Biomedical subjects

C Pothoulakis

Publications and source records attributed to C Pothoulakis.

At least 73 records · Page 4Linked to original sources

PubMed1994

Neuronal involvement in the intestinal effects of Clostridium difficile toxin A and Vibrio cholerae enterotoxin in rat ileum.

BACKGROUND/AIMS: Activation of intestinal mast cells and neurons is involved in intestinal inflammation and diarrhea. This study compared the effects of neuronal inhibitors and inhibition of intestinal sensory afferent nerves on the intestinal actions of Clostridium difficile toxin A, an inflammatory enterotoxin, and cholera toxin, a noninflammatory enterotoxin. METHODS: The effects of lidocaine, hexamethonium, atropine, and long-term pretreatment of capsaicin on fluid secretion, mannitol permeability, myeloperoxidase (MPO) activity, and release of rat mast cell protease II (RMCPII) were measured in toxin A- and cholera toxin-exposed loops in vivo. RESULTS: Lidocaine, hexamethonium, and capsaicin, but not atropine, inhibited toxin A-mediated secretion and MPO activity, but only capsaicin reduced mannitol permeability. Lidocaine, but not capsaicin, reduced secretion and permeability caused by cholera toxin. Toxin A caused release of RMCPII from rat ileum in vivo and in vitro; this was inhibited by lidocaine or capsaicin, whereas cholera toxin had no effect on release of RMCPII. CONCLUSIONS: Neuronal mechanisms are important in the in vivo effects of these two enterotoxins. Capsaicin-sensitive sensory afferent neurons and mast cells are involved in the intestinal mechanism of toxin A, but not cholera toxin.

Animals↗

PubMed1994

IL-8 secretion and neutrophil activation by HT-29 colonic epithelial cells.

This study examines the ability of HT-29 human colonic epithelial cells to stimulate neutrophil migration and adhesion. Interleukin-8 (IL-8), a potent neutrophil chemoattractant, was detected in conditioned media from both unstimulated (1.1 ng/ml) and IL-1 beta-stimulated (16.1 ng/ml) HT-29 cultures. Conditioned medium from IL-1 beta-exposed HT-29 cells stimulated neutrophil migration (395% of control, P < 0.01), and this effect was completely inhibited by anti-IL-8 antibody. HT-29 medium also induced shedding of neutrophil L-selectin and increased expression of neutrophil CD11/CD18 adhesion receptors. Coculture of HT-29 cells with human endothelial cell monolayers resulted in increased neutrophil transendothelial migration (169% of control, P < 0.01), which was blocked by both anti-IL-8 and anti-CD18 antibody. Northern hybridization analysis demonstrated increased levels of mRNA for IL-8 and intercellular adhesion molecule-1 (ICAM-1) in cytokine-treated HT-29 cells. Cytokine stimulation of HT-29 monolayers was also associated with increased neutrophil adhesion to these cells. Neutrophil-HT-29 cell adhesion was blocked by monoclonal antibodies to neutrophil CD18 or to ICAM-1 on the HT-29 cells (86% and 56% inhibition, respectively, P < 0.01 for both). These data suggest that IL-8 secretion by activated colonic epithelial cells may contribute to neutrophil extravasation and tissue infiltration in intestinal inflammation.

Blotting, Northern↗

PubMed1994

Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit.

Neutrophil infiltration is a prominent feature of Clostridium difficile-associated enteritis and colitis. The aim of this study was to examine the importance of neutrophil recruitment and neutrophil-mediated tissue damage in C. difficile toxin A-induced enteritis. Competitive binding experiments using purified 3H-toxin A demonstrated the presence of a single class of medium affinity receptors on rabbit neutrophils (Kd 7 x 10(-8) M). Pertussis toxin and the nonhydrolyzable GTP analog GTPgamma S both inhibited 3H-toxin A binding (by 56 and 65%, respectively), indicating that the rabbit neutrophil toxin A receptor is G protein linked. Toxin A elicited a dose-dependent (25-200 micrograms/ml) stimulation of neutrophil migration in vitro, and this functional effect was also pertussis toxin sensitive (69% inhibition). Treatment of neutrophils with R15.7, a blocking monoclonal antibody to the leuocyte adhesion molecule CD18, inhibited toxin A-stimulated neutrophil migration by 85% in vitro. Pretreatment of rabbits with R15.7 also prevented neutrophil infiltration of toxin A-exposed ileal loops in vivo as determined by histologic examination and by ileal tissue myeloperoxidase levels. Furthermore, R15.7 effected a substantial inhibition of fluid secretion (by 65%), mannitol permeability (by 66%), and histologic damage in toxin A-exposed ileal loops. Anti-CD18 (R15.7) had no inhibitory effect on cholera toxin enterotoxicity. These data demonstrate that C. difficile toxin A is a proinflammatory toxin whose enterotoxic effects are substantially dependent upon neutrophil recruitment.

Animals↗

PubMed1994

Clostridium difficile toxin A-induced microvascular dysfunction. Role of histamine.

Clostridium difficile toxin A (Tx-A) mediates secretion and inflammation in experimental enterocolitis. Intravital video microscopy was used to define the mechanisms that underlie the inflammatory reactions elicited by direct exposure of the microvasculature to Tx-A. Leukocyte adherence and emigration, leukocyte-platelet aggregation, and extravasation of FITC-albumin were monitored in rat mesenteric venules exposed to Tx-A. Significant increases in leukocyte adherence and emigration (LAE) and albumin leakage were noted within 15-30 min of Tx-A exposure. These responses were accompanied by mast cell degranulation and the formation of platelet-leukocyte aggregates. The Tx-A-induced increases in LAE and albumin leakage were significantly attenuated by pretreatment with either monoclonal antibodies (mAbs) directed against the leukocyte adhesion glycoproteins, CD11/CD18, intercellular adhesion molecule-1, and P-selectin (but not E-selectin) or with sialyl Lewis x, a counter-receptor for P-selectin. The mast cell stabilizer, lodoxamide, an H1- (but not an H2-) receptor antagonist, and diamine oxidase (histaminase) were also effective in reducing the LAE and albumin leakage elicited by Tx-A. The platelet-leukocyte aggregation response was blunted by an mAb against P-selectin, sialyl Lewis x, and the H1-receptor antagonist. These observations indicate that Tx-A induces a leukocyte-dependent leakage of albumin from postcapillary venules. Mast cell-derived histamine appears to mediate at least part of the leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation by engaging H1-receptors on endothelial cells and platelets to increase the expression of P-selectin. The adhesion glycoproteins CD11/CD18 and intercellular adhesion molecule-1 also contribute to the inflammatory responses elicited by toxin A.

Animals↗

PubMed1993

Effects of purified Clostridium difficile toxin A in the small intestine of the rat in vivo.

The action of highly purified Clostridium difficile toxin A was studied in the jejunum of rats in vivo. C. difficile toxin A reversed dose-dependently net fluid absorption into net fluid secretion, accompanied by an increase in prostaglandin E2 but not 5-hydroxytryptamine output into the gut lumen. Accordingly, indomethacin but not the 5-hydroxytryptamine receptor antagonists ketanserin plus tropisetron were able to inhibit toxin A-induced fluid secretion. Atropine and hexamethonium were without effect on the action of toxin A, such excluding a nervous mechanism. The cyclic nucleotides cyclic AMP and cyclic GMP appear not to be involved in the mediation of the secretory response. The reduced cyclic GMP levels are most likely the result of a complete destruction of the villus membranes, where the guanylate cyclase is located. Histological studies revealed massive damage to intestinal villi, whereas the majority of the crypts seem to be unaffected. In conclusion, toxin A-induced intestinal fluid secretion appears to be caused mainly by severe mucosal damage. PGE2-release may be the consequence of the inflammation accompanying this damage. The mechanism seems to be completely different to those of cholera toxin or Escherichia coli heat stable enterotoxin.

Animals↗

PubMed1993

Saccharomyces boulardii inhibits Clostridium difficile toxin A binding and enterotoxicity in rat ileum.

BACKGROUND: Saccharomyces boulardii is a nonpathogenic yeast used for the prevention and treatment of Clostridium difficile-associated diarrhea and colitis. However, the mechanism by which S. boulardii exerts its protective effects remains unclear. METHODS: The binding of [3H]toxin A to its brush border receptor preincubated with S. boulardii-cultured suspension or filtered conditioned medium was measured in vitro. The effect of toxin A on secretion, epithelial permeability, and morphology in rat ileal loops in vivo was also examined in rats pretreated with S. boulardii. RESULTS: S. boulardii reduced [3H]toxin A-receptor binding in a dose-dependent fashion. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of ileal brush border exposed to S. boulardii-conditioned medium revealed a diminution of all brush border proteins. Treatment of rats with S. boulardii suspension reduced fluid secretion and mannitol permeability caused by toxin A. CONCLUSIONS: S. boulardii may reduce some of the enterotoxic effects of toxin A by inhibiting toxin A-receptor binding. This effect appears to be manifested by a secreted product of the yeast, possibly a protease.

Animals↗

PubMed1993

Ketotifen inhibits Clostridium difficile toxin A-induced enteritis in rat ileum.

BACKGROUND: Clostridium difficile toxin A is the principal mediator of inflammatory enterocolitis in experimental animals. The purpose of this study was to explore the effect of ketotifen, an anti-inflammatory drug, on toxin A-induced enterotoxicity in rat ileum. METHODS: The effects of intragastric administration of ketotifen on secretion, mannitol permeability, histological damage, and mucosal levels of leukotriene B4, leukotriene C4, and platelet activating factor in toxin A-exposed rat ileal loops were measured in vivo. The effects of ketotifen on toxin A-mediated release of rat mast cell protease II (rat mucosa mast cell product) release were also measured in rat ileal explants in vitro. The effect of ketotifen on neutrophil migration in vitro was also evaluated. RESULTS: Ketotifen pretreatment inhibited toxin A-associated intestinal secretion by 42.5% and mannitol permeability by 56.3% and reduced epithelial cell inflammation and necrosis. These effects were associated with reduced levels of leukotriene B4 by 65.8%, leukotriene C4 by 88.8%, platelet activating factor by 77.8%, and inhibition of rat mast cell protease II by 58.4%. In addition, pretreatment of neutrophils with ketotifen inhibited neutrophil migration in vitro. CONCLUSIONS: The protective effect of ketotifen in this animal model was associated with significant inhibition of release of mast cells and neutrophil derived mediators, supporting their involvement in C. difficile enteritis.

Animals↗

PubMed1993

Clostridium difficile colitis and diarrhea.

Clostridium difficile is now regarded as the most prevalent nosocomial pathogen, infecting as many as a quarter of hospitalized patients. The pathophysiology of infection with this unusual enteric pathogen involves alteration of the normal enteric flora by antibiotics, ingestion of spores, and colonization by C. difficile. The organism then releases potent exotoxins that produce an inflammatory colitis and diarrhea. A spectrum of host responses occurs, ranging from the asymptomatic carrier state to life-threatening pseudomembranous colitis. Effective therapy with vancomycin or metronidazole is available, but relapses occur in 15% to 20% of patients and may necessitate multiple courses of therapy.

Clostridioides difficile↗

PubMed1992

Clostridium difficile toxin B disrupts the barrier function of T84 monolayers.

The contribution of toxin B to Clostridium difficile-associated infection is undefined. Toxin B induces dramatic phenotypic alterations (cytotoxic effects) in cultured mesenchymal and nonintestinal epithelial cells, yet its effects on intestinal epithelial cells are not clearly understood. The alterations induced by toxin B in nonintestinal cells appear to be secondary to toxin-induced redistribution of filamentous actin. It has not been determined whether toxin B exerts similar effects on cultured intestinal epithelial cells or whether such phenotypic alterations are of any physiological consequence. To address these questions, we examined the effect of C. difficile toxin B on the phenotype and barrier function of T84 cell monolayers. Our studies show that the cytotoxic effects of toxin B, i.e., cell rounding, do extend to cultured intestinal epithelial cells (T84). In addition, toxin B dramatically reduces the barrier function of T84 monolayers grown on collagen-coated filters. Toxin B-induced redistribution of filamentous actin appears to be responsible for the alterations in both intestinal epithelial cell phenotype and barrier function. Specifically, filamentous actin comprising the perijunctional actomyosin ring, known to be important in regulating tight junction permeability, is condensed into discrete plaques. Flux studies confirm that the permeability defect is at the level of the tight junction. We conclude that toxin-induced changes in actin distribution perturb intercellular junctional contacts and thereby ablate epithelial barrier function. There was no evidence of cell death as determined by lactate dehydrogenase release assays.

Actins↗

PubMed1992

Human colonic aspirates containing immunoglobulin A antibody to Clostridium difficile toxin A inhibit toxin A-receptor binding.

Clostridium difficile toxin A, a 308-kilodalton protein exotoxin, is the principal causative agent of antibiotic-associated, C. difficile-induced colitis. In the current study, the prevalence of specific human serum and secretory antibody to toxin A and the possible protective effect of secretory, intestinal anti-toxin A antibody are examined. Serum (n = 35), colonic aspirates (n = 35), and duodenal aspirates (n = 20) were collected from adults at diagnostic endoscopy. Patients with evidence of colitis or a history of recent antibiotic use were excluded from the study. Specific serum immunoglobulin (Ig) A and IgG antitoxin A antibodies were detected in 60% and 57% of subjects, respectively, by enzyme-linked immunosorbent assay. Fifty-seven percent of colonic aspirates contained IgA antitoxin, whereas only 10% of duodenal aspirates were positive (P = 0.002). Binding of toxin A to its intestinal receptor was studied using [3H]toxin A and purified rabbit ileal brush border membranes. Toxin A binding was significantly inhibited by colonic aspirates with high IgA anti-toxin A antibody levels (0.503 +/- 0.055 pmol toxin A bound per milligram of brush border membrane protein, mean +/- SE) in comparison with antitoxin A-negative aspirates (0.778 +/- 0.089 pmol; P = 0.02) and control (0.766 +/- 0.004 pmol; P = 0.03). In the current study, a specific intestinal secretory IgA antibody response to C. difficile toxin A in humans is reported. This antibody response is more evident in the colon, the site of C. difficile infection, than in the upper intestinal tract. Our data suggest that human colonic IgA antitoxin may protect against C. difficile colitis by inhibiting the binding of toxin A to its intestinal epithelial cell receptor.

Adult↗

PubMed1992

Diminished Clostridium difficile toxin A sensitivity in newborn rabbit ileum is associated with decreased toxin A receptor.

Human infants are relatively resistant to Clostridium difficile-associated diarrhea and colitis compared to adults. In that toxin A is the major cause of intestinal damage with this organism, we compared toxin A receptor binding and biological effects in newborn vs adult rabbit ileum. Purified toxin A (M(r) 308 kD) was labeled with tritium or biotin with full retention of biologic activity. Appearance of specific toxin A brush border (BB) binding was strongly age dependent with minimal [3H]toxin A specific binding at 2 and 5 d of life, followed by gradual increase in binding to reach adult levels at 90 d. Absence of toxin A binding sites in newborn and presence in adult rabbits was confirmed by immunohistochemical studies using biotinylated toxin A. Toxin A (50 ng to 20 micrograms/ml) inhibited protein synthesis in 90-d-old rabbit ileal loops in a dose-dependent fashion. In contrast, inhibition of protein synthesis in 5-d-old rabbit ileum occurred only at the highest toxin A doses (5 and 20 micrograms/ml) and at all doses tested was significantly less than the adult rabbit ileum. In addition, toxin A (5 micrograms/ml) caused severe mucosal damage in adult rabbit ileal explants but had no discernable morphologic effect on 5-d-old rabbit intestine. Our data indicate that newborn rabbit intestine lacks BB receptors for toxin A. The absence of the high-affinity BB receptor for toxin A in the newborn period may explain lack of biologic responsiveness to purified toxin, and the absence of disease in human infants infected with this pathogen.

Age Factors↗

PubMed1991

Treatment with intravenously administered gamma globulin of chronic relapsing colitis induced by Clostridium difficile toxin.

We tested the hypothesis that children with chronic relapsing colitis induced by Clostridium difficile toxin have defective antibody responses to C. difficile toxins as a cause of their underlying illness. Six such children were tested for serum IgG and IgA antibody to C. difficile toxin A. These six children had lower IgG anti-toxin A levels than 24 healthy children (p = 0.026) and 18 healthy adults (p = 0.0008). Five patients treated with 400 mg intravenously administered gamma-globulin per kilogram every 3 weeks had significant increases in IgG (p = 0.01) but not IgA anti-toxin A (p = 0.406) levels, and all five had clinical resolution of their gastrointestinal symptoms as well as clearing of C. difficile cytotoxin B from their stools. These observations suggest that a deficiency of IgG anti-toxin A may predispose children to the development of chronic relapsing C. difficile-induced colitis. In such cases, intravenous gamma-globulin therapy may be effective in producing clinical remission.

Antibodies, Bacterial↗

PubMed1991

Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.

The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.

Animals↗

PubMed1991

The chemotactic response of human granulocytes to Clostridium difficile toxin A is age dependent.

Elderly patients are at high risk for developing diarrhea and colitis as a complication of antimicrobial therapy. Clostridium difficile, the causative agent of antibiotic-associated diarrhea and colitis produces an enterotoxin (toxin A) and a cytotoxin (toxin B). Of these two exotoxins, toxin A appears to be largely responsible for the inflammatory phenomena of C. difficile colitis, because it produces secretion, pronounced granulocytic infiltration, and epithelial cell necrosis and ulceration in ligated ileal loops of experimental animals. We have recently demonstrated that the inflammatory effects of C. difficile toxin A in the intestine may be related to its ability to mobilize intracellular calcium and elicit a chemotactic response by human granulocytes. In this study, in order to explain why the elderly are at greater risk for developing antibiotic-associated colitis, we investigated the effects of toxin A on activation of granulocytes from healthy elderly and young subjects. Highly purified toxin A and the chemotactic factor N-formyl-Met-Leu-Phe (FMLP) at concentrations of 10(-7) M both elicited a significant (p less than 0.001) and comparable chemotactic and chemokinetic response in human granulocytes from both age groups. A significantly (p less than 0.001) increased chemotactic effect in elderly subjects compared with young subjects was elicited by toxin A and not by FMLP. These findings suggest that the enhanced intestinal inflammatory effects of C. difficile in the elderly, compared with the young, may be related to the ability of its enterotoxin to elicit a more pronounced chemotactic response by granulocytes.

Adult↗

PubMed1990

Macrophage-dependent stimulation of T cell-depleted spleen cells by Clostridium difficile toxin A and calcium ionophore.

Clostridium difficile toxin A causes severe intestinal inflammation and fluid secretion in rabbit ileum and is chemotactic for neutrophils in vitro. The mechanism of intestinal injury produced by toxin A appears to involve direct epithelial cell damage as well as recruitment of an inflammatory cell response. The current study was undertaken to determine if toxin A can directly stimulate a proliferative response in lymphocytes. Highly purified toxin A, in the presence of the calcium ionophore, ionomycin, stimulated substantial [3H]thymidine incorporation by murine splenic lymphocytes, which was maximal at 10(-9) M toxin A and 800 ng/ml ionomycin. Removal of T cells with anti-Thy-1.2 antibody plus complement had no effect on the proliferative response induced by toxin A. However, [3H]thymidine incorporation in response to toxin A was significantly inhibited (P less than 0.001) by the removal of macrophages from splenocyte suspensions and was restored by the addition of peritoneal macrophages or cell-free supernatant from toxin A-treated macrophage cultures. Analysis of the toxin A-treated macrophage supernatants showed high levels of IL-1, but not IL-2 or IL-4. The combination of recombinant IL-1 plus ionomycin was found to stimulate [3H]thymidine incorporation by T cell-depleted splenic lymphocytes. These results suggest that toxin A stimulates the release of IL-1, and possibly other factors, from macrophages which can costimulate murine B lymphocytes.

Animals↗

PubMed1990

C. difficile toxin A increases intestinal permeability and induces Cl- secretion.

Mucosal sheets of guinea pig ileum mounted in Ussing chambers were used to determine effects of highly purified Clostridium difficile toxin A on intestinal structure and barrier function in the absence of recruited neutrophils and blood flow. With the use of standard electrophysiological and morphological techniques, our results indicate that 5 micrograms/ml mucosal toxin A induces substantial alteration in epithelial permeability and in structure of absorptive cells. Transepithelial fluxes of mannitol (3.6 A) and inulin (11.5 A) increased 20-80 min after toxin A exposure (63 +/- 13 vs. 149 +/- 14 and 2.33 +/- 0.43 vs. 7.03 +/- 1.0 nmol.cm-2.h-1 for mannitol and inulin; controls vs. toxin exposed, respectively, both P less than 0.01). Toxin A exposure diminished short-circuit current (153 +/- 8 vs. 77 +/- 6 microA/cm2 for control and toxin exposed, respectively, P less than 0.001), decreased transepithelial electrical resistance (61.6 +/- 3 vs. 50.1 +/- 3.9 omega.cm2 for control and toxin exposed, respectively, P less than 0.05), increased passive permeation of Na+ (19.1 +/- 0.9 vs. 27.4 +/- 0.9 mumol.cm-2.h-1 for control and toxin exposed, respectively), and induced a Cl- secretory response (net flux 3.65 +/- 3.0 vs. -4.62 +/- 2.6 mumol.cm-2.h-1, for control and toxin exposed, respectively, P less than 0.05) over the 2-h experimental time course. Toxin A-induced structural alterations of villus tip absorptive cells were strikingly similar to those induced by the actin-binding agent cytochalasin D. Specifically, cells displayed constricted subjunctional zones, flared microvillus brush borders, condensation of microfilaments in the zone of the perijunctional actomyosin ring, and breakdown of intercellular tight junctions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Comparative study of Clostridium difficile toxin A and cholera toxin in rabbit ileum.

The purpose of this study was to compare the effects of Clostridium difficile toxin A and cholera toxin on fluid secretion, intestinal permeability, and arachidonate metabolites in rabbit ileum. Injection of 25 micrograms of either purified toxin into 10-cm ileal loops caused significant increases in fluid secretion and intestinal permeability to mannitol as well as release of prostaglandin E2 into the lumen. Toxin A, but not cholera toxin, caused a severe inflammatory reaction of the lamina propria and necrosis of enterocytes as well as increased release of leukotriene B4. The toxin A-mediated increases in prostaglandin E2 and leukotriene B4 could be blocked by prior instillation of 10 mg of 5-aminosalicylic acid into ileal loops. 5-Aminosalicylic acid also significantly diminished the expected increase in mannitol permeability after both toxins, but had no significant inhibitory effect on fluid secretion or, in the case of toxin A, intestinal inflammation. Our results indicate that C. difficile and cholera enterotoxins differ substantially in their effects on the rabbit intestine. Clostridium difficile toxin A, an inflammatory toxin, produces a striking infiltration of the lamina propria with neutrophils that is associated with increased release of leukotriene B4. In contrast, cholera toxin does not cause inflammation or leukotriene B4 release. Increased release of prostaglandin E2 occurs after exposure to both toxins and appears to be correlated with increased intestinal permeability.

Animals↗

PubMed1989

Effect of purified Clostridium difficile toxins on intestinal smooth muscle. I. Toxin A.

In these studies we determined the effects of purified Clostridium difficile toxin A, an enterotoxin, on the electrophysiological and contractile properties of rabbit intestinal circular smooth muscle and correlated these effects with changes of smooth muscle morphology. Simultaneous measurements of intracellular membrane potential and contractility were determined in excised ileal muscle strips after administration of toxin A in vivo (60 micrograms/ml) into an isolated rabbit ileal loop or directly in vitro (0.1-60 micrograms/ml) to a normal muscle strip. Toxin A injection in vivo resulted in membrane depolarization and increased slow wave and action potential frequency. Toxin A injection in vivo also caused increased amplitude of spontaneous and carbachol-induced phasic contractions. The electrophysiological effects of in vivo administration of toxin A were correlated with an inflammatory infiltrate of the lamina propria, but no light or electron microscopic evidence of injury to smooth muscle cells was seen. In contrast to the in vivo studies, direct in vitro exposure of normal ileal muscle strips to toxin A had no effect on either spontaneous or carbachol-induced electromechanical activity. Our results indicate that in vivo administration of C. difficile toxin A into a rabbit ileal loop, but not direct in vitro exposure, causes significant alterations of smooth muscle excitation-contraction coupling that may be mediated by products of local inflammatory cells.

Action Potentials↗