Chloride Secretory Response Research Articles

Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.

Taurodeoxycholate Modulates Apical Cl−/OH− Exchange Activity in Caco2 Cells

Bile acid malabsorption has been shown to be associated with diarrhea in cases such as ileal resection Crohn's disease of the ileum, and radiation enteritis. The mechanisms of bile acid-induced diarrhea are not fully understood. Although the induction of colonic chloride secretion in response to bile acids has been extensively investigated, to date the direct effect of bile acids on intestinal chloride absorption has not been well defined. Therefore, the current studies were undertaken to investigate the effect of bile acids on the apical Cl(-)/OH(-) exchange process utilizing Caco2 monolayers as an in vitro cellular model. Cl(-)/OH(-) exchange activity was measured as DIDS-sensitive pH gradient-driven (36)Cl uptake. The results are summarized as follows: (i) short-term exposure (20 min) of Caco2 cells to taurodeoxycholate (TDC; 200 microM) and glycochenodeoxycholate (GCDC; 200 microM) acids significantly inhibited apical Cl(-)/OH(-) exchange (by approximately 60-70%); (ii) the Ca(2+) chelator BAPTA-AM blocked the inhibition by TDC; (iii) the reduction in Cl(-)/OH(-) exchange by TDC was reversed by the PKC inhibitor, chelerythrine chloride; (iv) functional and inhibitor studies indicated that TDC induced inhibition of Cl(-)/OH(-) exchange was mediated via the activation of the PKC beta I isoform; (v) the effect of TDC on apical Cl(-)/OH(-) exchange was completely blocked by the PI3 kinase inhibitor LY294002 (5 microM); and (vi) the PKA inhibitor, RpcAMP, had no effect on TDC induced inhibition of Cl(-)/OH(-) exchange. In conclusion, our studies provide direct evidence for inhibition of human intestinal apical Cl(-)/OH(-) exchange activity by bile acids via Ca(2+)-, PI3 kinase-, and PKC beta I-dependent pathways in Caco2 cells.

How do the rotavirus NSP4 and bacterial enterotoxins lead differently to diarrhea?

Rotavirus is the major cause of infantile gastroenteritis and each year causes 611 000 deaths worldwide. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur with no visible tissue damage and, conversely, the histological lesions can be asymptomatic. Rotavirus impairs activities of intestinal disaccharidases and Na+-solute symports coupled with water transport. Maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption can lead to a malabsorption component of diarrhea. Since the discovery of the NSP4 enterotoxin, diverse hypotheses have been proposed in favor of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of diarrhea, but the mechanisms appear to be quite different from those used by bacterial enterotoxins that cause pure secretory diarrhea. Rotavirus failed to stimulate Cl- secretion in crypt, whereas it stimulated Cl- reabsorption in villi, questioning, therefore, the origin of net Cl- secretion. A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection. Also, the overall chloride secretory response is regulated by a phospholipase C-dependent calcium signaling pathway induced by NSP4. However, the overall response is weak, suggesting that NSP4 may exert both secretory and subsequent anti-secretory actions, as did carbachol, hence limiting Cl- secretion. All these characteristics provide the means to make the necessary functional distinction between viral NSP4 and bacterial enterotoxins.

The Cdc42 inhibitor secramine B prevents cAMP-induced K + conductance in intestinal epithelial cells

Cyclic AMP- (cAMP) and calcium-dependent agonists stimulate chloride secretion through the coordinated activation of distinct apical and basolateral membrane channels and ion transporters in mucosal epithelial cells. Defects in the regulation of Cl − transport across mucosal surfaces occur with cystic fibrosis and V. cholerae infection and can be life threatening. Here we report that secramine B, a small molecule that inhibits activation of the Rho GTPase Cdc42, reduced cAMP-stimulated chloride secretion in the human intestinal cell line T84. Secramine B interfered with a cAMP-gated and Ba 2+-sensitive K + channel, presumably KCNQ1/KCNE3. This channel is required to maintain the membrane potential that sustains chloride secretion. In contrast, secramine B did not affect the Ca 2+-mediated chloride secretion pathway, which requires a separate K + channel activity from that of cAMP. Pirl1, another small molecule structurally unrelated to secramine B that also inhibits Cdc42 activation in vitro, similarly inhibited cAMP-dependent but not Ca 2+-dependent chloride secretion. These results suggest that Rho GTPases may be involved in the regulation of the chloride secretory response and identify secramine B an inhibitor of cAMP-dependent K + conductance in intestinal epithelial cells.

Mutual Enhancement of Virulence by Enterotoxigenic and Enteropathogenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) are common causes of diarrhea in children in developing countries. Dual infections with both pathogens have been noted fairly frequently in studies of diarrhea around the world. In previous laboratory work, we noted that cholera toxin and forskolin markedly potentiated EPEC-induced ATP release from the host cell, and this potentiated release was found to be mediated by the cystic fibrosis transmembrane conductance regulator. In this study, we examined whether the ETEC heat-labile toxin (LT) or the heat-stable toxin (STa, also known as ST) potentiated EPEC-induced ATP release. We found that crude ETEC culture filtrates, as well as purified ETEC toxins, did potentiate EPEC-induced ATP release in cultured T84 cells. Coinfection of T84 cells with live ETEC plus EPEC bacteria also resulted in enhanced ATP release compared to EPEC alone. In Ussing chamber studies of chloride secretion, adenine nucleotides released from the host by EPEC also significantly enhanced the chloride secretory responses that were triggered by crude ETEC filtrates, purified STa, and the peptide hormone guanylin. In addition, adenosine and LT had additive or synergistic effects in inducing vacuole formation in T84 cells. Therefore, ETEC toxins and EPEC-induced damage to the host cell both enhance the virulence of the other type of E. coli. Our in vitro data demonstrate a molecular basis for a microbial interaction, which could result in increased severity of disease in vivo in individuals who are coinfected with ETEC and EPEC.

The role of epithelial P2Y2 and P2Y4 receptors in the regulation of intestinal chloride secretion

UTP-induced chloride secretion by the intestinal mucosa mounted in Ussing chambers was assessed by measurement of the short-circuit current (I(sc)) in the presence of phloridzin in the case of jejunum or amiloride in the case of colon to eliminate any contribution of electrogenic Na(+) movement to the net ionic transport. Since we have previously demonstrated the absence of chloride-secretory response to apical UTP in the jejunum from P2Y(4)-null mice, in the present study we studied the response to basolateral UTP in the jejunum and to either apical or basolateral UTP in the colon, in both P2Y(2)- and P2Y(4)-deficient mice. In the jejunum, the chloride-secretory response to basolateral UTP was partially reduced in both P2Y(2)- (40%) and P2Y(4)- (60%) null mice. In the colon, both apical or basolateral UTP increased the I(sc). That response was abolished in a chloride-free medium. The colonic chloride-secretory response to either basolateral or apical UTP was abolished in P2Y(4)-deficient mice, but not significantly affected in P2Y(2)-deficient mice. The chloride-secretory response to forskolin was potentiated by prior basolateral addition of UTP and this potentiation was abolished in P2Y(4)-null mice. The jejunum of mice homozygous for the DeltaF508 mutation of cystic fibrosis transmembrane conductance regulator was responsive to UTP, but the magnitude of that response was smaller than in the wild-type littermates. In conclusion, the P2Y(4) receptor fully mediates the chloride-secretory response to UTP in both small and large intestines, except at the basolateral side of the jejunum, where both P2Y(2) and P2Y(4) receptors are involved.

Endocytosis of cholera toxin by human enterocytes is developmentally regulated

Many secretory diarrheas including cholera are more prevalent and fulminant in young infants than in older children and adults. Cholera toxin (CT) elicits a cAMP-dependent chloride secretory response in intestinal epithelia, which accounts for the fundamental pathogenesis of this toxigenic diarrhea. We have previously reported that the action of this bacterial enterotoxin is excessive in immature enterocytes and under developmental regulation. In this study, we tested the hypothesis that enhanced endocytosis by immature human enterocytes may, in part, account for the excessive secretory response to CT noted in the immature intestine and that enterocyte endocytosis of CT is developmentally regulated. To test this hypothesis, we used specific inhibitors to define endocytic pathways in mature and immature cell lines. We showed that internalization of CT in adult enterocytes is less and occurs via the caveolae/raft-mediated pathway in contrast to an enhanced immature human enterocyte CT uptake that occurs via a clathrin pathway. We also present evidence that this clathrin pathway is developmentally regulated as demonstrated by its response to corticosteroids, a known maturation factor that causes a decreased CT endocytosis by this pathway.

Investigation of the effect of black walnut extract on in vitro ion transport and structure of equine colonic mucosa

To examine the secretory response (in the presence and absence of prostaglandin inhibition) in vitro and structural alterations of colonic mucosa in horses after intragastric administration of black walnut extract (BWE). 14 adult horses. Seven horses were administered BWE intragastrically and monitored for 11 hours. Tissue samples were obtained from the right ventral, left ventral, and right dorsal colons (RVC, LVC, and RDC, respectively) of the 7 BWE-treated and 7 control horses. Tissue samples were examined via light microscopy, and the extent of hemorrhage, edema, and granulocytic cellular infiltration (neutrophils and eosinophils) was graded. Colonic mucosal segments were incubated with or without flunixin meglumine (FLM) for 240 minutes; spontaneous electrical potential difference and short-circuit current (Isc) were recorded and used to calculate mucosal resistance. Colonic tissues from BWE-treated horses (with or without FLM exposure) had an overall greater Isc during the 240-minute incubation period, compared with tissues from control horses. The resistance pattern in RVC, LVC, and RDC samples (with or without FLM exposure) from BWE-treated horses was decreased overall, compared with control tissues (with or without FLM exposure). Histologically, colonic mucosal tissues from BWE-treated horses had more severe inflammation (involving primarily eosinophils), edema, and hemorrhage, compared with tissue from control horses. In horses, BWE administration appears to cause an inflammatory response in colonic mucosal epithelium that results in mucosal barrier compromise as indicated by decreased mucosal resistance with presumed concomitant electrogenic chloride secretory response, which is not associated with prostaglandin mediation.

The Effect of Varying Tonicity on Nasal Epithelial Ion Transport in Cystic Fibrosis

There is reasonable evidence that the fluid layer of the airway epithelium is exposed to changes in tonicity. The inspiration of cool, dry air causes an increased tonicity, whereas this tonicity may be decreased by glandular secretions. We hypothesized that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the responses to changes in tonicity and that these may be altered in cystic fibrosis (CF). Using nasal potential difference (PD) protocols in 8 subjects with CF and 10 subjects without CF, we investigated the effects of hyper- and hypotonicity on ion transport processes. We found significant differences between the two groups. In response to a hypertonic challenge (mannitol 500 mM), there was a decreased PD in both groups, suggesting decreased sodium absorption. However, after the prior inhibition of sodium transport using amiloride, there was an increased PD in the non-CF group alone, suggesting CFTR-mediated chloride secretion in response to luminal hypertonicity. For the hypotonic solution, we found that hypotonicity inhibited CFTR-mediated chloride secretion in the non-CF group. These data suggest that CFTR plays a role in the recognition and regulation of airway fluid tonicity.

Chemokine receptor CCR6 transduces signals that activate p130Cas and alter cAMP-stimulated ion transport in human intestinal epithelial cells.

Human colon epithelial cells express the G protein-coupled receptor CCR6, the sole receptor for the chemokine CCL20 (also termed MIP-3alpha). CCL20 produced by intestinal epithelial cells is upregulated in response to proinflammatory stimuli and microbial infection, and it chemoattracts leukocytes, including CCR6-expressing immature myeloid dendritic cells, into sites of inflammation. The aim of this study was to determine whether CCR6 expressed by intestinal epithelial cells acts as a functional receptor for CCL20 and whether stimulation with CCL20 alters intestinal epithelial cell functions. The human colon epithelial cell lines T84, Caco-2, HT-29, and HCA-7 were used to model colonic epithelium. Polarized intestinal epithelial cells constitutively expressed CCR6, predominantly on the apical side. Consistent with this, apical stimulation of polarized intestinal epithelial cells resulted in tyrosine phosphorylation of the p130 Crk-associated substrate (Cas), an adaptor/scaffolding protein that localizes in focal adhesions and has a role in regulating cytoskeletal elements important for cell attachment and migration. In addition, CCL20 stimulation inhibited agonist-stimulated production of the second messenger cAMP and cAMP-mediated chloride secretory responses by intestinal epithelial cells. Inhibition was abrogated by pertussis toxin, consistent with signaling through Galphai proteins that negatively regulate adenylyl cyclases and cAMP production. These data indicate that signaling events, occurring via the activation of the apically expressed chemokine receptor CCR6 on polarized intestinal epithelial cells, alter specialized intestinal epithelial cell functions, including electrogenic ion secretion and possibly epithelial cell adhesion and migration.

A 5-HT3 receptor agonist induces a chloride secretory response in the presence of neural blockade in rat distal colon

Abstract Introduction: Serotonin (5-HT) is a well-described intestinal secretagogue. The 5-HT3 receptor is one of many 5-HT subtypes and is generally accepted to reside at the level of the enteric neuron. We hypothesize that a further subtype of the 5-HT3 receptor may be found on a nonneural cell in rat colon. Methods: Partial-thickness sheets of rat distal colon (n = 9) were mounted in Ussing chambers and short-circuited. The selective 5-HT3 receptor agonist, 2-met-5-HT (50 micromol), was added in the presence or absence of 1 micromol of the neural sodium channel blocker, tetrodotoxin (TTX). Control tissue received no drug. Changes in short-circuit current (Isc), a measure of electrogenic chloride secretion, was measured continuously. Results: Histologic examination of the stripped colon revealed intact mucosa and muscularis mucosa, scant submucosa, and virtually no neural ganglia (HE ANOVA) or TTX alone (p Conclusions: In this in vitro preparation of rat colon, the selective 5-HT3 receptor agonist, 2-met-5-HT, induced a chloride secretory response that was TTX-insensitive. These findings suggest the presence of a nonneural 5-HT3 receptor located at the level of the mucosa. TableMean (+/− SEM) changes in Isc from baseline in microamps/cm 2 MinControl2Met5HT∗2Met5HT + TTX∗TTX5−1.3 ± 0.911.3 ± 2.28.1 ± 3.0−0.3 ± 0.210−1.8 ± 1.912.0 ± 3.37.3 ± 1.9−0.8 ± 0.315−3.3 ± 3.09.3 ± 1.86.7 ± 1.6−0.8 ± 0.430−4.3 ± 4.18.6 ± 1.95.2 ± 1.2−0.9 ± 0.660−5.0 ± 4.35.5 ± 2.12.0 ± 0.8−1.1 ± 0.7∗p

Modulation of chloride secretory responses and barrier function of intestinal epithelial cells by the Salmonella effector protein SigD.

The Salmonella effector protein SigD is an inositol phosphate phosphatase that inhibits phosphatidylinositol 3-kinase-dependent signaling. Because epidermal growth factor (EGF) inhibits chloride secretion via phosphatidylinositol 3-kinase, we explored whether Salmonella infection might modify the inhibitory effect of EGF. As expected, EGF inhibited chloride secretion induced by carbachol in T(84) epithelial cells. Infection with wild-type (WT) but not sigD(-) mutant S. typhimurium SL1344 decreased CCh-stimulated chloride secretion. Moreover, WT but not sigD(-) Salmonella reduced the inhibitory effect of EGF on carbachol-stimulated chloride secretion. Complementation of sigD restored the ability of mutant Salmonella to reverse the inhibitory effect of EGF. EGF-induced EGF receptor phosphorylation was similar in cells infected with either WT or mutant Salmonella, and neither WT nor sigD(-) Salmonella altered recruitment of the p85 subunit of phosphatidylinositol 3-kinase to EGF receptor, implying that SigD acts downstream of these signaling events. Furthermore, transepithelial resistance fell more rapidly in cells infected with WT vs. sigD(-) Salmonella, indicating an early role for SigD in reducing barrier function, perhaps via activation of protein kinase C. We conclude that the Salmonella bacterial effector protein SigD may play critical roles in the pathogenesis of disease caused by this microorganism.

Inhibition of neural nitric oxide synthase attenuates the chloride secretory response to stroking in human jejunum

Background. Stroking of human jejunal mucosa induces serotonin release and a rise in short-circuit current (ΔIsc). Nitric oxide is known to function as a nonadrenergic, noncholinergic neurotransmitter in response to neural serotonin receptor activation in the rat. We hypothesize that neural nitrergic mechanisms mediate the chloride secretory response to mucosal stroking in human jejunum. Methods. Segments of normal proximal jejunum were obtained from patients having gastric bypass surgery for obesity. Muscle-stripped segments of jejunum were mounted in Ussing chambers under short-circuit conditions. The neural nitric oxide synthase inhibitor, l-thiocitrulline, was added to experimental tissues. Mucosal stroking of control and experimental segments was then performed. Results. Pretreatment with l-thiocitrulline attenuated the short circuit rise seen after stroking in the experimental group when compared with the control (5.4 ± 1.5 μA/cm2 vs 8.0 ± 1.6 μA/cm2; P <.05, Student t test, paired data, n = 11), but did not affect baseline Isc before stroking. Serotonin released by stroking was not different in experimental versus control tissue. Conclusions. The significantly reduced ΔIsc in the group pretreated with the neural nitric oxide synthase inhibitor suggests that nitric oxide liberated from enteric neurons participates in the chloride secretory response to stroking in human jejunum in vitro. (Surgery 2003;134:255-9.)

Loss of Nucleotide Regulation of Epithelial Chloride Transport in the Jejunum of P2Y4-Null Mice

The P2Y(4) receptor is responsive to UTP in human and to ATP and UTP in rodents. With the aim of identifying its pharmacotherapeutic interest, we generated P2Y(4)-null mice by a classic gene targeting method. The proportion of genotypes was consistent with X-linked Mendelian transmission. Gene inactivation was checked by the complete disappearance of P2Y(4) receptor mRNA from liver, stomach, and intestine. The P2Y(4)-null mice had a grossly normal behavior, growth, and reproduction. Chloride secretion by the jejunal epithelium was assessed in Ussing chambers by the measurement of the short circuit current in the presence of phlorizin. We show here that the UTP- and ATP-induced chloride secretory responses observed in wild-type mice are abolished in P2Y(4)-null mice. This is the first clearcut demonstration of a biological role of the P2Y(4) receptor.

Activation of proteinase‐activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase‐and cyclo‐oxygenase‐dependent pathway

Proteinase-activated receptor 1 (PAR-1) is activated by thrombin and induces chloride secretion by intestinal epithelial cells. To elucidate further the mechanisms whereby PAR-1 stimulates secretion, monolayers of SCBN intestinal epithelial cells were studied in modified Ussing chambers. Short circuit current responses were determined after basolateral application of thrombin and the PAR-1-activating peptide, Ala-parafluoro-Phe-Arg-cyclohexyl-Ala-Citrulline-Tyr (Cit-NH2) in the presence or absence of a variety of signal transduction and cyclo-oxygenase (COX) pathway inhibitors. Increased kinase activity was monitored by immunoprecipitation and Western blot analysis of target phosphoproteins. The PAR-1-induced chloride secretory response was significantly attenuated by inhibitors of the EGF receptor tyrosine kinase, Src-kinase, MEK1/2, as well as by inhibitors of cytosolic phospholipase (cPL) A2, COX-1 and COX-2. PAR-1-induced activation of cPLA2, as shown by Western blot of phosphoserine residues, was blocked in cells treated with the MEK inhibitor U0126, indicating that the MEK-ERK1/2 MAP kinase pathway mediated PAR-1-induced cPLA2 phosphorylation. Our data show that PAR-1-induced chloride secretion in SCBN cells involves Src, EGF receptor trans-activation, activation of a MAPK pathway, phosphorylation of cPLA2, COX activity, but not PGF2alpha or PGE2. These findings may be of clinical importance in inflammatory diseases of the intestine where secretory dysfunction is evident and thrombin levels are elevated.

Functional human CFTR produced by a stable minichromosome.

Artificial chromosomes have been claimed to be the ideal vector for gene therapy, but their use has been hampered by an inability to produce stable and well designed molecules. We have used a structurally defined minichromosome to clone the human cystic fybrosis transmembrane conductance regulator (CFTR) locus. To guarantee the presence of the proper regulatory elements, we used the 320 kb yeast artificial chromosome (YAC) 37AB12 with the intact CFTR gene and upstream sequences. The resulting minichromosome was analyzed for the presence of the entire CFTR gene and for its functional activity by molecular and functional methods. We have identified clones showing the presence of both the transcript and the CFTR protein. Moreover, in the same clones, a chloride secretory response to cAMP was detected. Mitotic and molecular stability after prolonged growth without selection demonstrated that the constructs were stable. This is the first example of a structurally known minichromosome made to contain an active therapeutic gene.

Release of ATP during host cell killing by enteropathogenic E. coli and its role as a secretory mediator.

Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5'-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added. In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5-2 microM 4 h after infection with wild-type EPEC strains. In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine. Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not. The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-(p-sulfophenyl)-theophylline and MRS1754. EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2b receptors. These findings provide new insight into how EPEC causes watery diarrhea.

Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium: role of iNOS and COX-2.

Various invasive pathogens cause diarrhea, but the mechanism(s) are poorly understood. We hypothesized that nitric oxide and prostaglandins might modulate chloride secretory and barrier properties of the infected intestinal epithelium and that diarrhea is caused, in part, by altered expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). Studies were conducted in human intestinal epithelial cell lines (HT29/cl.19A, Caco-2, and T84). Cells were infected with enteroinvasive Escherichia coli (EIEC 029:NM) or Salmonella dublin (SD), or nonpathogenic, noninvasive bacteria (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]). Infected cells and controls were tested for transepithelial resistance, chloride secretion, prostaglandin E2, guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate, and protein expression. Cells infected with EIEC or SD, but not uninfected controls or ST/LA-exposed monolayers, showed a progressive reduction in transepithelial resistance starting at 6-12 hours. Infected HT29/cl.19A and Caco-2 cells, but not T84 cells, also showed an increase in total nitrite. Expression of iNOS, and consequently COX-2, was also increased, followed by increased production of prostaglandins and cyclic nucleotides. Furthermore, basal and stimulated chloride secretory responses to various agonists were enhanced in HT29/cl.19A and Caco-2 cells after infection with enteroinvasive bacteria, and this effect was reversed for some agonists by iNOS or COX-2 inhibitors. Increased expression of cystic fibrosis transmembrane conductance regulator and NKCC1 was also observed in EIEC or SD-infected cells vs. controls, secondary to NO synthase activity. Up-regulation of iNOS and COX-2 by enteroinvasive bacteria can modulate chloride secretion and barrier function in intestinal epithelial cells. Thus, these enzymes represent possible therapeutic targets in infectious diarrhea.

Both serotonin and a nitric-oxide donor cause chloride secretion in rat colonocytes by stimulating cGMP

Background. Previous studies have demonstrated that an antagonist of nitric oxide synthase inhibits neurally mediated chloride secretion in response to serotonin (5-HT). The purpose of this study was to demonstrate that chloride secretion in rat colonocytes that were caused by stimulation of neural 5-HT receptors is mediated by way of a nitrergic pathway that involves the activation of guanylate cyclase. Methods. The nitric oxide (NO) donor, diethylenetriamine/NO (DNO), was added to an enriched suspension of rat colonocytes that were preloaded with 36Cl−. In parallel experiments, DNO (1 μmol/L) was added to cells that were pretreated with the specific inhibitor of soluble guanylate cyclase, NS2028 (2 μmol/L). In additional studies, the neural 5-HT3 receptor agonist, 2-methyl-5-HT (10 μmol/L), was added to the serosal surface of muscle-stripped sheets of rat colonic mucosa that were mounted in Ussing chambers under voltage clamp conditions, both in the absence and presence of NS2028 (20 μm). Results. DNO induced 18.0% ± 8.0% greater 36Cl− efflux than controls (P <.05; n = 14 animals). This efflux was abolished by previous treatment with NS2028. In the chamber experiments, 2-methyl-5-HT induced electrogenic chloride secretion that was significantly inhibited by previous treatment with NS2028 (2.2 ± 0.5 μA/cm2 vs 13.1 ± 2.1 μA/cm2; P <.001; n = 9 animals). Conclusions. The predominant secretomotor neurotransmitter that mediates the chloride secretory effects of 5-HT in vitro is nitric oxide. Both the secretory effect initiated at the 5-HT3 receptor on enteric neurons and at the NO− receptor on the rat colonocytes are mediated through the activation of intracellular guanylate cyclase and the production of cyclic guanosine monophosphate. (Surgery 2001;130:236-41.)

Nitric oxide is a neurotransmitter in the chloride secretory response to serotonin in rat colon

Background: Serotonin (5-hydroxytryptamine [5-HT]) has been shown to induce chloride secretion through a nonadrenergic/noncholinergic neural pathway, mediated by a 5-HT3 receptor. We hypothesized that 5-HT3–induced Cl– secretion is ultimately mediated by nitric oxide (NO). Methods: Unstripped sheets of rat distal colon were mounted in Ussing chambers and short-circuited. The 5-HT3 receptor agonist, 2-methyl-5-HT, was added in the absence and presence of the NO synthase inhibitor, L-NAME. Companion studies involved the addition of sodium nitroprusside to tissue that was incubated with or without tetrodotoxin. Results: L-NAME caused a significant reduction in the 2-methyl-5-HT–induced change in circuit current, in a concentration-dependent manner. Sodium nitroprusside caused a change in circuit current over baseline in 5 minutes. The addition of tetrodotoxin did not significantly alter the change in circuit current; however, the apical Cl– channel blocker, anthracene-9-carboxylic acid, abolished this response. Conclusions: Neurally mediated Cl– secretion in response to 2-methyl-5-HT is inhibited by an NO synthase inhibitor. Exogenous NO mimics this response, which is unaffected by tetrodotoxin. These data suggest that neurally mediated serotoninergic Cl– secretion is, in part, mediated by NO. The ability of exogenous NO to induce a change in circuit current in the presence of tetrodotoxin suggests that NO is a final neurotransmitter in this neural-mucosal reflex and therefore acts directly on the enterocyte to induce secretion. (Surgery 2000;128:240-5.)