Sangeeta Tyagi

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 36Cl uptake. The results are summarized as follows: (i) short-term exposure (20 min) of Caco2 cells to taurodeoxycholate (TDC; 200 μM) and glycochenodeoxycholate (GCDC; 200 μM) acids significantly inhibited apical Cl−/OH− exchange (by ∼60–70%); (ii) the Ca2+ 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 PKCβI isoform; (v) the effect of TDC on apical Cl−/OH− exchange was completely blocked by the PI3 kinase inhibitor LY294002 (5 μM); 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 Ca2+-, PI3 kinase-, and PKCβI-dependent pathways in Caco2 cells.

B-cell CLL/Lymphoma 10 (BCL10) Is Required for NF-κB Production by Both Canonical and Noncanonical Pathways and for NF-κB-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)-κB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IκB-kinase (IKK)α−/− mouse embryonic fibroblasts, increases in phospho-IκBα, nuclear NF-κB 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. In IKKβ−/− cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-κB-inducing kinase (NIK), cytoplasmic NF-κB p100, and nuclear NF-κB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-κB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKβ−/− and IKKα−/− cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKγ, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-κB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.

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. Basolateral membrane vesicles (BLMV) were purified from mucosal scrapings of organ donors, utilizing a Percoll continuous density gradient centrifugation technique. The results showed [3H] thiamine uptake into BLMV to be: (1) markedly stimulated in the presence of an outwardly directed H+ gradient (pH 5.5in/7.5out); (2) significantly inhibited by amiloride in a dose-dependent manner; (3) sensitive to temperature and medium osmolarity and insensitive to changes in membrane potential; (4) not influenced by the addition of 1 mM Mg2+-ATP, inside and outside the vesicles in the presence of Na+ and K+; (5) inhibited by structural analogs—amprolium, oxythiamin, and unlabeled thiamine (100 μM); (6) not affected by organic cations, eg, TEA, N-methyl-nicotinamide (NMN), and choline. (7) saturable as a function of concentration (apparent Km of 0.76 ± 0.21 μM and a Vmax of 1.38 ± 0.35 pmol/mg protein/10 sec). These results indicate the presence of a proton gradient-dependent specialized carrier-mediated exchange mechanism for thiamine transport across the human jejunum basolateral membranes.

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‐&agr; (TNF‐&agr;) on the expression and activity of NHE2, a Na+/H+ exchanger (NHE) that is involved in transepithelial Na+ absorption in intestinal epithelial cells. Methods: NHE2 regulation was examined in TNF‐&agr;‐treated C2BBe1 cells by reverse‐transcription polymerase chain reaction (RT‐PCR), reporter gene assays, and Western blot analysis. NHE isoform activities were measured as ethyl‐isopropyl‐amiloride‐ and HOE694‐sensitive 22Na‐uptake. In vitro and in vivo protein‐DNA interactions were assessed by gel mobility shift assays and chromatin immunoprecipitation studies. Results: TNF‐&agr; treatment of C2BBe1 cells led to repression of NHE2 promoter activity, mRNA, and protein levels; and inhibited both NHE2 and NHE3 mediated 22Na‐uptake. 5′‐deletion analysis of the NHE2 promoter‐reporter constructs identified basepair −621 to −471 as the TNF‐&agr;‐responsive region (TNF‐RE). TNF‐&agr; activated NF‐&kgr;B subunits, p50 and p65, and their DNA‐binding to a putative NF‐&kgr;B motif within TNF‐RE. Mutations in the NF‐&kgr;B motif abolished NF‐&kgr;B‐DNA interactions and abrogated TNF‐&agr;‐induced repression. Ectopic overexpression of NF‐&kgr;B resulted in repression of NHE2 expression. Two functionally distinct inhibitors of NF‐&kgr;B blocked the inhibitory effect of TNF‐&agr;. Conclusions: The human NHE2 isoform is a direct target of transcription factor NF‐&kgr;B. TNF‐&agr;‐mediated activation of NF‐&kgr;B decreases the expression and activity of NHE2 in the intestinal epithelial cell line, C2BBe1. These findings implicate NF‐&kgr;B in the modulation of Na+ absorption during intestinal inflammatory conditions such as IBD where a high level of TNF‐&agr; is detected. (Inflamm Bowel Dis 2011;)

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. Our results showed that NPY (100 nM, 30 min) significantly increased Cl⁻/HCO₃⁻(OH⁻) exchange activity (∼2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl⁻/HCO₃⁻(OH⁻) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl⁻/HCO₃⁻(OH⁻) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (∼45%) by NPY compared with control. MβCD significantly decreased Cl⁻/HCO₃⁻(OH⁻) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive ³⁶Cl⁻ uptake (∼50%). Our results demonstrate that NPY modulates Cl⁻/HCO₃⁻(OH⁻) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl⁻/HCO₃⁻(OH⁻) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine.

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. A 1,120 bp (p−964/+156) SLC26A6 promoter fragment cloned upstream to the luciferase reporter gene in pGL2‐basic exhibited high promoter activity when transfected in Caco2 cells. Progressive deletions of the 5′‐flanking region demonstrated that −214/−44 region of the promoter harbors cis‐acting elements important for maximal SLC26A6 promoter activity. Since, diarrhea associated with inflammatory bowel diseases is attributed to increased secretion of pro‐inflammatory cytokines, we examined the effects of IFNγ (30 ng/ml, 24 h) on SLC26A6 function, expression and promoter activity. IFNγ decreased both SLC26A6 mRNA and function and repressed SLC26A6 promoter activity. Deletion analysis indicated that IFNγ response element is located between −414/−214 region and sequence analysis of this region revealed the presence of potential Interferon Stimulated Responsive Element (ISRE), a binding site (−318/−300 bp) for interferon regulatory factor‐1 transcription factor (IRF‐1). Mutations in the potential ISRE site abrogated the inhibitory effects of IFNγ. These studies provided novel evidence for the involvement of IRF‐1 in the regulation of SLC26A6 gene expression by IFNγ in the human intestine. J. Cell. Biochem. 105: 454–466, 2008.

Evidence for a Na+–H+ Exchange Across Human Colonic Basolateral Plasma Membranes Purified from Organ Donor Colons

The mechanism(s) of electrolyte transport across the human colonic contraluminal domain is not well understood. Current studies were undertaken to develop a technique for the isolation and purification of the human colonic basolateral membrane vesicles (BLMV) and to examine the presence of a Na+–H+ exchange process in these membranes. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll density gradient centrifugation technique, and Na+ transport was examined utilizing a rapid filtration, technique. Our data demonstrate that purified basolateral membranes were enriched 10- to 11-fold in Na+, K+-ATPase activity compared to crude homogenate. Results consistent with the Na+–H+ exchange in BLMV are as follows: (1) an outwardly directed H+ gradient stimulated 22Na uptake; (2) 22Na uptake was markedly inhibited by EIPA and amiloride; (3) H+-gradient-stimulated 22Na uptake was not inhibited by bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; (4) 22Na uptake was voltage insensitive; (5) 22Na uptake demonstrated saturation kinetics; (6) 22Na uptake was markedly inhibited by Na+ and Li+ but was unaffected by N-methyl glucamine+, choline+, and NH4+. Immunoblotting studies demonstrated this Na+–H+ exchanger isoform to be represented by NHE1. In conclusion, a technique has been established for the purification of functional human proximal colonic BLMV, and an electroneutral Na+–H+ exchange process has been demonstrated in these membranes.

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. Human colonic basolateral membrane vesicles (BLMVs) were isolated and purified from mucosal scrapings of organ donor colons, utilizing a technique developed in our laboratory. 45Ca uptake was measured by a rapid filtration technique. 45Ca uptake represented transport into the intravesicular space as evidenced by an osmolarity study and by the demonstration of Ca2+ efflux from calcium preloaded vesicles by Ca2+ ionophore A23187. Calcium uptake was stimulated by Mg2+ ATP. The kinetic parameters for ATP-dependent Ca2+ uptake revealed saturation kinetics with Michaelis constant (Km) of 0.22 ± 0.04 μM and a maximum rate of uptake (Vmax) of 0.38 ± 0.12 nmol/mg protein/min. The Km of ATP concentration required for half maximal Ca2+ uptake was 0.39 ± 0.04 mM. ATP-stimulated calcium uptake into these vesicles was further stimulated in the presence of calmodulin and was inhibited by calmodulin antagonist, trifluoperazine. Uptake of 45Ca into BLMVs was markedly inhibited by cis-Na+ but was significantly stimulated by trans-Na+ (40–50% stimulation). Our results demonstrate the presence of a Mg2+/ATP-dependent calmodulin-regulated Ca2+ transport system and a Na+–Ca2+ exchange process in the human colonic basolateral membranes.

Modulation of Cl•/OH• exchange activity in Caco2 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. Our results suggest that NO-induced inhibition of Cl-/OH- exchange may play an important role in the pathophysiology of diarrhea associated with inflammatory bowel diseases.

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. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.