Effect of Clostridium perfringens Alpha Toxin on Contraction of Isolated Guinea-Pig Diaphragm

The effect of Clostridium perfringens alpha toxin on contraction induced by-electric stimulation of isolated guinea-pig diaphragm was investigated. The toxin inhibited electrically stimulated contraction of the tissue in a dose- and incubation time-dependent manner. Tetrodotoxin resulted in no effect of the action of the toxin. Nifedipine dose-dependently delayed the action of the toxin, but verapamil and diltiazem did not. On the other hand, treatment of the toxin with N-acetylimidazole caused significant reduction of the inhibitory activity of the toxin on contraction, but did not cause significant loss of phospholipase C activity (PN activity) as measured by hydrolysis of p-nitrophenylphosphorylcholine. The data showed that the toxin impairs contraction of isolated guinea-pig diaphragm.     

胰岛素对离体大鼠胸主动脉血管张力的影响

制备离体大鼠胸主动脉环,分有内皮组和去内皮组,采用离体血管灌流技术,观察胰岛素对去氧肾上腺素（PE）和氯化钾（KCl）预收缩的胸主动脉环收缩张力的影响。结果表明胰岛素对PE预收缩的胸主动脉环产生浓度依赖性的舒张作用,且有内皮组和去内皮组间无显著差异。胰岛素对KCl预收缩的胸主动脉环没有显著影响。胰岛素对PE预收缩的胸主动脉环有非内皮依赖性舒张作用。     

SCORPION TOXIN-INDUCED CATECHOLAMINE RELEASE FROM SYNAPTOSOMES

Abstract— A toxin purified from crude venom of the scorpion L. quinquestriatus releases [3H]norepinephrine from synaptosomes prepared from rat brain. The toxin-induced release is dependent on duration of exposure and concentration of toxin in the medium. The absence of calcium in the medium diminishes toxin-induced release but does not abolish it. Toxin-induced release is diminished by tetrodotoxin or, to a lesser extent, by desmethylimipramine. Since the released tritium is present predominantly as norepinephrine, it appears that toxin-induced release is similar to that produced by veratradine or tyramine and is distinct from reserpine induced release.     

Prevention of toxin-induced cytoskeletal disruption and apoptotic liver cell death by the grapefruit flavonoid, naringin

The protein phosphatase-inhibitory algal toxins, okadaic acid and microcystin-LR, induced overphosphorylation of keratin and disruption of the keratin cytoskeleton in freshly isolated rat hepatocytes. In hepatocyte cultures, the toxins elicited DNA fragmentation and apoptotic cell death within 24 h. All these toxin effects could be prevented by the grapefruit flavonoid, naringin. The cytoprotective effect of naringin was apparently limited to normal hepatocytes, since the toxin-induced apoptosis of hepatoma cells, rat or human, was not prevented by the flavonoid.     

Contraction induced by Clostridium perfringens epsilon toxin in the isolated rat ileum

Clostridium perfringens epsilon toxin caused contraction of the isolated ileum of the rat in a dose-dependent manner. The contraction caused by the toxin was inhibited by a low Na medium, tetrodotoxin (TTX), atropine, mecamylamine or tetraethylammonium (TEA). Furthermore, the contractile response induced by the toxin was abolished by incubation in Ca-free medium, and completely restored by and addition of Ca2+. In addition, verapamil inhibited contraction induced by the toxin in a dose-dependent manner. These data suggest that epsilon toxin induces contraction of the isolated ileum and that the toxin-elicited contraction is the result of an indirect action mediated through the nervous systems.     

Mechanism of vasorelaxation caused by N-Benzylsecoboldine in rat thoracic aorta

The vasorelaxing effect of N-benzylsecoboldine on the rat thoracic aorta was investigated, and we also compare it with nifedipine and cromakalim. In high K⁺ (60 mM) medium, Ca²⁺ (0.03–3 mM)-induced vasoconstriction was inhibited concentration-dependently by N-benzylsecoboldine, whereas this contraction was not altered by cromakalim. Cromakalim relaxed aortic rings precontracted with 15 but not 60 mM of K⁺. N-benzylsecoboldine and nifedipine were more potent and effective in producing relaxation in 60 mM than in 15 mM K⁺-induced contraction. N-benzylsecoboldine was found to be an ₁-adrenoceptor-blocking agent in rat thoracic aorta as revealed by its competitive antagonism of phenylephrine (PE)-induced contraction (pA₂=6.31 ± 0.04, pA₁₀=5.41 ± 0.03). This relaxing effect of N-benzylsecoboldine was not antagonized by indomethacin or methylene blue, and still persisted in endothelium-denuded aorta or in the presence of nifedipine (1 µM). The increase of inositol monophosphate caused by PE in rat aorta was significantly suppressed by N-benzylsecoboldine, but not by nifedipine or cromakalim. High concentration of N-benzylsecoboldine (100 µM) did not affect the contraction induced by B-HT 920, serotonin or PGF₂. Glibenclamide and charybdotoxin did not affect the relaxation of N-benzylsecoboldine in aortic rings precontracted with PE. Neither cGMP nor cAMP levels were changed by N-benzylsecoboldine. We suggest that N-benzyl-secoboldine relaxes rat thoracic aorta by suppressing the Ca²⁺ influx and also has antagonistic effect on ₁-adrenoceptors.     

Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent

Levobupivacaine is a long-acting local anesthetic that intrinsically produces vasoconstriction in isolated vessels. The goals of this study were to investigate the calcium-dependent mechanism underlying levobupivacaine-induced contraction of isolated rat aorta in vitro and to elucidate the pathway responsible for the endothelium-dependent attenuation of levobupivacaine-induced contraction. Isolated rat aortic rings were suspended to record isometric tension. Cumulative levobupivacaine concentration-response curves were generated in either the presence or absence of the antagonists verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, Gd(3+), N(W)-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and methylene blue, either alone or in combination. Verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, low calcium concentrations, and calcium-free Krebs solution attenuated levobupivacaine-induced contraction. Gd(3+) had no effect on levobupivacaine-induced contraction. Levobupivacaine increased intracellular calcium levels in vascular smooth muscle cells. L-NAME, ODQ, and methylene blue increased levobupivacaine-induced contraction in endothelium-intact aorta. SKF-96365 attenuated calcium-induced contraction in a previously calcium-free isotonic depolarizing solution containing 100?mmol/L KCl. Levobupivacaine-induced contraction of rat aortic smooth muscle is mediated primarily by calcium influx from the extracellular space mainly via voltage-operated calcium channels and, in part, by inositol 1,4,5-trisphosphate receptor-mediated release of calcium from the sarcoplasmic reticulum. The nitric oxide - cyclic guanosine monophosphate pathway is involved in the endothelium-dependent attenuation of levobupivacaine-induced contraction.     

Vasodilatory action mechanisms of apigenin isolated from Apium graveolens in rat thoracic aorta.

The effect of apigenin, isolated from Apium graveolens, on the contraction of rat thoracic aorta was studied. Apigenin inhibited the contraction of aortic rings caused by cumulative concentrations of calcium (0.03-3 mM) in high potassium (60 mM) medium, with an IC50 of about 48 microM. After pretreatment it also inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contraction in a concentration (35-140 microM)-dependent manner with an IC50 of 63 microM. At the plateau of NE-induced tonic contraction, addition of apigenin caused relaxation. This relaxing effect of apigenin was not antagonized by indomethacin (20 microM) or methylene blue (50 microM), and still existed in endothelial denuded rat aorta or in the presence of nifedipine (2-100 microM). Neither cAMP nor cGMP levels were changed by apigenin. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine in the Ca(2+)-free solution were unaffected by apigenin. 45Ca2+ influx caused by either NE or K+ was inhibited by apigenin concentration-dependently. It is concluded that apigenin relaxes rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage- and receptor-operated calcium channels.     

Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxin-induced cytotoxicity

The lethal toxin ofBacillus anthracis is central to the pathogenesis of anthrax. Using primary cultures of mouse peritoneal macrophages, we have demonstrated that intracellular calcium release inhibitors protect against anthrax lethal toxin-induced cytotoxicity. The cytolytic effect of anthrax lethal toxin was markedly reduced by dantrolene, an inhibitor of calcium release from intracellular calcium stores. Pretreatment of macrophages with cyclosporin A, which has been shown to be a potent inhibitor of calcium release from mitochondria, also protected cells against cytotoxicity. These results indicate that calcium release from intracellular store may be an essential step for the propagation of anthrax lethal toxin-induced cell damage in macrophages. Thus our findings suggest that dantrolene, cyclosporin A, and possibly other drugs affecting intracellular calcium pools might be effectively preventing the toxicity from anthrax lethal toxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vasorelaxant activity of cyclic peptide, cyclosquamosin B, from Annona squamosa

A cyclic octapeptide, cyclosquamosin B (2), isolated from the seeds of Annona squamosa showed a vasorelaxant effect on rat aorta. It showed a slow relaxation activity against norepinephrine (NE)-induced contractions of rat aorta with/without endothelium. It showed inhibition effect on vasocontraction of depolarized aorta with high concentration potassium, but moderately inhibition effect on NE-induced contraction in the presence of nicardipine. These results showed that the vasorelaxant effect by 2 might be attributed mainly to inhibition of calcium influx from extracellular space through voltage-dependent calcium channels.     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

Cycloheximide potentiation of prostaglandin E1- and cholera toxin-stimulated cyclic AMP accumulation in NG108-CC15 neuroblastoma-glioma hybrid cells

Previous studies have demonstrated that catecholamine responsiveness in a variety of cells can be altered by inhibitors of RNA and protein synthesis. The neuroblastoma-glioma hybrid, NG108-CC15, which lacks catecholamine-stimulated accumulation of cyclic AMP, was investigated to determine if the responsiveness to prostaglandin E1 (PGE1) could be modified by inhibitors of protein synthesis. Cycloheximide in a time-dependent manner potentiated the ability of prostaglandin E1 to stimulate accumulation of intracellular cyclic AMP. However, the alpha-adrenergic inhibition of the prostaglandin response was not affected by cycloheximide. Withdrawal of norepinephrine following a long-term incubation resulted in a potentiation of subsequent PGE1-stimulated cyclic AMP accumulation. Cycloheximide enhanced this norepinephrine withdrawal effect. Our previous studies have shown that cholera toxin induces refractoriness to beta-adrenergic agonists in C6-2B rat astrocytoma cells and that cycloheximide blocked this action of cholera toxin. In an analogous manner cholera toxin caused refractoriness to subsequent prostaglandin-stimulated cyclic AMP production in NG108-CC15 cells, and cycloheximide reduced cholera toxin-induced prostaglandin refractoriness. Thus cycloheximide potentiates the prostaglandin stimulatory effect, has no effect on the ability of alpha-agonists to inhibit the prostaglandin response, increases the stimulatory effect of PGE1 after norepinephrine withdrawal, and reduces cholera toxin-induced PGE1 refractoriness. these observations suggest that PGE1-stimulated cyclic AMP accumulation in NG108-CC15 cells contains components which are regulated by de novo protein synthesis.     

Calmodulin inhibitors suppress calcium signaling from serotonin receptors in smooth muscle cells and abolish vasoconstrictive response on intravenous introduction of serotonin

Comparative study of the effect of calmodulin inhibitors trifluoperazine, W-12, and W-13 and the TRPV1 channel blocker capsazepine on receptor-dependent calcium metabolism in smooth muscle cells of the rat aorta and on the contraction of the isolated rat aorta was performed. Trifluoperazine almost completely abolishes an increase in free cytoplasmic calcium concentration in smooth muscle cells isolated from the rat aorta and smooth muscle cells of the A7r5 line in response to serotonin and does not affect cellular reaction to vasopressin and angiotensin II. W-12 and W-13 also do not attenuate responses to vasopressin and angiotensin II and reduces by two times free cytoplasmic calcium concentration elevation in response to serotonin. The efficiency of calcium metabolism suppression by calmodulin inhibitors correlates with the degree of inhibition of the aorta contractile response to serotonin. It was demonstrated that the inhibitory action of calmodulin antagonists on calcium metabolism in smooth muscle cells and the contractility of the isolated rat aorta during the activation of serotonin vasoconstrictive receptors are realized by a TRPV1-independent mechanism. It was demonstrated in experiments in vivo that trifluoperazine does not influence hypotensive reaction in rats (normally observed in response to intravenous serotonin injection), but removes the hypertensive effect of this neurotransmitter in rats after chronic introduction of dexamethasone. The results obtained confirm the hypothesis (that we previously stated) about the direct involvement of calmodulin in signal transmission from vasoconstrictive serotonin receptors.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells

Clostridium perfringens beta toxin is an important agent of necrotic enteritis. Of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin. The toxin induced swelling and lysis of the cell. Treatment of the cells with the toxin resulted in K+ efflux from the cells and Ca2+, Na+, and Cl- influxes. These events reached a maximum just before the cells were lysed by the toxin. Incubation of the cells with the toxin showed the formation of toxin complexes of about 191 and 228 kDa, which were localized in the domains that fulfilled the criteria of lipid rafts. The complex of 228 kDa was observed until 30 min after incubation, and only the complex of 191 kDa was remained after 60 min. Treatment of the cells with methyl-beta-cyclodextrin or cholesterol oxidase blocked binding of the toxin to the rafts and the toxin-induced K+ efflux and swelling. The toxin-induced Ca2+ influx and morphological changes were inhibited by an increase in the hydrodynamic diameter of polyethylene glycols from 200 to 400 and markedly or completely inhibited by polyethylene glycol 600 and 1000. However, these polyethylene glycols had no effect on the toxin-induced K+ efflux. The toxin induced carboxyfluorescein release from phosphatidyl-choline-cholesterol liposomes containing carboxyfluorescein and formed an oligomer with 228 kDa in a dose-dependent manner but did not form an oligomer with the 191-kDa complex. We conclude that the toxin acts on HL 60 cells by binding to lipid rafts and forming a functional oligomer with 228 kDa.     

Non-specific antagonism against thromboxane A2 of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) in contraction of isolated smooth muscles

7-Ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was reported as an antagonist of thromboxane (Tx) A₂ in the contraction of rabbit aorta. It was, however, observed that EG-626 did inhibit the contraction of superfused rabbit aorta, but also did inhibit that of rabbit coeliac artery, rat stomach strip and rat colon induced by TxA₂, PG endoperoxides, angiotensin II and PGF_2α in non-specific manner. EG-626 had no effect on the biosynthesis of PG endoperoxides as well as TxA₂. These results indicate that EG-626 is not a TxA₂ antagonist, but has a general inhibitory effect on the smooth muscles. This inhibitory effect of EG-626 may be explained by the inhibition of phosphodiesterase.     

Non-specific antagonism against thromboxane A2 of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) in contraction of isolated smooth muscles

7-Ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was reported as an antagonist of thromboxane (Tx) A2 in the contraction of rabbit aorta. It was, however, observed that EG-626 did inhibit the contraction of superfused rabbit aorta, but also did inhibit that of rabbit coeliac artery, rat stomach strip and rat colon induced by TxA2, PG endoperoxides, angiotensin II and PGF2 alpha in non-specific manner. EG-626 had no effect on the biosynthesis of PG endoperoxides as well as TxA2. These results indicate that EG-626 is not a TxA2 antagonist, but has a general inhibitory effect on the smooth muscles. This inhibitory effect of EG-626 may be explained by the inhibition of phosphodiesterase.     

Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: evidence against a role for prostaglandins in cholera toxin-induced secretion.

The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin. Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation. Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin. These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo.     

Inhibitory effect of ibuprofen on the cholera toxin-induced cyclic AMP response in Chinese hamster ovary cells

Abstract Ibuprofen, an inhibitor of prostaglandin synthesis in eukaryotic cells, was shown to inhibit the accumulation of 3',5'-cyclic adenosine monophosphate (cyclic AMP) in Chinese hamster ovary (CHO) cells exposed to cholera toxin. The inhibition was dose dependent, with a dose of 100 μg/ml reducing the cholera toxin response by approximately 50%, and maximal inhibition was observed when the drug was applied to the cells simulataneously with or 1 h before the toxin. Although ibuprofen also inhibited adenylate cyclase stimulation by forskolin, suggesting a nonspecific effect, the drug had no effect on cholera toxin-induced cyclic AMP accumulation when added to the culture medium 15 min or more after the toxin.     

Pharmacological characteristics of novel putative purinoceptors in vascular endothelium

Using an isolated rat aorta with intact endothelium, a functional bioassay system was created as follows: pre-contract the isolated rat aorta with intact endothelium in the presence of 0.62 micromol/l of norepinephrine, and then add acetylcholine to obtain maximal endothelium-dependent relaxation. In the addition of low concentration of adenosine, a P(1) agonist or ATP, a P(2) agonist to this system, the rat aorta smooth muscle contraction was observed. This observation could not be explained in terms of the classic P(1) and P(2) receptor properties, suggesting that there may be a novel subtype of purinoceptors in the endothelium of the rat aorta. The P(1) and P(2) agonists-induced rat aorta smooth muscle contraction could be blocked by the pretreatments of the bioassay system with a G protein (Gi/o) inhibitor, PTX, and EDNO synthesis inhibitor, L-NAME and cyclooxygenase inhibitor, indomethacine. The data strongly support that this novel purinoceptor may couple with PTX-sensitive G protein, Gi or Go, and the novel purinoceptor-mediated rat aorta smooth muscle contraction is dependent on the endogenous nitric oxide and prostaglandin synthesis. In an additional observation, pathophysiological conditions, such as hypertension, altered the characteristics of the novel receptor. Hypertension caused the novel receptor insensitive to adensone and uncoupling to PTX-sensitive G-proteins and lost of ability to initiate the receptor-mediated prostaglandin synthesis. All together, the novel putative endothelial purinoceptor may play an important role in the control of vascular tone in physiological or pathophysiological statues.