1 Effects of okadaic acid, a toxin isolated from marine sponges, on smooth muscle contraction and platelet activation were examined. 2 Contractions in rabbit aorta induced by high concentrations of K+ and noradrenaline were inhibited by 0.1-1 yM okadaic acid in a concentration-dependent manner. Spontaneous rhythmic contractions as well as high K+-induced contraction in guinea-pig taenia caeci were also inhibited by 1 gM okadaic acid. 3 High K+-induced contraction in rabbit aorta was accompanied by increased Ca2+ influx measured with 45Ca2+ and increased cytosolic Ca2+ ([Ca2+]C) measured with fura-2-Ca2+ fluorescence. Okadaic acid inhibited the contraction without inhibiting Ca2+ influx and produced only a small decrease in [Ca2 +]cyt. 4 In a saponin-skinned taenia, Ca2 +-induced contraction was not inhibited but rather potentiated by okadaic acid.5 Okadaic acid, 1 y, inhibited aggregation, ATP release and increase in [Ca2 +] yt induced by thrombin in washed rabbit platelets. Okadaic acid itself did not change the platelet activities. 6 Okadaic acid did not change the cyclic AMP content of rabbit aorta although the inhibitory effects of okadaic acid were similar to those of cyclic AMP. 7 Although the mechanism of the inhibitory effect of okadaic acid was not clarified in the present experiments, it is suggested that okadaic acid acts by inhibiting protein phosphatases resulting in an indirect activation of cyclic AMP-dependent protein phosphorylation.

The effects of carbachol on muscle tension and cytosolic Ca2+ concentrations ([Ca2+]cyt), measured with fura-2, were examined in the guinea pig intestinal smooth muscle. Carbachol induced an initial transient increase followed by a sustained increase in [Ca2+]cyt and muscle tension. Higher concentrations of carbachol induced larger transient changes and smaller sustained changes. In the presence of carbachol, application of Ca2+ to a Ca2(+)-depleted muscle induced a contraction that was smaller in the presence of higher concentrations of carbachol. High concentrations of carbachol inhibited the high-K(+)-stimulated muscle tension and [Ca2+]cyt. Contractile and inhibitory effects of carbachol were inhibited by a muscarinic M2 antagonist. Increase in the external Ca2+ concentration or addition of BAY K 8644, a Ca2(+)-channel activator, antagonized the inhibitory effect. There was a linear correlation between log [Ca2+]cyt and muscle tension under the conditions employed in the present experiments (r = 0.949). These results suggest that lower concentrations of carbachol increase [Ca2+]cyt and induce contraction, whereas high concentrations of carbachol have an additional effect to decrease [Ca2+]cyt and inhibit contraction by a Ca2(+)-channel blocker-like action.

1 Effects of phorbol esters on the cytosolic Ca2" level ([Ca2+]j) and muscle tension in the intestinal smooth muscle of guinea-pig taenia caeci were examined.2 12-Deoxyphorbol 13-isobutyrate (DPB, 1 gM) did not change the [Ca2+]i and tension in resting muscle.3 In high K+-stimulated muscle, 1 gM DPB transiently augmented the contraction and decreased [Ca2+]. 12-Deoxyphorbol 13-isobutyrate 20-acetate (1 JAM) and phorbol 12, 13-dibutyrate (1 JAM) showed similar effects to DPB whereas phorbol 12-myristate 13-acetate (1 JM) and phorbol 12, 13-didecanoate (1 JM) were ineffective.4 DPB (1 JM) inhibited both [Ca2+]i and tension stimulated by 300 nM carbachol or 3 JAM histamine. In the presence of a higher concentration of carbachol (1 JM), DPB decreased [Ca2+]i and transiently increased muscle tension. 5 In the muscle strips permeabilized with bacterial a-toxin, 1 JAM DPB shifted the Ca2+-tension curve to the left. An inhibitor of protein kinase C, H-7 (30 JAM), inhibited the effect of DPB. 6 DPB did not change the high K+-induced contraction in the muscle strips pretreated with 3 JAM phorbol 12-myristate 13-acetate for 24 h. 7 These results suggest that activation of protein kinase C has dual effects; it augments contraction by increasing the Ca2+ sensitivity of the contractile elements and it inhibits contraction by decreasing [Ca2+ i.

2 A noradrenaline (10 pM)-induced sustained contraction was associated with a sustained increase in the fura-PE3 signal, or a transient increase followed by small sustained increase in the aequorin signal. A high K+-induced contraction was associated with a sustained increase in both the fura-PE3 and aequorin signals. 3 A second application of noradrenaline or high K+ induced reproducible contractions and fura-PE3 signals. In contrast, the aequorin signal resulting from a second application of noradrenaline or high K+ was much smaller than the first signal. 4 Following a 13 h but not a 3 h resting period, the aequorin signal stimulated by noradrenaline or high K+ recovered, without any change in the contractile response.5 In Ca2"-free solution, high K+ was ineffective, whereas noradrenaline induced only a small aequorin signal and contraction compared to those obtained in the presence of external Ca2". After the addition of Ca2", the first application of noradrenaline induced a large aequorin signal and a large contraction, although a second application induced a much smaller aequorin signal accompanied by a large contraction.6 These results suggest that high K+ and noradrenaline increase Ca2" in at least two cytosolic compartments; a compartment that is coupled to the contractile mechanism ('contractile' Ca2+ compartment; major portion of cytoplasm containing contractile elements) and a compartment that is not coupled to contractile mechanisms ('non-contractile' Ca2+ compartment; small sub-membrane area that does not contain contractile elements). On stimulation, the Ca2+ level in the 'contractile' compartment may increase to a level high enough to stimulate myosin light chain kinase but not so high as to consume aequorin rapidly. In contrast, the Ca2+ level in the 'non-contractile' compartment may increase so greatly that aequorin in this compartment is rapidly consumed. These two compartments may be separated by a diffusion barrier and, during a resting period, aequorin may slowly diffuse from the 'contractile' compartment to the 'non-contractile' compartment and thus restore the full aequorin signal. An increase in Ca2+ in the 'non-contractile' compartment seems to be dependent mainly on Ca2+ influx and partly on Ca2+ release.