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Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (PDBu, 1 μM) induced sustained contractions with no increase in [Ca2+]i in nonpregnant and pregnant human myometria. The contractile effects of PDBu in pregnant myometrium were much greater than those in nonpregnant myometrium, and the contractions in pregnant myometrium were accompanied by an increase in myosin light chain (MLC) phosphorylation at Ser19. The contraction induced by PDBu in pregnant myometrium was inhibited by the inhibitors of conventional PKC isoforms, bisindolylmaleimides and indolocarbazole, such as Go6976, Go6983, and Go6850 (1 μM). LY333531 (1 μM), a specific inhibitor of PKCβ, also inhibited the PDBu-induced contraction in the pregnant myometrium. In the pregnant myometrium permeabilized with α-toxin, PDBu increased the contractions induced at fixed Ca2+ concentration (0.3 μM) both in nonpregnant and pregnant myometria, indicating Ca2+ sensitization of contractile elements. Western immunoblot analysis indicated that pregnant myometrium contained PKC isozymes such as conventional PKC (α, β, γ), novel PKC (δ, ɛ, θ), and atypical PKC (ζ but not ι and λ). RT-PCR and real-time RT-PCR analysis indicated that, among the conventional PKC, the levels of mRNA of β isoform in pregnant human myometrium were greater than those in nonpregnant myometrium. CPI-17 is a substrate for PKC, and the phosphorylated CPI-17 is considered to inhibit myosin phosphatase. The levels of CPI-17 mRNA and protein expression were also greater in the pregnant myometrium. These results suggest that the PKC-mediated contractile mechanism is augmented in human myometrium after gestation, and that this augmentation may be attributable to the increased activity of the β PKC isoform and CPI-17. British Journal of Pharmacology (2003) 140, 1303–1312. doi:10.1038/sj.bjp.0705552

1 Phorbol ester decreases muscle tension in the rat myometrium, and the effect is more potent in latepregnant myometrium than in nonpregnant myometrium. In the present study, we have examined the contribution of protein kinase C (PKC) isoforms to the phorbol ester-induced inhibition of tension in rat uterine smooth muscle. 2 Thymeleatoxin (THX), a selective activator of conventional-type PKC (cPKC), and 12deoxyphorbol 13-isobutyrate (DPB), an activator of pan PKC, inhibited the tension induced by high K + , and inhibitions were significantly increased in pregnant myometrium compared to nonpregnant myometrium. The inhibition by DPB and THX of high K + -induced tension was significantly attenuated when PKC was downregulated by long-term pretreatment with THX and inhibited by Go6976, a cPKC inhibitor. 3 Of the cPKCs, PKCa is predominantly expressed in the rat myometrium, as detected by Western blot analysis. The expression of PKCa gradually increases from the beginning of gestation, reaching a maximum at day 21 of pregnancy. Treatment with DPB induced PKCa to translocate from the cytosol to the membrane in the pregnant myometrium. PKCe and PKCz, other dominant PKC isoforms in the rat myometrium, decrease during gestation, reaching a minimum in late pregnancy. 4 These results suggest that cPKC may be at least partly involved in the PKC-mediated inhibition of muscle tension in the rat myometrium. British Journal of Pharmacology (2003) 139, 408 – 414. doi:10.1038/sj.bjp.0705237

1 Xestospongin-C isolated from a marine sponge, Xestospongia sp., has recently been shown to be a membrane-permeable IP3 receptor inhibitor. In this study we examined the eAects of this compound on smooth muscle from guinea-pig ileum. 2 In guinea-pig ileum permeabilized with a-toxin, xestospongin-C (3 mM) inhibited contractions induced by Ca 2+ mobilized from sarcoplasmic reticulum (SR) with IP3 or carbachol with GTP, but not with caAeine. 3 In intact smooth muscle tissue, xestospongin-C (3‐10 mM) inhibited carbachol- and high-K + induced increases in [Ca 2+ ]i and contractions at sustained phase. 4 It also inhibited voltage-dependent inward Ba 2+ currents in a concentration-dependent manner with an IC50 of 0.63 mM. Xestospongin-C (3‐10 mM) had no eAect on carbachol-induced inward Ca 2+ currents via non-selective cation channels; but it did reduce voltage-dependent K + currents in a concentration-dependent manner with an IC50 of 0.13 mM. 5 These results suggest that xestospongin-C inhibits the IP3 receptor but not the ryanodine receptor in smooth muscle SR membrane. In intact smooth muscle cells, however, xestospongin-C appears to inhibit voltage-dependent Ca 2+ and K + currents at a concentration range similar to that at which it inhibits the IP3 receptor. Xestospongin-C is a selective blocker of the IP3 receptor in permeabilised

1. We evaluated the role of the cross-linking of Fc epsilon RI-mediated inositol 1,4,5-triphosphate (IP(3)) in the increase in cytosolic Ca(2+) level ([Ca(2+)](i)) using xestospongin C, a selective membrane permeable blocker of IP(3) receptor, in RBL-2H3 mast cells. 2. In the cells sensitized with anti-dinitrophenol (DNP) IgE, DNP-human serum albumin (DNP-HSA) and thapsigargin induced degranulation of beta-hexosaminidase and a sustained increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited both of these changes that were induced by DNP-HSA without changing those induced by thapsigargin. 3. In the absence of external Ca(2+), DNP-HSA induced a transient increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited this increase in [Ca(2+)](i). 4. In the cells permeabilized with beta-escin, the application of IP(3) decreased Ca(2+) in the endoplasmic reticulum (ER) as evaluated by mag-fura-2. Xestospongin C (3 - 10 microM) inhibited the effect of IP(3). 5. After the depletion of Ca(2+) stores due to stimulation with DNP-HSA or thapsigargin, the addition of Ca(2+) induced capacitative calcium entry (CCE). Xestospongin C (3 - 10 microM) inhibited the DNP-HSA-induced CCE, whereas it did not affect the thapsigargin-induced CCE. 6. These results suggest that Fc epsilon RI-mediated generation of IP(3) contributes to Ca(2+) release not only in the initial phase but also in the sustained phase of the increase in [Ca(2+)](i), resulting in prolonged Ca(2+) depletion in the ER. The ER Ca(2+) depletion may subsequently activate CCE to achieve a continuous [Ca(2+)](i) increase, which is necessary for degranulation in the RBL-2H3 mast cells. Xestospongin C may inhibit Ca(2+) release and consequently may attenuate degranulation.

We investigated the chronic effects of doxorubicin (DXR) on morphological and functional changes in the rabbit mesenteric artery using an organ culture system. In arteries cultured with 0.3 μM DXR for 7 days, the contractions induced by noradrenaline, but not those induced by endothelin-1 or high K+, were strongly inhibited. This reaction was followed by a decrease in the induction of the α1A-adrenoceptor without any change in the mRNA level. Inhibition of noradrenaline-induced contractions by DXR was attenuated by superoxide dismutase, and α1A-adrenoceptor protein expression recovered. In the arteries cultured with 1 μM DXR for 7 days, contractions induced by endothelin-1 or high K+ and absolute force in the permeabilized muscles were also inhibited. Morphological examinations revealed the existence of concentrated nuclei and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)-positive smooth muscle cells, and internucleosomal DNA fragmentation was also detected, indicating the induction of apoptosis. In the arteries cultured with 10 μM DXR for 7 days, nuclear swelling, karyolysis and random DNA fragmentation indicative of necrosis were observed, and muscle contractility was abolished. These results suggest that 0.3 μM DXR selectively down-regulates the α1A-adrenoceptor protein expression, resulting in a decrease in the noradrenaline-induced contraction. This down-regulation may be at least partly due to the production of a superoxide radical. DXR also caused a decrease in muscle contractility followed by apoptotic changes at 1 μM and necrotic changes at 10 μM. These changes might be responsible for the disturbance of the circulatory system that is often observed during the course of repetitive chemotherapy. British Journal of Pharmacology (2001) 132, 1365–1373; doi:10.1038/sj.bjp.0703959

1 Recent evidence has indicated that the mitogen platelet-derived growth factor (PDGF) can act acutely to regulate arterial tone. In this study we demonstrate that the BB isoform of PDGF (PDGF-BB) can cause endothelium-independent relaxation of rabbit isolated mesenteric arteries. 2 In endothelium-denuded arteries, PDGF-BB (40 pM‐8.0 nM) caused concentration-dependent relaxation of noradrenaline-induced tone. The relaxation to PDGF-BB was abolished by a cyclooxygenase inhibitor, indomethacin (10 mM) and by the PDGF receptor-associated tyrosine kinase inhibitor, tyrphostin AG1295 (50 mM), but not by N G -monomethyl-L-arginine (L-NMMA, 200 mM), an inhibitor of nitric oxide (NO) synthase. 3 PDGF-BB (4.0 nM) significantly increased the release of prostacyclin (PGI2) in endotheliumdenuded arteries. Exogenously applied iloprost (1 mM), a stable analogue of PGI2, relaxed the endothelium-denuded artery. PDGF-BB (4.0 nM) significantly increased the cyclic AMP content. 4 In the absence of Ca 2+ , PDGF-BB (4.0 nM) failed to relax the artery, and the release of PGI2 was almost completely suppressed. In addition, the release of PGI2 by PDGF-BB (4.0 nM) was significantly reduced in the presence of endothelium. The eAect of endothelium was eliminated by LNMMA (200 mM) and PGI2 release increased. 5 These data indicate that in rabbit mesenteric arteries, PDGF-BB can evoke endotheliumindependent relaxation by stimulating the synthesis of PGI2. The PDGF-BB-induced prostaglandin synthesis is dependent on both Ca 2+ and tyrosine phosphorylation of the PDGF receptor, and is attenuated by endothelium-derived NO. British Journal of Pharmacology (2000) 131, 1546‐1552

We evaluated the role of the inositol 1,4,5-triphosphate (IP(3)) receptor-mediated Ca(2+) release on the positive inotropic effects of alpha-adrenergic stimulation using a novel, potent, selective membrane-permeable blocker of IP(3) receptor, xestospongin C. Guinea-pig papillary muscle permeabilized with saponin exhibited spontaneous oscillatory contractions in solution buffered with pCa(2+) 6.5 by a low concentration of EGTA. The oscillatory activity was increased by adding 100 microM IP(3) and abolished by 1 microM ryanodine or 30 microM cyclopiazonic acid. Xestospongin C (3 microM) inhibited the IP(3)-induced increase in the oscillatory contractions without affecting basal oscillations. In intact papillary muscle, xestospongin C (3 microM) inhibited the positive inotropic effects of phenylephrine, resulting in a rightward and downward shift of the concentration-response curve for phenylephrine. On the contrary, xestospongin C did not affect the concentration-response curve for phenylephrine obtained in the presence of ryanodine (1 microM). On the other hand, xestospongin C affected neither basal contractions nor the positive inotropic effects of a high extracellular Ca(2+) concentration (3.2 mM) or that of isoprenaline (1 and 10 nM). These results suggest that the IP(3)-mediated increase in Ca(2+) release is involved in the positive inotropic effects of alpha-adrenergic stimulation in the guinea-pig cardiac muscle.

It has been reported that endothelium-dependent relaxation is impaired in pulmonary hypertensive vessels. The underlying mechanisms for this phenomenon, however, have not yet been identified. In this study, the mechanisms responsible for decreased endothelium-dependent relaxation in the pulmonary artery isolated from monocrotaline (MCT)-induced pulmonary hypertensive rat (MCT rat) were examined. MCT (60 mg kg−1), or its vehicle was administered by a single subcutaneous injection to 6-week-old male Sprague Dawley rats. Endothelium-dependent relaxation induced by carbachol or ionomycin in the MCT rat artery was significantly smaller than that in vehicle-treated rat (control rat) artery. Cyclic GMP levels, measured by enzyme-immunoassay, under resting or stimulation with carbachol or ionomycin were also smaller in the MCT rat artery. However, sodium nitroprusside-induced cyclic GMP accumulation in the endothelium-denuded artery was similar in control and MCT rats. These results suggest that MCT treatment decreases endothelial nitric oxide (NO) production. Resting endothelial Ca2+ levels ([Ca2+]i) in the fura-PE3-loaded MCT rat artery, were not different from those in the control rat. However, the increase in endothelial [Ca2+]i elicited by carbachol was attenuated in the MCT rat. In quantitative RT–PCR analysis, the expression of mRNA encoding endothelial NO synthase was rather increased in the MCT rat artery, suggesting an up-regulation of eNOS expression. These results provide evidence that impaired NO-mediated arterial relaxation in the MCT rat is due to dissociation between eNOS expression and NO production. This dissociation may be derived from an inhibition of receptor-mediated Ca2+ metabolism and also from the apparent decrease in Ca2+ sensitivity of eNOS. British Journal of Pharmacology (1999) 128, 1098–1104; doi:10.1038/sj.bjp.0702878

Treatment with acetylcholine (ACh) of a beta-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca2+ concentration was clamped at 1 microM. The ACh-induced Ca2+ sensitization of myofilaments was significantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was significantly increased in the airway hyperresponsive rats. Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist-induced, Rho-mediated Ca2+ sensitization of myofilaments.

Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K+(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC50 of 88 μM. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca2+ (3 and 10 μM)-induced contractions with an IC50 of 46 μM for 3 μM Ca2+ and 105 μM for 10 μM Ca2+. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca2+. ST-A had no effect on this contraction. ST-A inhibited Mg2+-ATPase activity in native actomyosin prepared from chicken gizzard with an IC50 of 25 μM. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC50 of 152 μM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin. ST-A inhibited calmodulin activity, assessed by Ca2+/calmodulin-dependent enzymes, (Ca2+-Mg2+)-ATPase of erythrocyte membrane, with an IC50 of 100 μM and phosphodiesterase prepared from bovine cardiac muscle with an IC50 of 52 μM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3–300 μM). Similar results were obtained in the presence or absence of Ca2+ suggesting that ST-A binds to calmodulin and that Ca2+ is not essential for the binding of ST-A to calmodulin. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.

1. The effects of a non-selective P2-receptor agonist ATP and a selective P2x-receptor agonist alpha,beta-methylene-ATP on intracellular free Ca2+ level ([Ca2+]i) and force were examined in rat isolated aorta without endothelium. 2. Both ATP (1-1000 microM) and alpha,beta-methylene-ATP (0.1-100 microM) induced transient increase followed by small sustained increase in [Ca2+]i in a concentration-dependent manner. Compared with the force induced by a high concentration of KCl, the force induced by alpha,beta-methylene-ATP was smaller and that induced by ATP was much smaller at a given [Ca2+]i. 3. An L-type Ca2+ channel blocker, verapamil (10 microM), completely inhibited the high K(+)-stimulated [Ca2+]i and force. Verapamil partially inhibited the transient and sustained increases in [Ca2+]i induced by 10 microM alpha,beta-methylene-ATP and the sustained increase but not the transient increase induced by 1 mM ATP. 4. In the absence of extracellular Ca2+ (with 0.5 mM EGTA) 1 mM ATP caused transient increase in [Ca2+]i while 10 microM alpha,beta-methylene-ATP was ineffective 5. ATP, but not alpha,beta-methylene-ATP, increased the tissue adenosine 3':5'-cyclic monophosphate (cyclic AMP) level. 6. These data suggest that ATP and alpha,beta-methylene-ATP increase [Ca2+]i by an activation of both L-type and non-L-type Ca2+ channels. In addition, ATP, but not alpha,beta-methylene-ATP, increases [Ca2+]i by a release of Ca2+ from an intracellular Ca2+ store. Possible reasons are discussed as to why the increase in [Ca2+]i due to ATP and alpha,beta-methylene-ATP resulted in only a small contraction.

1. Inhibitory effects of a novel smooth muscle relaxant, KT-362 (5-[3-([2-(3,4-dimethoxyphenyl)-ethyl]amino)-1-oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate), on contraction and the cytosolic Ca2+ level ([Ca2+]cyt) in isolated vascular smooth muscle of rat aorta were examined. 2. KT-362 inhibited the contractions induced by high K+ and noradrenaline. The inhibitory effect was antagonized by an increase in external Ca2+ concentration. A Ca2+ channel activator, Bay K 8644, did not change the effect of KT-362 on high K+-induced contraction. 3. [Ca2+]cyt, measured with fura-2-Ca2+ fluorescence, increased during the contractions induced by high K+ or noradrenaline. KT-362 decreased [Ca2+]cyt and muscle tension stimulated by high K+ or noradrenaline. By contrast, a Ca2+ channel blocker, verapamil, inhibited the noradrenaline-induced increase in [Ca2+]cyt with only partial inhibition of the noradrenaline-induced contraction and KT-362 inhibited the verapamil-insensitive portion of the contraction without changing [Ca2+]cyt. 4. In a Ca2(+)-free solution, noradrenaline and caffeine induced a transient contraction following a transient increase in [Ca2+]cyt. KT-362 inhibited the increments due to noradrenaline but not those induced by caffeine. 5. These results suggest that KT-362 inhibits vascular smooth muscle contraction by inhibiting Ca2+ channels, receptor-mediated Ca2+ mobilization, and receptor-mediated Ca2+ sensitization of contractile elements.

Tracheal smooth muscle precontracted with carbachol relaxes upon the addition of 3 μM okadaic add. Although cytosolic Ca2+ concentrations decrease, myosin light chain remains highly phosphorylated (50%). In smooth muscle treated with carbachol alone or carbachol plus okadaic acid 32P is incorporated into a single peptide on myosin light chain which corresponds to the site phosphorylated by myosin light chain kinase. Treatment with okadaic acid alone does not result in myosin light chain phosphorylation or tension development. These results suggest that a cellular mechanism other than myosin light chain phosphorylation can regulate contractile tension.

1. The effects of procaine on muscle tension and 45Ca2+ movements were investigated in vascular smooth muscle of the rabbit aorta and intestinal smooth muscle of the taenia isolated from guinea-pig caecum. 2. Procaine (10 mM) induced a contraction in the taenia but had little effect on the resting tension in the aorta. 3. Procaine, 0.5-10 mM, relaxed the sustained contractions induced by 65.4 mM KCl and 10(-6) M noradrenaline in the aorta, and by 45.4 mM KCl, 10(-6) M carbachol and 10(-6) M histamine in the taenia. The inhibitory effect of procaine on the high K+-induced contractions was antagonized by external Ca2+ but not by the Ca2+ channel activators, Bay K 8644 and CGP 28,392. 4. 45Ca2+ uptake was increased by high K+ or noradrenaline in the aorta and by high K+ or carbachol in the taenia. The increments were inhibited by procaine at the concentrations needed to inhibit the muscle contractions. 5. In a Ca2+-free solution, noradrenaline and caffeine induced a transient contraction in the aorta, whereas a second application of each stimulant was almost ineffective. Addition of 1-10 mM procaine shortly before the first application of the stimulant inhibited the contraction. After washing the muscle with a Ca2+-free solution without procaine, the second application of the stimulant induced a greater contraction than that in control muscle without procaine pretreatment. 6. Noradrenaline and caffeine released 45Ca2+ from a cellular site in the aorta. Procaine inhibited the effects of these stimulants. 7. It was concluded that procaine may inhibit both the opening of Ca2+ channels and the release of Ca2 + from cellular stores and the former but not the latter effect may be attributable to a local anaesthetic action.

The effects of verapamil and sodium nitroprusside on muscle tension and 45Ca uptake activated in different ways were compared in rabbit aorta, rat aorta and guinea-pig taenia coli. In rabbit aorta, K-induced contraction was specifically inhibited by verapamil and noradrenaline-induced contraction by sodium nitroprusside. In rat aorta, both K-induced and noradrenaline-induced contractions were inhibited by verapamil or by sodium nitroprusside also. In taenia, both K- and histamine-induced sustained contractions were inhibited by verapamil but not by sodium nitroprusside. The effect of verapamil was competitively antagonized by external Ca, while that of sodium nitroprusside was not. High K, noradrenaline and histamine increased the rate of 45Ca uptake in aortae and taenia. In rabbit aorta the increment in response to high K was specifically inhibited by verapamil and the increment induced by noradrenaline was specifically inhibited by sodium nitroprusside. In rat aorta, increments induced by both high K and noradrenaline were inhibited by verapamil and by sodium nitroprusside. In taenia, the increments induced by high K and by histamine were inhibited by verapamil but not by sodium nitroprusside. These results suggest different characteristics of Ca entry systems in these smooth muscles. In rabbit aorta, there seem to be two Ca channels, one of which is activated by high K and inhibited by verapamil, while the other is activated by noradrenaline and inhibited by sodium nitroprusside. In rat aorta, both K- and noradrenaline-activated Ca pathways are sensitive to both verapamil and sodium nitroprusside whereas, in taenia, both K- and histamine-activated Ca pathways are sensitive only to verapamil.

Nitroglycerine induced biphasic relaxation in the rat aorta, previously contracted by noradrenaline; a rapid decrease in tension was followed by a gradual increase reaching a steady level below the control contractile tension. No initial transient relaxation was induced by nitroglycerine in high K-stimulated muscle. The initial transient relaxation, but not the sustained relaxation, was dependent on the concentration of external K; maximum relaxation was observed in the presence of 2.7 mM K solution and only a slight relaxation was observed in 0 mM or 10.8 mM K solution. The initial transient relaxation was also inhibited by ouabain or low Na solution. On an appropriate increase in the concentration of external K, noradrenaline-induced contraction was transiently relaxed. Previous application of nitroglycerine potentiated this K-induced relaxation. Pretreatment of the muscle with methylene blue, an inhibitor of guanylate cyclase, inhibited both the initial transient and the sustained relaxations induced by nitroglycerine, but not the K-induced transient relaxation. It is suggested that the nitroglycerine-induced initial transient relaxation, but not the sustained relaxation, may be due to a stimulation of an electrogenic Na pump. Both relaxation phases may be mediated by cyclic GMP.

Age-related changes in the sensitivity to verapamil and sodium nitroprusside were examined in isolated aortic strips of the rabbit. In the aortae of newborn rabbits within 10 days of birth, the resting tone of the muscle was strongly reduced by sodium nitroprusside but not by either Ca-deficient solution or by verapamil. High K-induced contraction and noradrenaline-induced contraction were both inhibited by verapamil or sodium nitroprusside. In the aortae of 24 day-old rabbits, resting tension was slightly reduced by sodium nitroprusside but not by verapamil. High K-induced contraction was less sensitive to sodium nitroprusside than to verapamil whereas noradrenaline-induced contraction was less sensitive to verapamil than to sodium nitroprusside. In the aortae isolated from 60 day-old or older rabbits, resting tension was not affected by either sodium nitroprusside or verapamil. High K-induced contraction was inhibited by verapamil whereas sodium nitroprusside showed only a weak inhibitory effect. Noradrenaline-induced contraction was inhibited by sodium nitroprusside although verapamil had only a slight inhibitory effect. In the aortae of 1 day-old and also in adult rabbits, noradrenaline induced an additional increase in muscle tension when applied during the sustained contraction induced by high K. It is suggested that, in the newborn rabbit aorta, the voltage-dependent Ca channel is sensitive to both verapamil and sodium nitroprusside and the sensitivity to sodium nitroprusside gradually decreases during maturation whereas the receptor-linked Ca channel is also sensitive to both of the inhibitors at birth but the sensitivity to verapamil gradually decreases with age.

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 microM 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 microM okadaic acid. 3. High K+-induced contraction in rabbit aorta was accompanied by increased Ca2+ influx measured with 45Ca2+ and increased cytosolic Ca2+ [( Ca2+]cyt) 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 microM, inhibited aggregation, ATP release and increased in [Ca2+]cyt 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.

1 Several putative calmodulin antagonists have been examined for their inhibitory action on muscle tension and cellular Ca content in the K-depolarized vascular and intestinal smooth muscles. 2 The 65.4 mM K-induced sustained contraction in the media-intimal layer of rabbit aorta and the 45.4 mM K-induced sustained contraction in guinea-pig taenia coli were inhibited by the calmodulin antagonists, prenylamine, chlorpromazine, N2-dansyl-L-arginine-4-t-butylpiperadine amide (No. 233), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), and also by the organic Ca antagonists, verapamil and diltiazem. 3 The cellular Ca content in rabbit aorta and guinea-pig taenia coli as measured by a modified lanthanum technique increased in the high-K solutions. The increments were inhibited by these antagonists at concentrations similar to those required to inhibit the K-induced contractions. However, W-7 did not change (in aorta) or only slightly decreased (in taenia coli) the K-induced increase in the cellular Ca content. 4 A high concentration (2 X 10(-4)M) of W-7 increased the resting cellular Ca content without increasing the muscle tension in aorta. The increment was inhibited by verapamil, sodium nitroprusside or hypoxia (N2 aeration). 5 It is suggested that the inhibitory effects of prenylamine, chlorpromazine and No. 233 may be attributed mainly to the Ca antagonistic effect whereas W-7 may inhibit the process beyond the transmembrane Ca influx.