tension in the presence of low concentrations (01-1 /LM) of Ca".2. The Ca2+-tension relation of the skinned taenia showed a high co-operativity (Hill coefficient, h = 5) in the presence of 02 /LM-calmodulin. The concentration of Ca2+ required to obtain half-maximal tension (ED50) was 1P8 /SM. OA (5/tM) reduced the co-operativity (h = 2 3) and increased the Ca21 sensitivity (ED50 = 0-92 ,gM-Ca21).OA further increased the tension produced with 30 /LM-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 cooperativity 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 gm1-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 ,tM-Ca2 . Complete relaxation did not occur in the presence of more than 1 ,uM-OA. The concentration of OA required to produce a 50% reduction (ID60) of the relaxation rate was 78 nM. 6. The phosphatase activity in the taenia extract was inhibited by OA (1-10 /IM) 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.

The influence of a purified holoenzyme form of polycation-modulable (PCM-) myosin phosphatase on Ca2+-dependent actin-myosin interactions was studied in detergent-skinned smooth muscle fibers from chicken gizzard. The concentration of Ca2+ required for half maximal isometric contraction (A0.5; 0.26 microM) of fibers incubated in the absence of phosphatase was increased 2-fold when PCM-phosphatase (13 U/ml) was included in the medium. Removal of the phosphatase restored A0.5 to control level showing that the enzyme-mediated decrease in Ca2+-sensitivity was reversible. Two-dimensional electrophoresis of fiber homogenates revealed that PCM-phosphatase decreased Ca2+-sensitivity for phosphorylation of the regulatory myosin light chains in parallel fashion. Ca2+-dependent increases in isometric force were directly correlated to increases in the extent of light chain phosphorylation up to about 0.35 mol PO4/mol light chain; further increases in phosphorylation were not associated with further increases in force. Addition of PCM-phosphatase to fibers which had been contracted with a suboptimal concentration of Ca2+ (0.35 microM) resulted in rapid relaxation. Unloaded shortening velocity, reflecting cross-bridge cycling rate, was reduced by 92% in the presence of PCM-phosphatase and light chain phosphorylation was decreased by 50%. These data show that both tension and unloaded shortening velocity may be related to Ca2+-dependent phosphorylation of the light chains. The results indicate that the level of phosphorylation attained in the fiber preparations studied probably reflects the ratio of myosin kinase to phosphatase activities. Since protein phosphatases are regulated enzymes the results also suggest that modulation of phosphatase activity may participate in control of smooth muscle contractility.

Since the Ca2+-regulatory mechanism for actin-myosin interaction in smooth muscle involves phosphorylation of the 20,000-Da myosin light chains, it was hypothesized that such interaction should be influenced by myosin phosphatase. Accordingly, we studied the effects of an aortic myosin light-chain phosphatase on Ca'+-dependent actin-myosin interaction in detergent-skinned porcine carotid artery and bovine aortic native actomyosin. In skinned preparations, the aortic phosphatase (16 U/ml) markedly inhibited the rate of isometric contraction in low Ca2+ (6.8 X lop7 M ) and responsiveness to Ca2+ (force attained with 6.8 X Ca2+/force attained with 1.6 X lop6 M Ca2+), whereas relaxation was accelerated. Ca2+dependent actomyosin ATPase activity and phosphorylation of the light chains were significantly and progressively depressed in the presence of increasing concentrations of phosphatase (0.1 -0.9 U/ml). The concentration of Ca2+ (1.1 X lop6 M ) required for half-maximal activation of either ATPase activity or light-chain phosphorylation increased by 70% in the presence of 0.1 U phosphatase/ml. Neither the maximal rate of Ca2+-sensitive ATP hydrolysis (39 2 0.8 nmole/ min/mg actomyosin) nor the extent of phosphorylation (0.68 k 0.05 mole PO,/mole light chain) was altered at >5 X M Ca2+. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 pM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca2+ 36