A toxin isolated from marine sponge, mycalolide‐B, inhibited smooth muscle contractions without changing cytosolic Ca 2+ levels. It also inhibited Ca 2+ ‐induced contraction in permeabilized smooth muscles. In native actomyosin prepared from chicken gizzard, mycalolide‐B inhibited superprecipitation and Mg 2+ ‐ATPase activity stimulated by Ca 2+ without changing myosin light chain phosphorylation. In the permeabilized muscle and native actomyosin preparation thiophosphorylated with ATPγS, mycalolide‐B inhibited ATP‐induced contraction and Mg 2+ ‐ATPase activity, respectively, in the absence of Ca 2+ . Mycalolide‐B also inhibited Mg 2+ ‐ATPase activity of skeletal muscle native actomyosin. Mycalolide‐B had no effect on calmodulin‐stimulated (Ca 2+ –Mg 2+ )‐ATPase activity of erythrocyte membranes. These results suggest that mycalolide‐B selectively inhibits actin—myosin interaction.
In the isolated rabbit trachea, electrical field stimulation (EFS) induced contraction that was inhibited by atropine or tetrodotoxin. Nonselective endothelin (ETA/ETB) receptor agonist, ET-1, relatively selective ETB receptor agonist, ET-3, and selective ETB receptor agonists, IRL 1620 and sarafotoxin S6c (STXc), augmented the EFS-induced contraction by 2- to 3-fold with similar EC50 (0.4-1 nM). These agonists also showed direct contractile effect in the trachea. However, the threshold concentration of ET-1 (3 fM) to augment the electrical field stimulation-induced contraction was 100,000 times lower than that needed to directly stimulate smooth muscle. In contrast, these agonists did not augment the contraction induced by stimulation of muscarinic receptor by carbachol. An ETA receptor antagonist, BQ-123, was almost ineffective in antagonizing the effects of ET-1, ET-3 and STXc although if weakly antagonized the effects of IRL 1620. An ETB receptor antagonist, RES-701-1, antagonized the effects of ET-3 and IRL 1620 without changing the effect of STXc and antagonized the effects of only lower concentrations of ET-1. In the trachea in which the ETB receptor was desensitized by strong activation, IRL 1620 and STXc were ineffective and ET-3 showed only small effect at higher concentrations. In contrast, the ETB desensitization inhibited the effects of only lower concentrations of ET-1. The effect of ET-1 in the ETB-desensitized trachea was partially, but not fully, antagonized by BQ-123. A potent ETB antagonist, BQ-788, showed similar effects to the ETB desensitization. These results suggest that ET-1 enhances nervous acetylcholine release by simultaneously activating the ET-1-selective ETA receptor and the isopeptide-nonselective ETB receptor (ETB1 subtype that is sensitive to both RES-701-1 and BQ-788 and the ETB2 subtype that is sensitive only to BQ-788).
Role of hi calponin on Ca2+-sensitivity of smooth muscle contraction was investigated using h1 calponin gene-deficient mice (CP−/−) and wild type mice (CP+/+). PGF2α induced a comparable force in intact aorta of CP+/+ and CP−/−. DPB showed similar effects to PGF2α. In membrane-permeabilized ileal smooth muscle, PDBu enhanced Ca2+-sensitivity of contraction comparably in CP+/+ and CP−/−. GTPγ-S showed similar effects. Our results suggest that hi calponin does not regulate Ca2+-sensitivity in the contractile mechanism of smooth muscle.
We compared the effects of dimeric marine toxins, bistheonellide A, and swinholide A, on actin polymerization. Bistheonellide A and swinholide A possess two identical side chains with similar structures to those of other marine toxins, mycalolide B, and aplyronine A. By monitoring changes in fluorescent intensity of pyrenyl-actin, bistheonellide A was found to inhibit polymerization of G-actin and to depolymerize F-actin in a concentration-dependent manner. The relationship between the concentration of bistheonellide A and its inhibitory activity on actin polymerization suggested that one molecule of bistheonellide A binds two molecules of G-actin. We demonstrated by SDS-PAGE that the complex of G-actin with bistheonellide A, swinholide A, or mycalolide B could not interact with myosin. No evidence was found that bistheonellide A severs F-actin at the concentrations examined (molar ratio to actin; 0.025–2.5), while swinholide A showed severing activity, although it was weaker than that of mycalolide B. We also demonstrated that the depolymerizing effect of bistheonellide A or mycalolide B is irreversible. Bistheonellide A increased, while swinholide A decreased, the rate of nucleotide exchange in G-actin, suggesting that binding of these toxins induces different conformational changes in the actin molecule. These results suggest that bistheonellide A intervenes between two actin molecules, forms a tertiary complex with each of its side chains bound to G-actin, and inhibits polymerization by sequestering G-actin from incorporation into F-actin. A difference in structure at the end of the side chain between dimeric macrolides and mycalolide B may account for the weak severing activity of the former.
