Clostridium difficile toxins A and B decrease intestinal SLC26A3 protein expression.

Clostridium difficile infection (CDI) is the primary cause of nosocomial diarrhea in the United States. Although C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C.

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions.

Stimulation of apical Cl⁻/HCO₃⁻(OH⁻) exchanger, SLC26A3 by neuropeptide Y is lipid raft dependent.

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl⁻/HCO₃⁻(OH⁻) exchange activity and the underlying mechanisms involved utilizing Caco2 cells.

Tumor necrosis factor-α represses the expression of NHE2 through NF-κB activation in intestinal epithelial cell model, C2BBe1.

Background: High levels of proinflammatory cytokines are linked to pathogenesis of diarrhea in inflammatory bowel disease (IBD). Na(+) absorption is compromised in IBD. The studies were designed to determine the effect of tumor necrosis factor-α (TNF-α) on the expression and activity of NHE2, a Na(+) /H(+) exchanger (NHE) that is involved in transepithelial Na(+) absorption in intestinal epithelial cells.

Mechanisms underlying modulation of monocarboxylate transporter 1 (MCT1) by somatostatin in human intestinal epithelial cells.

Somatostatin (SST), an important neuropeptide of the gastrointestinal tract has been shown to stimulate sodium chloride absorption and inhibit chloride secretion in the intestine. However, the effects of SST on luminal butyrate absorption in the human intestine have not been investigated. Earlier studies from our group and others have shown that monocarboxylate transporter (MCT1) plays an important role in the transport of butyrate in the human intestine.

B-cell CLL/lymphoma 10 (BCL10) is required for NF-kappaB production by both canonical and noncanonical pathways and for NF-kappaB-inducing kinase (NIK) phosphorylation.

B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-kappaB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IkappaB-kinase (IKK)alpha(-/-) mouse embryonic fibroblasts, increases in phospho-IkappaBalpha, nuclear NF-kappaB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol.

Characterization of the 5'-flanking region and regulation of expression of human anion exchanger SLC26A6.

SLC26A6 (putative anion transporter 1, PAT1) has been shown to play an important role in mediating the luminal Cl(-)/OH(-)(HCO(3)(-)) exchange process in the intestine. Very little is known about the molecular mechanisms involved in the transcriptional regulation of intestinal SLC26A6 gene expression in the intestine. Current studies were, therefore, designed to clone and characterize the 5'-regulatory region of the human SLC26A6 gene and determine the mechanisms involved in its regulation.

The probiotic Lactobacillus acidophilus stimulates chloride/hydroxyl exchange activity in human intestinal epithelial cells.

Probiotics are viable nonpathogenic microorganisms that are considered to confer health benefits to the host. Recent studies indicated that some Lactobacillus species function as probiotics and have been used as alternative treatments for diarrhea, which occurs due to increased secretion, decreased absorption, or both. However, the direct effects of probiotics on intestinal electrolyte absorption are not known.

Role of Fyn and PI3K in H2O2-induced inhibition of apical Cl-/OH- exchange activity in human intestinal epithelial cells.

H(2)O(2) is a highly reactive oxygen metabolite that has been implicated as an important mediator of inflammation-induced intestinal injury associated with ischaemia/reperfusion, radiation and inflammatory bowel disease. Previous studies have shown that H(2)O(2) inhibits NaCl absorption and activates Cl(-) secretion in the rat and rabbit colon. To date, however, almost no information is available with respect to its effect on the human intestinal apical anion exchanger Cl(-)/OH(-) (HCO(3)(-)).

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.

Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells.

Background & Aims: Increased serotonin levels have been implicated in the pathophysiology of diarrhea associated with celiac and inflammatory diseases. However, the effects of serotonin on Na+ /H+ exchange (NHE) activity in the human intestine have not been investigated fully. The present studies examined the acute effects of 5-hydroxytryptamine (5-HT) on NHE activity using Caco-2 cells as an in vitro model.

Involvement of c-Src and protein kinase C delta in the inhibition of Cl(-)/OH- exchange activity in Caco-2 cells by serotonin.

Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter and intercellular messenger regulating various gastrointestinal functions, including electrolyte transport. To date, however, no information is available with respect to its effects on the human intestinal apical anion exchanger Cl(-)/OH- (HCO3-). The present studies were therefore undertaken to examine the direct effects of serotonin on OH- gradient-driven 4,4'-diisothiocyanato-stilbene-2, 2'-disulfonic acid-sensitive 36Cl- uptake utilizing the post-confluent transformed human intestinal epithelial cell line Caco-2.

Cholesterol modulates human intestinal sodium-dependent bile acid transporter.

Bile acids are efficiently absorbed from the intestinal lumen via the ileal apical sodium-dependent bile acid transporter (ASBT). ASBT function is essential for maintenance of cholesterol homeostasis in the body. The molecular mechanisms of the direct effect of cholesterol on human ASBT function and expression are not entirely understood.

Differential regulation of Na+/H+ exchange isoform activities by enteropathogenic E. coli in human intestinal epithelial cells.

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal foodborne pathogen associated with diarrhea, especially in infants and young children. Although EPEC produces characteristic attaching and effacing lesions and loss of microvilli, the pathophysiology of EPEC-associated diarrhea, particularly during early infection, remains elusive. The present studies were designed to examine the direct effects of EPEC infection on intestinal absorption via Na(+)/H(+) exchanger (NHE) isoforms.

Evidence for a carrier-mediated mechanism for thiamine transport to human jejunal basolateral membrane vesicles.

Recent studies from our laboratory have demonstrated the presence of a pH-dependent, amiloride-sensitive, electroneutral carrier-mediated exchange for thiamine absorption in the human small intestinal brush-border membrane vesicles. However, the mechanism of thiamine transport across the human small intestinal basolateral membrane is not understood. The present study was aimed to characterize the mechanism of thiamine transport across the basolateral membranes of the human jejunum.

Mechanisms of calcium transport in human colonic basolateral membrane vesicles.

Human colon has been suggested to play an important role in calcium absorption especially after extensive disease or resection of the small intestine. We have previously demonstrated the presence of a carrier-mediated calcium uptake mechanism in the human colonic luminal membrane vesicles. Current studies were, therefore, undertaken to investigate the mechanism(s) of calcium exit across the basolateral membrane domain of the human colon.

Inhibition of apical Cl-/OH- exchange activity in Caco-2 cells by phorbol esters is mediated by PKCepsilon.

The present studies were undertaken to examine the possible regulation of apical membrane Cl-/OH- exchanger in Caco-2 cells by protein kinase C (PKC). The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA), an in vitro PKC agonist, on OH- gradient-driven 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl uptake in Caco-2 cells was assessed. The results demonstrated that PMA decreased apical Cl-/OH- exchanger activity via phosphatidylinositol 3-kinase (PI3-kinase)-mediated activation of PKCepsilon.

Regulation of NHE3 by nitric oxide in Caco-2 cells.

The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10(-3) M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a approximately 45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside.

Modulation of Cl-/OH- exchange activity in Caco-2 cells by nitric oxide.

The present studies were undertaken to determine the direct effects of nitric oxide (NO) released from an exogenous donor, S-nitroso-N-acetyl pencillamine (SNAP) on Cl-/OH- exchange activity in human Caco-2 cells. Our results demonstrate that NO inhibits Cl-/OH- exchange activity in Caco-2 cells via cGMP-dependent protein kinases G (PKG) and C (PKC) signal-transduction pathways. Our data in support of this conclusion can be outlined as follows: 1) incubation of Caco-2 cells with SNAP (500 microM) for 30 min resulted in approximately 50% inhibition of DIDS-sensitive 36Cl uptake; 2) soluble guanylate cyclase inhibitors Ly-83583 and (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one significantly blocked the inhibition of Cl-/OH- exchange activity by SNAP; 3) addition of 8-bromo-cGMP (8-BrcGMP) mimicked the effects of SNAP; 4) specific PKG inhibitor KT-5823 significantly inhibited the decrease in Cl-/OH- exchange activity in response to either SNAP or 8-BrcGMP; 5) Cl-/OH-exchange activity in Caco-2 cells in response to SNAP was not altered in the presence of protein kinase A (PKA) inhibitor (Rp-cAMPS), demonstrating that the PKA pathway was not involved; 6) the effect of NO on Cl-/OH- exchange activity was mediated by PKC, because each of the two PKC inhibitors chelerythrine chloride and calphostin C blocked the SNAP-mediated inhibition of Cl-/OH- exchange activity; 7) SO/OH- exchange in Caco-2 cells was unaffected by SNAP.