Effect of ethanol on cholecystokinin-induced enzyme secretion from isolated rat pancreatic acini

Cholecystokinin octapeptide (CCK8)-stimulated amylase release in isolated rat pancreatic acini was inhibited over 30% by 600 mM ethanol. The configuration of the dose-response curve for CCK8, however, in the presence of ethanol was similar to that of the control. Amylase release elicited by maximal concentrations of CCK8 (300 pM) was inhibited by increasing concentrations of ethanol (0.3 to 1.3 M), and this inhibition was concentration dependent. In addition, the binding of [125I]CCK33 to specific membrane receptors on acini was inhibited by ethanol in a dose-dependent manner. A positive correlation between the inhibitory effects of ethanol on CCK binding and CCK-induced amylase release was observed. Furthermore, these inhibitory effects of ethanol were reversible. Basal amylase release, however, was increased 20-50% by ethanol between the concentrations of 0.3 and 1.3 M; higher concentrations caused a leakage of amylase from the acini both in the absence and presence of 300 pM CCK8. This is confirmed by 51Cr release from prelabeled acini which revealed no significant damage to acinar cell membrane between 0.3 and 1.6 M ethanol, but significant damage to acini at higher concentrations. These data suggest that the 600 mM ethanol-induced inhibition of CCK action in acini is due to reversible perturbation of the acinar cell membrane.     

Irreversible inhibition by acetaldehyde of cholecystokinin-induced amylase secretion from isolated rat pancreatic acini

Acetaldehyde inhibited both amylase secretion induced by maximal concentrations (300 pM) of cholecystokinin octapeptide and the binding of radioiodinated cholecystokinin to receptors on isolated rat pancreatic acini. This inhibition was concentration dependent (10 mM to 1 M for amylase secretion and 100 mM to 1 M for binding). However, a correlation between the two inhibitory effects could not be obtained. Furthermore, the inhibitory effects were not reversible. Acetaldehyde did not alter the basal amylase secretion between 6 and 45 mM concentrations. However, 60, 100 and 300 mM acetaldehyde significantly decreased basal amylase secretion; no significant change in amylase secretion was observed at 600 mM and 1 M. Higher concentrations of acetaldehyde produced a 2- to 10-fold increase in basal amylase secretion. 51Cr release from prelabeled acini revealed no significant cell membrane damage between 10 and 600 mM acetaldehyde. These data suggest that acetaldehyde inhibition of cholecystokinin-induced amylase secretion is intracellularly mediated.     

Absence of intestinal secretion on supernatants from macrophages stimulated with Clostridium difficile toxin B on rabbit ileum.

Several studies have documented the involvement of both Clostridium difficile, toxins, A and B in the pathogenesis of antibiotic-associated diarrhea. Recently, we demonstrated that IL-1 beta is the intestinal secretory factor released by macrophages stimulated with toxin A. The aim of this study was to evaluate the importance of macrophages stimulated with toxin B on rabbit ileal ion transport. The changes in ion transport were analyzed by studying the short-circuit current of the rabbit ileal mucosa mounted in Ussing chambers. The supernatants of macrophages treated with toxin B (3.6 x 10(-7) M) had no effect on the ion transport (change in short-circuit current =28.0+/-9.2 vs. control=26.8+/-3.6 microA cm(-2)). Supernatants of macrophages stimulated with toxin A (3.2 x 10(-7) M), our positive control, induced a significant change in ileal ion transport (delta I(sc)=55.2+/-5.7 mA cm(-2)). It was also observed that, like toxin A, toxin B stimulated macrophages to produce TNF-alpha (555.0+/-37.9 pg/ml vs. control=182.0+/-39.8 pg/ml; p<0.05). Nevertheless, in contrast to toxin A, toxin B did not stimulate IL-1 beta synthesis (28.0+/-7.5 pg/ml vs. control=40. 0+/-14.4 pg/ml; p>0.05). We conclude that the supernatants of macrophages stimulated with toxin B are not able to stimulate ion transport and that both toxins stimulate the genesis of TNF-alpha, but only toxin A induces the synthesis of IL-1 beta, which, we have earlier reported, causes an electrogenic intestinal response in rabbit ileum.     

艰难梭菌A毒素的纯化

目的建立一种纯化艰难梭菌A毒素的方法。方法将艰难梭菌VPI 10 4 6 3菌株经透析培养 ,5 0 %饱和硫酸胺盐析 ,酸沉淀 ,再经DEAE Toyopearl 6 5 0M离子交换色谱。结果精制的艰难梭菌A毒素Native PAGE及对流免疫电泳均为单一带 ,相对分子质量为 5 5 0 0 0 0 ,小鼠毒力为 1× 10 6MLD/ml,Vero细胞毒性为 1× 10 7CU/ml。凝血活性为 1∶5 12 ,肠袢试验为阳性。结论该法简便、实用、可行。     

Comparative study of immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L.

We compared the immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L. These two cytotoxins are immunologically related in that the cytotoxic effect of either toxin can be neutralized by the polyclonal antiserum prepared against either cytotoxin. On the other hand, polyclonal antiserum prepared against Clostridium difficile enterotoxin A did not cross-react with the cytotoxins B and L when tested by cytotoxic neutralization test nor by double immunodiffusion assay. However, despite this immunological relationship between toxins B and L, the morphological modifications observed in MacCoy cells induced by treatment with these cytotoxins are clearly distinct. We describe the first quantitative analysis of specific cellular parameters which illustrates the morphological differences induced by these cytotoxins. Moreover, immunocytochemical experiments show that, whereas disruption of microfilaments is observed with toxin B- and L-treatments, alterations of F-actin network are different in the cells treated with toxin B or L. The observation that the cellular modifications induced by toxin B- and toxin L-treatment differ suggests that the molecular mechanisms involved in the respective cytotoxicities are also likely to be different.     

Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network. Received: 28 October 1997 / Accepted: 19 January 1998     

Clostridium difficile toxin in chronic idiopathic colitis

Clostridium difficile toxin was isolated from the stools of three patients with chronic idiopathic colitis. Two patients were known to have chronic idiopathic colitis before Cl difficile toxin was isolated. The third patient was subsequently found to have ulcerative colitis after presentation with Cl difficile toxin in the stool. Two patients were on sulphasalazine at the time of diagnosis of Cl difficile infection and one had taken sulphasalazine two months previously. Only one patients had antibiotic exposure and that was at least three months before presentation. In each patient, treatment with vancomycin was accompanied by symptomatic improvement and disappearance of the toxin. The underlying colitis remained unaffected. In patients with inflammatory bowel disease in relapse, the presence of Cl difficile toxin should be sought as this may be a factor in the relapse. In any patient presenting with diarrhoea, the presence of Cl difficile toxin may obscure the presence of underlying inflammatory bowel disease.     

Immunochemical and structural similarities in toxin A and toxin B of Clostridium difficile shown by binding to monoclonal antibodies

Clostridium difficile toxins A and B were shown to share immunochemical and structural features, including shared sequential epitopes. Nineteen hybridomas generated after immunization of mice with a mixture of toxoids produced monoclonal antibodies, all IgM(x), which bound to toxin A and toxin B in a solid-phase radioimmunoassay (RIA). None of the antibodies neutralized the cytotoxicity of either toxin, alone or in pairs, nor did they neutralize mouse lethality. The antibodies did not inhibit hemagglutination by toxin A, and none of those tested neutralized the toxin's enterotoxic activity. Studies of binding of antibodies to native toxins in the RIA showed that the antibodies differed in their recognition of the toxins. Many of the antibodies bound with higher avidity to toxin A than to toxin B. In Western blots, all the antibodies recognized both toxins in the native state; in addition, some antibodies recognized the minor cytotoxic species of toxin B. When the toxins were denatured and reduced, five antibodies bound to both toxins, five to A only, and nine to neither, demonstrating that the antibodies had different epitope specificities. Further structural comparisons were made by investigation of mol. wts, subunit structures and amino acid compositions. The native mol. wts of toxin A and toxin B, as determined by electrophoresis to equilibrium in 4-30% polyacrylamide gel electrophoresis (PAGE), were 430,000 and 368,000, respectively. Denatured and reduced toxins each had a single subunit of 315,000. Both toxins had about 50% hydrophobic amino acids.     

Difference in F-Actin Depolymerization Induced by Toxin B from the Clostridium difficile Strain VPI 10463 and Toxin B from the Variant Clostridium difficile Serotype F Strain 1470

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the causative agent of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis (PMC). TcdB from the C. difficile strain VPI10463 mono-glucosylates (thereby inactivates) the small GTPases Rho, Rac, and Cdc42, while Toxin B from the variant C. difficile strain serotype F 1470 (TcdBF) specifically mono-glucosylates Rac but not Rho(A/B/C). TcdBF is related to lethal toxin from C. sordellii (TcsL) that glucosylates Rac1 but not Rho(A/B/C). In this study, the effects of Rho-inactivating toxins on the concentrations of cellular F-actin were investigated using the rhodamine-phalloidin-based F-actin ELISA. TcdB induces F-actin depolymerization comparable to the RhoA-inactivating exoenzyme C3 from C. limosum (C3-lim). In contrast, the Rac-glucosylating toxins TcdBF and TcsL did not cause F-actin depolymerization. These observations led to the conclusion that F-actin depolymerization depends on the toxin’s capability of glucosylating RhoA. Furthermore, the integrity of focal adhesions (FAs) was analyzed using paxillin and p21-activated kinase (PAK) as FA marker proteins. Paxillin dephosphorylation was observed upon treatment of cells with TcdB, TcdBF, or C3-lim. In conclusion, the Rho-inactivating toxins induce loss of cell shape by either F-actin depolymerization (upon RhoA inactivation) or the disassembly of FAs (upon Rac1 inactivation).     

Clostridium difficile toxin A and its effects on cells

Clostridium difficile toxin A in its native form is a high molecular weight (520-540 K) aggregate with five major biological activities. It is lethal, enterotoxic, cytotoxic and cytotonic, and induces hemagglutination of rabbit red blood cells. Possibly these activities are contained in separate components. A major subunit of c. 230-310 K has been defined but lower molecular weight components cannot be excluded. The major component has been cloned, and sequence analysis indicated a complicated pattern of repeating sequences in the C-terminal third of the molecule. This review deals mainly with the effects of toxin A on cultured cells. Most mammalian cells are sensitive to toxin A whose major effect is to stop cell division irreversibly. The toxin binds via its repeat sequences to a trisaccharide receptor expressed on rabbit red cells and on brush border membranes from hamster intestine. This receptor seems to be functional in the hemagglutination reaction and the enterotoxicity. Its role in the cytotoxic effect of the toxin is not clear, but no other receptor structure has as yet been identified. In order to exert its cytotoxic (antiproliferative) effect toxin A must first be internalized by endocytosis. Thus a latency period of at least 30 min after toxin binding to cells is consistently observed, and all cytotoxic effects can be prevented by blocking the endocytosis pathway. The first microscopically visible signs of cytotoxicity consist in retraction and rounding of intoxicated cells. In addition the nucleus becomes polarized to one side of the cell while other cell organelles are not significantly affected. These morphological changes seem to be the consequence of a cytoskeletal rearrangement, mainly involving some components of the microfilament system. Inhibition of macromolecular syntheses as well as permeabilization of the plasma membrane may follow the early cytoskeletal effects and finally lead to cell death. Attempts to identify metabolic pathways of significance in the cytotoxicity suggest that the cytosolic level of Ca2+ is not important, thus excluding certain mechanisms for cell killing. In this respect the cytotoxic mode of action of toxin A clearly differs from that of toxin B. However, the biochemical basis for the antiproliferative effect of toxin A remains unknown.     

Mechanism of action of nicotine on amylase release by isolated pancreatic acini

The effects of nicotine on the pH of acinar suspension, amylase release and on amylase response stimulated by carbachol were examined in isolated rat pancreatic acini. Additions of nicotine at concentrations ranging from 10 microM to 30 mM caused dose-dependent increases in pH of acinar suspension with simultaneous amylase release (p less than 0.05). There was no increase in amylase release when acinar cells were incubated with nicotine adjusted to pH 7.40. Carbachol alone released amylase whereas nicotine (pH 7.40) at a concentration of 10 mM caused a significant and nonparallel inhibition of amylase release in response to graded doses of carbachol. At concentrations ranges between 3 microM and 10 mM, nicotine at pH 7.40 inhibited amylase release stimulated by 1 microM carbachol, with a half maximal inhibition at 0.8 +/- 0.2 mM. These results indicate that in isolated rat pancreatic acini nicotine at pH 7.40 has no effect on basal nonstimulated amylase release but it inhibits carbachol-stimulated amylase response in a noncompetitive manner. These observations may have direct implications in underlying mechanism of pancreatic disorders.     

Enhancement of cell-mediated cytotoxicity by Clostridium difficile toxin A: an in vitro study

Cells from the immune system exhibiting cytotoxic activity are able to kill tumor or infected cells in a major histocompatibility complex-restricted (cytotoxic lymphocytes) or non-restricted (natural killer cells) manner. In order to exert such a cytotoxicity they have to bind the target cell and release cytotoxic factors able to induce target cell death. Treatment of human peripheral blood mononuclear cells with toxin A from Clostridium difficile induced an enhancement of the cytotoxic efficiency of these effector cells. Morphological analysis of effector/target cell pairs seems to suggest that this could be related to an increased ability of cytotoxic effectors to establish close and intertwined contacts with target cells. These contacts involve adhesion molecules and lead to the formation of a "closed chamber" which probably improves the efficacy of lytic factors and results in an increased cytotoxicity.     

Acetaldehyde inhibition of protein synthesis in isolated rat pancreatic acini

Exposure of isolated dispersed pancreatic acini to increasing concentrations of ethanol (5 to 500 mM) or acetaldehyde (0.5 to 100 mM) produced a progressive inhibition of [3H]leucine incorporation into both "cellular" (those remaining in the cell) and "secretory" (those released into the medium) proteins. Whereas 500 mM ethanol caused 90-95% inhibition in the synthesis of "cellular" and "secretory" proteins, the concentration of acetaldehyde needed to produce a similar inhibition was found to be 50 mM. All subsequent experiments were performed with 12.5 mM acetaldehyde, a concentration that consistently inhibited acinar protein synthesis by about 50%. The acetaldehyde-mediated inhibition of acinar protein synthesis was partially normalized when this metabolite was removed after 30 min during a 90-min incubation period. In the presence of acetaldehyde, the secretion of 3H-pulse-labeled proteins, but not amylase, trypsinogen, or chymotrypsinogen, was greatly depressed. Acetaldehyde also caused a marked reduction in [3H]uridine incorporation into acinar RNA. The entry of [3H]uridine, [3H]leucine, and [3H]aminoisobutyric acid into isolated acini was found to be slightly (15-25%) decreased by acetaldehyde. It is concluded that acetaldehyde exerts a direct toxic effect on isolated dispersed pancreatic acini as evidenced by diminution of both protein and RNA synthesis and decreased secretion of the newly synthesized proteins. This inhibitory effect of acetaldehyde could be partially reversed.     

Preformulation studies of Clostridium difficile toxoids A and B

To enhance the physical stability of Clostridium difficile toxoids A and B, screening for stabilizing compounds was performed. The screening of 30 GRAS compounds at various concentrations and in several combinations was performed in two parts. First, a high-throughput aggregation assay was used to screen for compounds which delayed or prevented aggregation of toxoids under stress conditions (toxoids at pH 5-5.5 were incubated at 55 degrees C for 55 or 75 min). Compounds which stabilized both proteins were further studied for their ability to delay unfolding under conditions leading to a presumably native-like folded state (pH 6.5). The thermal stability of the toxoids on the surface of Alhydrogel was monitored with DSC and also showed significant improvement in the presence of certain excipients. This study has generated information concerning the free and adjuvant bound toxoids behavior under a range of conditions (temperature, solutes) that can be used to design pharmaceutical formulations of enhanced physical stability.     

A role for Rho in receptor- and G protein-stimulated phospholipase C Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B

Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂)-hydrolyzing phospholipase C (PLC) enzymes by activated or free subunits of the relevant G proteins. To study whether low molecular weight G proteins of the Rho family are involved in receptor signalling to PLC, we examined the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) subtype. Toxin B treatment of HEK cells did not affect basal PLC activity, but potently and efficiently inhibited mAChR-stimulated inositol phosphate formation. PLC activation by the endogenously expressed thrombin receptor and by the direct G protein activators, AlF _inf4 ^sup– and guanosine 5-[-thio]triphosphate (GTPS), studied in intact and permeabilized cells, respectively, were also inhibited by toxin B treatment. C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTPS-stimulated PLC activity. Finally, both toxin B and C3 exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P₂ in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate. Accordingly, when PLC activity was measured with exogenous PtdIns(4,5)P₂ as enzyme substrate, Ca²⁺- as well as GTPS- or A1F _inf4 ^sup– -stimulated PLC activities were not altered by prior toxin B treatment. In conclusion, evidence is provided that toxin B and C3 exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled receptor signalling to PLC, most likely by reducing the cellular substrate supply.     

Release of digestive enzymes from isolated rat pancreatic acini following muscarinic stimulation: a comparative study with enzyme release and receptor binding.

Effect of cholinergic stimulation on the release of digestive enzymes from isolated rat pancreatic acini prepared by collagenase digestion was investigated. The release of enzymes (amylase, chymotrypsinogen, lipase) increased linearly with the time of incubation in the absence of secretagogues. Carbachol, a muscarinic agonist, induced a remarkable increase in the release of these enzymes. This carbachol-stimulated amylase release showed a biphasic curve, and its maximal response was observed at 10(-5) M. The release patterns of chymotrypsinogen and lipase were similar to that of amylase. This carbachol-stimulated amylase release was inhibited by atropine in a dose-dependent manner, with an ED50 value of 1.17 X 10(-8) M. Other cholinergic agonists such as methacholine also stimulated the release of these enzymes, and these increases in the release were inhibited by atropine. Scatchard analysis for [3H]quinuclidinyl benzilate ([3H]QNB) binding to isolated pancreatic acini revealed that the binding site of [3H]QNB was a single component with a Kd value of 0.09 nM and a Bmax value of 89.3 fmol/mg protein, respectively. The effect of other cholinergic antagonists, pirenzepine and AF-DX 116 (11-[[2-[(diethylamino) methyl]1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one), on pancreatic acini was also investigated. Based on these results, it has been concluded that isolated rat pancreatic acini have muscarinic receptors and are useful for analyzing the mechanism of pancreatic enzyme secretion.     

The effect of ethanol on enzyme synthesis and secretion in isolated rat pancreatic lobules

This study investigates the effect of ethanol on enzyme synthesis and secretion in rat pancreatic lobules. Ethanol caused a dose-dependent inhibition of 3H-leucine incorporation into total protein. Examination of the time dependence showed that ethanol inhibited protein synthesis at each time point. Removal of ethanol partially reversed this inhibition. An autoradiograph of the newly synthesized proteins separated on SDS-PAGE showed that ethanol inhibited synthesis of all proteins. 14C-cycloleucine uptake was not altered by ethanol, excluding inhibition of amino acid uptake as the mechanism for the decreased protein synthesis induced by ethanol. Electron microscopy revealed no ultrastructural damage. Ethanol had no effect on the stimulated release of (i) amylase from zymogen granules nor (ii) newly synthesized pulse labelled enzymes. Acetaldehyde had no inhibitory effect on enzyme synthesis or secretion indicating that ethanol per se and not its metabolite is inhibitory. The decreased synthesis after acute exposure to ethanol with preservation of exocytosis would limit the autodigestive potential of pancreatic tissue. This may explain why isolated toxic doses of ethanol are rarely if ever associated with pancreatitis.     

Selective activation by photodynamic action of cholecystokinin receptor in the freshly isolated rat pancreatic acini

1 Sulphonated aluminium phthalocyanine (SALPC) photodynamic action induces amylase secretion and permanent calcium oscillation in rat pancreatic acinar cells, because of the activation of phospholipase C or signalling proteins upstream. The aim of the present study was to investigate the involvement of muscarinic acetylcholine and cholecystokinin (CCK) receptors. 2 Muscarinic receptor antagonist atropine (10 micro M) blocked amylase secretion induced by bethanechol (100 micro M), and CCK(1) receptor antagonist (S)-N-[1-(2-fluorophenyl)-3,4,6,7-tetrahydor-4-oxo-pyrrolo-[3,2,1-jk][1,4] benzodiazepine-3yl]-1H-indole-2-carboxamide (FK480) (1 micro M) blocked amylase secretion induced by CCK (100 pM). 3 Amylase secretion induced by SALPC photodynamic action was not inhibited when atropine and FK480 were present during photodynamic action. However, addition of FK480 1 micro M after initiation of photodynamic action inhibited photodynamic amylase secretion. Bethanechol (10, 100 micro M) added after photodynamic action resulted in a full secretory response. 4 Atropine (10 nM) abolished calcium oscillation induced by bethanechol (5 micro M), and FK480 (10 nM) blocked calcium oscillation induced by CCK (10 pM). 5 Atropine up to 10 micro M was without effect on Ca(2+) oscillation triggered by photodynamic action, but these oscillations were abolished by FK480 (10 nM). FK480 (10 nM) had no effect on calcium oscillations induced by bethanechol (5 micro M). Bethanechol 5 micro M, added after FK480 blockade of photodynamic calcium oscillation, still triggered regular calcium oscillation. 6 It is concluded that SALPC photodynamic action selectively and permanently activates CCK receptor in rat pancreatic acini. Such permanent and selective modulation of signalling proteins has important implications for the treatment of pancreatitis, prion diseases, and neurodegenerative disorders.     

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

Clostridium perfringens alpha toxin caused contraction of the isolated rat ileum in a dose-dependent manner. The contraction caused by the toxin was inhibited by verapamil, nifedipine, cinnarizine, but not by tetrodotoxin or incubation in a low Na+ medium. The toxin stimulated Ca2+ uptake in the ileum in a dose-dependent manner. Nifedipine completely inhibited the Ca2+ uptake into the ileum induced by the toxin. Furthermore, TMB-8, trifluoperazine, W-7 and H-7 significantly blocked the toxin-induced contraction. On the other hand, the toxin activated de novo production of phosphatidic acid and phosphatidylinositol in the tissue. These data suggest that the toxin-induced contraction is due to an increased Ca2+ permeability across the tissue, and activation of phospholipid metabolism.