The Influence of Low Doses of Zearalenone and T-2 Toxin on Calcitonin Gene Related Peptide-Like Immunoreactive (CGRP-LI) Neurons in the ENS of the Porcine Descending Colon.

The enteric nervous system (ENS) can undergo adaptive and reparative changes in response to physiological and pathological stimuli. These manifest primarily as alterations in the levels of active substances expressed by the enteric neuron. While it is known that mycotoxins can affect the function of the central and peripheral nervous systems, knowledge about their influence on the ENS is limited. Therefore, the aim of the present study was to investigate the influence of low doses of zearalenone (ZEN) and T-2 toxin on calcitonin gene related peptide-like immunoreactive (CGRP-LI) neurons in the ENS of the porcine descending colon using a double immunofluorescence technique. Both mycotoxins led to an increase in the percentage of CGRP-LI neurons in all types of enteric plexuses and changed the degree of co-localization of CGRP with other neuronal active substances, such as substance P, galanin, nitric oxide synthase, and cocaine- and amphetamine-regulated transcript peptide. The obtained results demonstrate that even low doses of ZEN and T-2 can affect living organisms and cause changes in the neurochemical profile of enteric neurons.

Curcumin Inhibits 5-Fluorouracil-induced Up-regulation of CXCL1 and CXCL2 of the Colon Associated with Attenuation of Diarrhoea Development.

The compound 5-fluorouracil (5-FU) is used in cancer chemotherapy and is known to cause diarrhoea. We recently reported that chemokine (C-X-C motif) ligand 1 (CXCL1) and neutrophils in the colonic mucosa were markedly increased by the administration of 5-FU in mice. Curcumin has anti-inflammatory, antitumour and antioxidant properties. Therefore, we examined the effect of curcumin on 5-FU-induced diarrhoea development and CXCL1 and CXCL2 up-regulation in the colon. Mice were given 5-FU (50 mg/kg, i.p.) daily for 4 days. Curcumin (100 or 300 mg/kg, p.o.) was administered on the day before the first administration of 5-FU and administered 30 min. before the administration of 5-FU. Gene expression levels of CXCL1 and CXCL2 in the colon were examined by real-time RT-PCR. Curcumin reduced the 5-FU-induced diarrhoea development. Under this condition, the CXCL1 and CXCL2 gene up-regulated by 5-FU administration was inhibited by curcumin. The gene expression of CXCL1 and CXCL2 was also enhanced by 5-FU application in vitro. The 5-FU-induced up-regulated CXCL1 and CXCL2 gene expressions were inhibited by curcumin, Bay-117082 and bortezomib, nuclear factor kappa B (NF-κB) inhibitors, C646, a p300/cyclic adenosine monophosphate response element-binding protein-histone acetyltransferase (HAT) inhibitor. In conclusion, these findings suggested that curcumin prevented the development of diarrhoea by inhibiting NF-κB and HAT activation.

Short-Term Effects of Relamorelin on Descending Colon Motility in Chronic Constipation: A Randomized, Controlled Trial.

BACKGROUND: The pentapeptide ghrelin agonist, relamorelin, accelerates colonic transit in patients with chronic constipation (CC). In a murine model, relamorelin decreased excitability of colonic circular smooth muscle cells and colonic intraluminal pressure. AIM: To determine short-term effects of relamorelin on colonic motility measured by barostat and multilumen manometry in CC. METHODS: In a placebo-controlled, single-dose, double-blind, randomized study in patients with CC, we investigated the motor effects of relamorelin, 100 µg, SQ (12 patients) compared to placebo SQ (six patients). A motility-barostat balloon assembly was used to measure colonic compliance; tone and phasic pressure activity were measured before and after a 1000-kcal milkshake meal (administered ~60 min post-medication). Overall "background" phasic pressure activity was assessed by: average amplitude and motility index (MI = ln[sum amplitudes × #contractions + 1]) over defined periods. High-amplitude propagating contractions (HAPCs) were characterized by amplitude >75 mmHg and propagating contractions >50 mmHg; both were propagated over at least 10 cm. Postprandial HAPCs were the primary end point. The study sample had 80% power to detect an increase of 3.3 HAPCs in the hour post-meal. RESULTS: Relamorelin, 100 µg, significantly induced more pre-meal propagated contractions [PCs of either >50 or >75 mmHg] compared to placebo (p < 0.05). Relamorelin also induced more post-meal PCs >50 or >75 mmHg than placebo. Relamorelin did not significantly alter colonic compliance, fasting or postprandial phasic pressure activity (20 min pre-meal fasting MI) or tone, and 60 min postprandial phasic pressure amplitude or MI, or tone. CONCLUSIONS: Relamorelin stimulates propagated colonic contractions without alteration of background irregular contractions in CC. ClinicalTrial.Gov registration number: NCT 01781104.

The probiotic Escherichia coli Nissle 1917 inhibits propagating colonic contractions in the rat isolated large intestine.

The objective of this research was to test an in vitro motility model by investigating whether a probiotic that reduces diarrhea in humans would reduce motility in the rat colon in vitro. The probiotic Escherichia coli Nissle 1917 (EcN) the active ingredient in Mutaflor® was used as an example probiotic because it is effective for treating infectious diarrheal diseases. The effect of EcN on motility was compared in two colonic preparations. In distal colon segments EcN extract decreased the tension of spontaneous contractions by 74% and frequency by 46% compared with pre-treatment controls. In the whole large intestine the number of synchronized spontaneous propagating contractions decreased by 86% when EcN extract was applied externally and 69% when applied via the lumen compared with pre-treatment. From the inhibition produced by EcN extract in the distal colon segment a myogenic action was inferred and in the whole large intestine neural involvement was implicated. Both are consistent with its anti-diarrheal effect in humans.

Progressive proximal-to-distal reduction in expression of the tight junction complex in colonic epithelium of virally-suppressed HIV+ individuals.

Effective antiretroviral therapy (ART) dramatically reduces AIDS-related complications, yet the life expectancy of long-term ART-treated HIV-infected patients remains shortened compared to that of uninfected controls, due to increased risk of non-AIDS related morbidities. Many propose that these complications result from translocated microbial products from the gut that stimulate systemic inflammation--a consequence of increased intestinal paracellular permeability that persists in this population. Concurrent intestinal immunodeficiency and structural barrier deterioration are postulated to drive microbial translocation, and direct evidence of intestinal epithelial breakdown has been reported in untreated pathogenic SIV infection of rhesus macaques. To assess and characterize the extent of epithelial cell damage in virally-suppressed HIV-infected patients, we analyzed intestinal biopsy tissues for changes in the epithelium at the cellular and molecular level. The intestinal epithelium in the HIV gut is grossly intact, exhibiting no decreases in the relative abundance and packing of intestinal epithelial cells. We found no evidence for structural and subcellular localization changes in intestinal epithelial tight junctions (TJ), but observed significant decreases in the colonic, but not terminal ileal, transcript levels of TJ components in the HIV+ cohort. This result is confirmed by a reduction in TJ proteins in the descending colon of HIV+ patients. In the HIV+ cohort, colonic TJ transcript levels progressively decreased along the proximal-to-distal axis. In contrast, expression levels of the same TJ transcripts stayed unchanged, or progressively increased, from the proximal-to-distal gut in the healthy controls. Non-TJ intestinal epithelial cell-specific mRNAs reveal differing patterns of HIV-associated transcriptional alteration, arguing for an overall change in intestinal epithelial transcriptional regulation in the HIV colon. These findings suggest that persistent intestinal epithelial dysregulation involving a reduction in TJ expression is a mechanism driving increases in colonic permeability and microbial translocation in the ART-treated HIV-infected patient, and a possible immunopathogenic factor for non-AIDS related complications.

Influence of phosphodiesterases on basal and 5-HT4 receptor facilitated cholinergic contractility in pig descending colon.

This study in pig colon descendens circular muscle investigated the possible role of phosphodiesterases (PDEs) (1) in the control of smooth muscle activity and (2) in the signal transduction of the 5-HT4 receptors located on the cholinergic neurons. Submaximal cholinergic contractions were electrically induced in colonic circular muscle strips and the influence of the non-selective PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) and selective inhibitors for the 5 classic PDE families (1-5) vinpocetine (PDE1), EHNA (PDE2), cilostamide (PDE3), rolipram (PDE4) and zaprinast (PDE5) was evaluated. IBMX and cilostamide concentration-dependently reduced the amplitude of the cholinergic contractions, as good as abolishing them at 30 and 0.3 µM respectively. EHNA only reduced the contractions significantly at the highest concentration tested (30 µM). IBMX and cilostamide also concentration-dependently inhibited submaximal cholinergic contractions induced with the muscarinic receptor agonist carbachol. The 5-HT4 receptor agonist prucalopride (1 µM) significantly enhanced the electrically induced cholinergic contractions. IBMX, vinpocetine and EHNA did not influence the facilitating effect of prucalopride but rolipram tended to enhance it. When rolipram was added after prucalopride, the facilitating effect of prucalopride was significantly enhanced. These results suggest that PDE3 is the main regulator of circular smooth muscle activity and that the signal transduction of 5-HT4 receptors on the cholinergic nerves towards the circular muscle layer is regulated by PDE4 in pig colon descendens.

Investigation of neurogenic excitatory and inhibitory motor responses and their control by 5-HT(4) receptors in circular smooth muscle of pig descending colon.

The aim of this study was to investigate whether the pig colon descendens might be a good model for the responses mediated via the different locations of human colonic 5-HT(4) receptors. The intrinsic excitatory and inhibitory motor neurotransmission in pig colon descendens was therefore first characterized. In circular smooth muscle strips, electrical field stimulation (EFS) at basal tone induced only in the combined presence of the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) and the SK channel blocker apamin voltage-dependent on-contractions. These on-contractions were largely reduced by the neuronal conductance blocker tetrodotoxin (TTX) and by the muscarinic receptor antagonist atropine, illustrating activation of cholinergic neurons. The 5-HT(4) receptor agonist prucalopride facilitated submaximal EFS-evoked cholinergic contractions and this effect was prevented by the 5-HT(4) receptor antagonist GR113808, supporting the presence of facilitating 5-HT(4) receptors on the cholinergic nerve endings innervating circular muscle in pig colon descendens. Relaxations were induced by EFS in strips pre-contracted with substance P in the presence of atropine. The responses at lower stimulation voltages were abolished by TTX. L-NAME or apamin alone did not influence or only moderately reduced the relaxations, but L-NAME plus apamin abolished the relaxations at lower stimulation voltages, suggesting that NO and ATP act as inhibitory neurotransmitters in a redundant way. Prucalopride did not influence the EFS-induced relaxations at lower stimulation voltage, nor did it per se relax contracted circular muscle strips. No evidence for relaxing 5-HT(4) receptors, either on inhibitory neurons or on the muscle cells was thus obtained in pig colon descendens circular muscle.

Anti-inflammatory effect of enzymatic hydrolysate of corn gluten in an experimental model of colitis.

OBJECTIVES: Intestinal bacteria are thought to be involved in the initiation and perpetuation of inflammatory bowel diseases. Prebiotics (non-digestable dietary carbohydrate) have beneficial properties that alter the intestinal flora and contain glutamine-rich protein. Glutamine significantly decreases indices of inflammation. In this study, an enzymatic hydrolysate of corn gluten (EHCG) was administered by gavage to Sprague-Dawley rats fed an elemental diet to determine whether EHCG can ameliorate experi- mental colitis. METHODS: Colitis was induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid after 10 days' daily oral administration of EHCG at 100 and 300 mg/kg. Macroscopic damage was assessed using a scoring system. The mucosa homogenate was sonicated and myeloperoxidase activity and histamine levels measured. KEY FINDINGS: Treatment with EHCG significantly decreased the severity of injury and reduced myeloperoxidase activity and histamine levels in the distal colon mucosa. CONCLUSIONS: EHCG may have therapeutic benefit as a supplement in enteral nutrition for patients with inflammatory bowel diseases.

Differential protein abundance and function of UT-B urea transporters in human colon.

Facilitative UT-B urea transporters enable the passage of urea across cell membranes. Gastrointestinal urea transporters are thought to play a significant role in the urea nitrogen salvaging process that occurs between mammalian hosts and their gut bacteria. This study investigated the expression of UT-B urea transporters in different segments of human colon. Immunoblot analysis showed that human colon expressed a 35-kDa glycosylated UT-B protein in the colonic mucosa. The 35-kDa UT-B transporter was predominantly located in plasma membrane-enriched samples (P < 0.001; n = 6), and its expression was greater in the ascending colon compared with the descending colon (P < 0.01; n = 3). At the cellular level, UT-B transporters were located throughout colonocytes situated in the upper portion of the colonic crypts. Bidirectional trans-epithelial urea transport was significantly greater in the ascending colon than the descending colon (P < 0.05; n = 6). In addition, the facilitative urea transporter inhibitor 1,3,dimethylurea significantly reduced urea transport in the ascending colon (P < 0.05; n = 6) but had no effect in the descending colon (NS; n = 6). These results illustrate differential protein abundance of functional UT-B protein in different sections of the human colon, strongly correlating to regions that contain the largest populations of intestinal bacteria. This study suggests an important role for UT-B urea transporters in maintaining the symbiotic relationship between humans and their gut bacteria.

Nicotine-induced neurogenic relaxation in the mouse colon: changes with dextran sodium sulfate-induced colitis.

Nicotine has been shown to reduce both tone and muscular activity in the human colon by releasing nitric oxide (NO) from nerves. To our knowledge, however, the effect of nicotine on mouse colon has not been elucidated, and the response in tissue from ulcerative colitis (UC) has not been investigated. We examined nicotine-induced responses in colon from control mice and mice with dextran sodium sulfate (DSS)-induced UC. In controls, bath application of nicotine caused a transient relaxation in longitudinal preparations from the transverse and distal colons but not from the rectum. The response was observed in the presence of bethanechol, abolished by treatment with tetrodotoxin and hexamethonium, and mediated partially (>50%) by the NO pathway. In longitudinal preparations of the distal colon from DSS-treated mice, spontaneous contractions decreased markedly, and nicotine caused contraction without relaxation in half of the preparations tested. Nicotine-induced relaxation in the presence of bethanechol was significantly decreased in the DSS-treated distal colon without changing bethanechol-induced contractions. These data suggest that 1) responses to nicotine differ dependent on colon regions, 2) DSS treatment predominantly caused nicotine-sensitive neurogenic changes in distal colon, and 3) DSS treatment may reverse the direction of nicotine-evoked responses in the colon, in mice.

Inflammatory bowel disease in rats: bacterial and chemical interaction.

AIM: To develop a novel model of colitis in rats, using a combination of iodoacetamide and enteropathogenic E. coli (EPEC), and to elucidate the pathophysiologic processes implicated in the development of ulcerative colitis (UC). METHODS: Male Sprague-Dawley rats (n = 158) were inoculated intrarectally on a weekly basis with 4 different combinations: (a) 1% methylcellulose (MC), (b) 100 microL of 6% iodoacetamide (IA) in 1% MC, (c) 200 microL containing 4 x 10(8) colony factor units (CFU) of EPEC, and (d) combined treatment of (IA) followed by bacteria (B) after 2 d. Thirty days post treatment, each of the four groups was divided into two subgroups; the inoculation was stopped for one subgroup and the other subgroup continued with biweekly inoculation until the end of the experiment. Colitis was evaluated by the clinical course of the disease, the macroscopic and microscopic alterations, activity of myeloperoxidase (MPO), and by TNF-alpha gene expression. RESULTS: Findings indicative of UC were seen in the combined treatment (IA + B) as well as the IA continued treatment groups: the animals showed slow rate of increase in body weight, diarrhea, bloody stools, high colonic ulcer score, as well as histological alterations characteristic of UC, with an extensive inflammatory reaction. During the course of the experiment, the MPO activity was consistently elevated and the TNF-alpha gene expression was upregulated compared to the control animals. CONCLUSION: The experimental ulcerative colitis model used in the present study resembles, to a great extent, the human disease. It is reproducible with characteristics indicative of chronicity.

Colon submucosal microdialysis: a novel in vivo approach in barrier function assessment - a pilot study in rats.

During shock, prognosis of a patient depends largely on intestinal barrier function. The potency of gut epithelium to represent an obstacle to toxins is determined by the blood supply. All established methods of mucosal function determination necessitate the functional involvement of bloodstream. Microdialysis allows monitoring of extracellular substances in the gut submucosa, but its potential use for gut barrier integrity assessment is unknown. Twelve rats underwent perfusion of the descending colon either with 20 % ethanol or control medium (vehicle). Both media contained equal amounts of a radioactive tracer substance ((51)Cr-EDTA). Mucosal permeability for (51)Cr-EDTA was assessed by microdialysate to luminal perfusate activity ratios. Sampling was performed using the colon submucosal microdialysis technique. The group subjected to ethanol treatment had profound macro- and microscopical alterations in perfused colonic segment associated with a significant increase in tracer permeability during ethanol exposure (2.354+/-0.298 % for ethanol as opposed to 0.209+/-0.102 % for control group, p 0.01), which remained elevated for 60 min after cessation of ethanol administration (3.352+/-0.188 % for ethanol compared to 0.140+/-0.0838 % for the control group, p 0.001). Submucosal microdialysis with radioactive tracer substance can be considered a feasible and advantageous alternative of gut barrier function estimation. Parallel monitoring of local tissue chemistry with this method remains a challenge in the future.

Restoration by VIP of the carbachol-stimulated Cl- secretion in TTX-treated guinea pig distal colon.

To determine if vasoactive intestinal peptide (VIP) restores neural activity from tetrodotoxin (TTX) blockade, we studied the effects of VIP and related agents on carbachol (Cch)-induced Cl(-) secretion in control-isolated guinea pig distal colon and in that treated with TTX. The short circuit current (I(sc)) increased dose-dependently after serosal applications of Cch (10(-6) - 2 x 10(-5) M) and VIP (5 x 10(-9) - 10(-7) M). But no additive or synergistic increase in I(sc) was observed. Cch- and VIP-induced I(sc) was completely abolished by a serosal application of TTX (10(-6) M). However, a serosal application, not mucosal, of VIP (10(-7) M) and 8-bromo-cAMP (10(-3) M) restored the Cch-stimulated, TTX-inhibited I(sc) by 113% and 75.8%, respectively. Furthermore, mucosal and serosal applications of forskolin (aden late cyclase activator) restored the I(sc) by 43.9% and 65.3%, respectively. The restored I(sc) was completely abolished by atropine (muscarinic receptor antagonist). These results suggest that VIP may restore the cholinergic activity by increasing the level of intracellular cAMP, and that cholinergic neuron is very likely to be responsible for the regulation of Cl(-) secretion at neuroepithelial junctions. The exact mechanism of VIP's effect on the TTX-inhibited epithelial Cl(-) secretion, and its possible usefulness in the treatment of TTX-induced pathophysiological conditions, remain to be determined.

Class I antiarrhythmics inhibit Na+ absorption and Cl- secretion in rabbit descending colon epithelium.

To clarify the mechanism of the diarrhea associated with the clinical use of antiarrhythmic drugs we assessed the effects of these agents on transepithelial Na+ absorption and Cl- secretion, on basolateral K+ conductance, and on the properties of single basolateral K+ channels of rabbit colon epithelium. Quinidine and propafenone, both at 10 microM, inhibited Na+ absorption by 27 and 38% respectively, compared with 50% with 5 mM Ba2+. The other tested class I antiarrhythmics disopyramide, mexiletine, lidocaine, and flecainide decreased Na+ current by 9-13%. Procainamide and the class III antiarrhythmics N-acetylprocainamide, sotalol, ibutilide, and amiodarone were no or were very weak inhibitors of Na+ absorption. Cl- secretion, stimulated with the adenosine analogue NECA (5'-N-ethylcarboxamide-adenosine), was reduced by 54% with quinidine and by 29% with propafenone compared with 100% with Ba2+. Mexiletine, lidocaine, and flecainide inhibited Cl- secretion by 10-23%, whereas the class III antiarrhythmics were no or were weak inhibitors. Those antiarrhythmics that inhibited Na+ and Cl- transport also reduced basolateral K+ conductance, determined in amphotericin B permeabilized epithelia. The activity of the high-conductance, Ca2+-activated, voltage-dependent K+ (BK(Ca)) channel, which is primarily responsible for basolateral K+ recycling during Na+ absorption, was inhibited by 10-30 microM quinidine or propafenone in the form of a rapidly dissociating block. Mexiletine and flecainide inhibited the single channel conductance at higher concentrations; disopyramide, lidocaine, and procainamide were ineffective. In conclusion, the present evidence suggests that the diarrhea caused by class I antiarrhythmic drugs such as quinidine and propafenone is a result of a reduction in basolateral K+ conductance and inhibition of BK(Ca) channels, thereby impeding transepithelial Na+ and water absorption.

Matrices for site-specific controlled-delivery of 5-fluorouracil to descending colon.

Colon-specific controlled-delivery 5-fluorouracil (5-FU) matrices for the treatment of colorectal carcinoma were prepared and evaluated. Matrices are destined to be introduced into enteric-coated capsules and thereby carried to and liberated in the ileum. There, drug release should be prevented until matrices reach descending colon where release should occur. Matrices (50 mg, diameter 0.6 mm) were prepared by compression of powders or of granules prepared by melt granulation. The ingredients comprised 30-70% w/w 5-FU, glyceryl palmitostearate as rate-controlling material and 5% w/w Aerosil as glidant. Drug release was measured by the rotating basket method. The matrix containing 60% w/w drug, prepared by compression of powders, was appropriate to make the planned system, in virtue of its fairly high drug load and its nearly constant and reasonable release rate. This matrix was spray-coated with Eudragit S100 (EUD). Subsequently, an external layer of chitosan hydrochloride (CH-HCl) was applied by a dipping-drying technique. When transit of coated matrix through ileum (phosphate buffer (PB) pH 7.4), ascending colon (PB pH 6 containing rat cecal contents) and descending colon (PB pH 7.4) was simulated in vitro, the pH 4.7 of the CH-HCl gel layer and the pH 6 of the ascending colon prevented dissolution of the protective EUD film until descending colon was reached, then controlled release started. The present small matrices can enter size no. 00 capsules. Considering that each capsule contains 10 matrices, the maximal dose is 300 mg.

Membrane permeability and antipyrine absorption in a rat model of ischemic colitis.

The aim of this study was to determine whether the duration of ischemia affects antipyrine absorption in the large intestine. This was carried out in a rat model of ischemic colitis in which ischemia and associated inflammation was induced by marginal vessel ligation. Blood flow was disrupted by positioning an o-ring around the distal rectum and ligating the marginal vessel at two locations in the hind-gut ligament artery region. Ligation was performed for 1, 2, 3, and 5h. We assessed large intestine damage by measuring key indicators of inflammation, myeloperoxidase (MPO) activity and thiobarbituric acid reactant substrates (TBARS) in the mucosa and by histological staining with hematoxylin-eosin stain. Antipyrine membrane permeability was assessed in Ussing-type diffusion chambers, and related pharmacokinetics were calculated from antipyrine plasma concentration measurements following colon administration of the drug. Vessel ligation caused some sloughing of epithelial cells and elevated the MPO and TBARS levels. Prolonged ligation failed to affect the apparent permeability coefficient (P(app)) of antipyrine. Prolonged ligation, however, gradually increased plasma antipyrine concentrations to near control levels. This increase was paralleled by increases in the absorption rate constant AUC and antipyrine bioavailability. Taken together, these results suggest that the absorption kinetics of antipyrine may depend on blood flow changes in the large intestine that occur with inflammation.

Mechanisms involved in carbachol-induced Ca(2+) sensitization of contractile elements in rat proximal and distal colon.

1. Mechanisms involved in Ca(2+) sensitization of contractile elements induced by the activation of muscarinic receptors in membrane-permeabilized preparations of the rat proximal and distal colon were studied. 2. In alpha-toxin-permeabilized preparations from the rat proximal and distal colon, Ca(2+) induced a rapid phasic and subsequent tonic component. After Ca(2+)-induced contraction reached a plateau, guanosine 5'-triphosphate (GTP) and carbachol (CCh) in the presence of GTP further contracted preparations of both the proximal and distal colon (Ca(2+) sensitization). Y-27632, a rho-kinase inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization more significantly in the proximal colon than in the distal colon. 3. Y-27632 at 10 microm had no effect on Ca(2+)-induced contraction or slightly inhibited phorbol-12,13-dibutyrate-induced Ca(2+) sensitization in either proximal or distal colon. Chelerythrine, a protein kinase C inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization in the distal colon, but not in the proximal colon. The component of Ca(2+) sensitization that persisted after the chelerythrine treatment was completely inhibited by Y-27632. 4. In beta-escin-permeabilized preparations of the proximal colon, C3 exoenzyme completely inhibited GTP plus CCh-induced Ca(2+) sensitization, but PKC(19-31) did not. In the distal colon, C3 exoenzyme abolished GTP-induced Ca(2+) sensitization. It inhibited CCh-induced sensitization by 50 % and the remaining component was inhibited by PKC(19-31). 5. These results suggest that both protein kinase C and rho pathways in parallel mediate the Ca(2+) sensitization coupled to activation of muscarinic receptors in the rat distal colon, whereas the rho pathway alone mediates this action in the proximal colon.