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

Abstract

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.

Effects of okadaic acid and ATPγS on cell length and Ca2+‐channel currents recorded in single smooth muscle cells of the guinea‐pig taenia caeci

“…The intracellular perfusion of either ATPyS (1 mM) or OA (50 pM) caused contraction of single cells of the taenia caeci, as has been reported in intact, or chemically permeabilized, smooth muscle strips (Ozaki et al, 1987;Bialojan et al, 1988;Karaki et al, 1989). In the present experiments, however, cells were perfused with 3mM EGTA and bathed in 7.5mM Ba2".…”

“…Contractions to noradrenaline (rabbit aorta; guinea-pig vas deferens), carbachol (bovine trachea) and ATP (guinea-pig vas deferens) are all blocked by OA (Karaki et al, 1989;Tansey et al, 1990;Shibata et al, 1991). In 'skinned' smooth muscle strips, OA (10-50/AM) can also induce contraction in the presence, or absence, of Ca2 + and calmodulin (Bialojan et al, 1988;Karaki et al, 1989). When OA (50/AM) was added to the pipette solution, single cells of the taenia caeci showed a time-dependent contraction to 29 + 5% (n = 4) of their initial length within 5 min of membrane rupture (Figure 5a).…”

“…Calcium sensitivity is conferred by the calcium-calmodulin dependence of the myosin light chain kinase (for review, see Paul, 1989). Inhibition of phosphoprotein phosphatases, however, induces contraction in various smooth muscles (Shibata et al, 1982;Bialojan et al, 1988) which are not necessarily dependent on Ca2+ or calmodulin (Ishihara et al, 1989;Obara et al, 1989).…”

Electrical effects of okadaic acid extracted from black sponge on rabbit sinus node

“…In saponin-treated 'skinned' smooth muscle in the absence of Ca2+ (Ozaki et al, 1987b), this agent causes a contraction. Recently, such a contraction has been attributed to myosin light-chain phosphorylation (Ozaki et al, 1987;Bialojan et al, 1988;Erdodi et al, 1988) mediated through inhibition of myosin phosphatase activity by okadaic acid (Bialojan et al, 1988;Erdodi et al, 1988). In cardiac muscle, okadaic acid also causes an increase in the contractile force induced by electrical stimuli (Kodama et al, 1986).…”

Synergistic Effects of Cyclic AMP-Related Vasodilators and the Phosphatase Inhibitor Okadaic Acid

“…Okadaic acid is a potent inhibitor of protein phos phatases, inhibiting type 2A phosphatase at lower concen trations and type 1 phosphatase at higher concentrations (1,2). In smooth muscle, okadaic acid has concentration dependent dual effects.…”