NUDT7 regulates total hepatic CoA levels and the composition of the intestinal bile acid pool in male mice fed a Western diet.

Nudix hydrolase 7 (NUDT7) is an enzyme that hydrolyzes CoA species, is highly expressed in the liver, and resides in the peroxisomes. Peroxisomes are organelles where the preferential oxidation of dicarboxylic fatty acids occurs and where the hepatic synthesis of the primary bile acids cholic acid and chenodeoxycholic acid is completed. We previously showed that liver-specific overexpression of NUDT7 affects peroxisomal lipid metabolism but does not prevent the increase in total liver CoA levels that occurs during fasting. We generated Nudt7-/- mice to further characterize the role that peroxisomal (acyl-)CoA degradation plays in the modulation of the size and composition of the acyl-CoA pool and in the regulation of hepatic lipid metabolism. Here, we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low-fat diet, but only in males fed a Western diet does the lack of NUDT7 activity increase total liver CoA levels. This effect is driven by the male-specific accumulation of medium-chain dicarboxylic acyl-CoAs, which are produced from the ß-oxidation of dicarboxylic fatty acids. We also show that, under conditions of elevated synthesis of chenodeoxycholic acid derivatives, Nudt7 deletion promotes the production of tauromuricholic acid, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion in male mice. These findings reveal that NUDT7-mediated hydrolysis of acyl-CoA pathway intermediates in liver peroxisomes contributes to the regulation of dicarboxylic fatty acid metabolism and the composition of the bile acid pool.

Dietary Na+ depletion up-regulates NKCC1 expression and enhances electrogenic Cl- secretion in rat proximal colon.

The corticosteroid hormone, aldosterone, markedly enhances K+ secretion throughout the colon, a mechanism critical to its role in maintaining overall K+ balance. Previous studies demonstrated that basolateral NKCC1 was up-regulated by aldosterone in the distal colon specifically to support K+ secretion-which is distinct from the more well-established role of NKCC1 in supporting luminal Cl- secretion. However, considerable segmental variability exists between proximal and distal colonic ion transport processes, especially concerning their regulation by aldosterone. Furthermore, delineating such region-specific effects has important implications for the management of various gastrointestinal pathologies. Experiments were therefore designed to determine whether aldosterone similarly up-regulates NKCC1 in the proximal colon to support K+ secretion. Using dietary Na+ depletion as a model of secondary hyperaldosteronism in rats, we found that proximal colon NKCC1 expression was indeed enhanced in Na+-depleted (i.e., hyperaldosteronemic) rats. Surprisingly, electrogenic K+ secretion was not detectable by short-circuit current (ISC) measurements in response to either basolateral bumetanide (NKCC1 inhibitor) or luminal Ba2+ (non-selective K+ channel blocker), despite enhanced K+ secretion in Na+-depleted rats, as measured by 86Rb+ fluxes. Expression of BK and IK channels was also found to be unaltered by dietary Na+ depletion. However, bumetanide-sensitive basal and agonist-stimulated Cl- secretion (ISC) were significantly enhanced by Na+ depletion, as was CFTR Cl- channel expression. These data suggest that NKCC1-dependent secretory pathways are differentially regulated by aldosterone in proximal and distal colon. Development of therapeutic strategies in treating pathologies related to aberrant colonic K+/Cl- transport-such as pseudo-obstruction or ulcerative colitis-may benefit from these findings.

Aldosterone up-regulates basolateral Na+ -K+ -2Cl- cotransporter-1 to support enhanced large-conductance K+ channel-mediated K+ secretion in rat distal colon.

Na+ -K+ -2Cl- cotransporter-1 (NKCC1) facilitates basolateral K+ and Cl- uptake, supporting their efflux across mucosal membranes of colonic epithelial cells. NKCC1 activity has also been shown to be critical for electrogenic K+ secretion induced by aldosterone, which is known to stimulate large-conductance K+ (BK) channel expression in mucosal membranes. This study was aimed to (1) identify whether aldosterone enhances NKCC1 expression specifically to support BK-mediated K+ secretion and (2) to determine whether increased NKCC1 supports electrogenic Cl- secretion in parallel to K+ secretion. Dietary Na+ depletion was used to induce secondary hyperaldosteronism in rats, or aldosterone was administered ex vivo to rat distal colonic mucosae. NKCC1-dependent electrogenic K+ or Cl- secretion was measured as a function of short circuit current (ISC ). qRT-PCR, western blot, and immunofluorescence analyses were performed using standard techniques. Aldosterone enhanced NKCC1 and BKα expression and electrogenic K+ secretion in the distal colon, which was inhibited by either serosal bumetanide (NKCC1 inhibitor) or mucosal iberiotoxin (IbTX; BK channel blocker), but not TRAM-34 (IK channel blocker). Expression of NKCC1 and BKα proteins was enhanced in crypt cells of hyper-aldosterone rats. However, neither NKCC1-dependent Cl- secretion nor CFTR (apical Cl- channel) expression was enhanced by aldosterone. We conclude that aldosterone enhances NKCC1 to support BK-mediated K+ secretion independently of Cl- secretion in the distal colon. The regulation of NKCC1 expression/K+ secretion by aldosterone may be a therapeutic target in treating gastrointestinal disorders associated with alterations in colonic K+ transport, such as colonic pseudo-obstruction, and hyperkalemia associated with renal disease.

Activation of KCNQ (KV7) K+ channels in enteric neurons inhibits epithelial Cl- secretion in mouse distal colon.

Voltage-gated Kv7 (KCNQ family) K+ channels are expressed in many neuronal populations and play an important role in regulating membrane potential by generating a hyperpolarizing K+ current and decreasing cell excitability. However, the role of KV7 channels in the neural regulation of intestinal epithelial Cl- secretion is not known. Cl- secretion in mouse distal colon was measured as a function of short-circuit current (ISC), and pharmacological approaches were used to test the hypothesis that activation of KV7 channels in enteric neurons would inhibit epithelial Cl- secretion. Flupirtine, a nonselective KV7 activator, inhibited basal Cl- secretion in mouse distal colon and abolished or attenuated the effects of drugs that target various components of enteric neurotransmission, including tetrodotoxin (NaV channel blocker), veratridine (NaV channel activator), nicotine (nicotinic acetylcholine receptor agonist), and hexamethonium (nicotinic antagonist). In contrast, flupritine did not block the response to epithelium-targeted agents VIP (endogenous VPAC receptor ligand) or carbachol (nonselective cholinergic agonist). Flupirtine inhibited Cl- secretion in both full-thickness and seromuscular-stripped distal colon (containing the submucosal, but not myenteric plexus) but generated no response in epithelial T84 cell monolayers. KV7.2 and KV7.3 channel proteins were detected by immunofluorescence in whole mount preparations of the submucosa from mouse distal colon. ICA 110381 (KV7.2/7.3 specific activator) inhibited Cl- secretion comparably to flupirtine. We conclude that KV7 channel activators inhibit neurally driven Cl- secretion in the colonic epithelium and may therefore have therapeutic benefit in treating pathologies associated with hyperexcitable enteric nervous system, such as irritable bowel syndrome with diarrhea (IBS-D).

Flupirtine enhances NHE-3-mediated Na+ absorption in rat colon via an ENS-dependent mechanism.

Recent studies in our lab have shown that the KV7 channel activator, flupirtine, inhibits colonic epithelial Cl- secretion through effects on submucosal neurons of the enteric nervous system (ENS). We hypothesized that flupirtine would also stimulate Na+ absorption as a result of reduced secretory ENS input to the epithelium. To test this hypothesis, unidirectional 22Na+ fluxes were measured under voltage-clamped conditions. Pharmacological approaches using an Ussing-style recording chamber combined with immunofluorescence microscopy techniques were used to determine the effect of flupirtine on active Na+ transport in the rat colon. Flupirtine stimulated electroneutral Na+ absorption in partially seromuscular-stripped colonic tissues, while simultaneously inhibiting short-circuit current (ISC; i.e., Cl- secretion). Both of these effects were attenuated by pretreatment with the ENS inhibitor, tetrodotoxin. The Na+/H+ exchanger isoform 3 (NHE-3)-selective inhibitor, S3226, significantly inhibited flupirtine-stimulated Na+ absorption, whereas the NHE-2-selective inhibitor HOE-694 did not. NHE-3 localization near the apical membranes of surface epithelial cells was also more apparent in flupirtine-treated colon versus control. Flupirtine did not alter epithelial Na+ channel (ENaC)-mediated Na+ absorption in distal colonic tissues obtained from hyperaldosteronaemic rats and had no effect in the normal ileum but did stimulate Na+ absorption in the proximal colon. Finally, the parallel effects of flupirtine on ISC (Cl- secretion) and Na+ absorption were significantly correlated with each other. Together, these data indicate that flupirtine stimulates NHE-3-dependent Na+ absorption, likely as a result of reduced stimulatory input to the colonic epithelium by submucosal ENS neurons.NEW & NOTEWORTHY We present a novel mechanism regarding regulation of epithelial ion transport by enteric neurons. Activation of neuronal KV7 K+ channels markedly stimulates Na+ absorption and inhibits Cl- secretion across the colonic epithelium. This may be useful in developing new treatments for diarrheal disorders, such as irritable bowel syndrome with diarrhea (IBS-D).

Increased DMT1 and FPN1 expression with enhanced iron absorption in ulcerative colitis human colon.

Iron deficiency anemia is a common complication of ulcerative colitis (UC) that can profoundly impact quality of life. Most iron absorption occurs in the duodenum via divalent metal transporter 1 (DMT1)-mediated uptake and ferroportin-1 (FPN1)-mediated export across the apical and basolateral membranes, respectively. However, the colon also contains iron transporters and can participate in iron absorption. Studies have shown increased duodenal DMT1 and FPN1 in patients with UC, but there is conflicting evidence about whether expression is altered in UC colon. We hypothesized that expression of colonic DMT1 and FPN1 will also increase to compensate for iron deficiency. Quantitative RT-PCR and Western blot analyses were performed on duodenal and colonic segmental (right colon, transverse colon, left colon, and rectum) biopsies obtained during colonoscopy. DMT1 mRNA and protein abundances in colonic segments were approximately equal to those in the duodenum, whereas colonic FPN1 mRNA and protein abundances of colonic segments were about one-quarter of those of the duodenum. DMT1 specific mRNA and protein abundances were increased twofold, whereas FPN1 mRNA and protein expressions were increased fivefold in UC distal colon. Immunofluorescence studies revealed enhanced expression of apical membrane- and basolateral membrane-localized DMT1 and FPN1 in UC human colon, respectively. Increased DMT1 expression was associated with enhanced 2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea (CISMBI, DMT1 specific inhibitor)-sensitive 59Fe uptake in UC human colon. We conclude from these results that patients with active UC have increased expression of colonic iron transporters and increased iron absorption, which may be targeted in the treatment of UC-related anemia.

Parallel intermediate conductance K+ and Cl- channel activity mediates electroneutral K+ exit across basolateral membranes in rat distal colon.

Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.

Zinc ameliorates intestinal barrier dysfunctions in shigellosis by reinstating claudin-2 and -4 on the membranes.

Whether zinc (Zn2+) regulates barrier functions by modulating tight-junction (TJ) proteins when pathogens such as Shigella alter epithelial permeability is still unresolved. We investigated the potential benefits of Zn2+ in restoring impaired barrier function in vivo in Shigella-infected mouse tissue and in vitro in T84 cell monolayers. Basolateral Shigella infection triggered a time-dependent decrease in transepithelial resistance followed by an increase in paracellular permeability of FITC-labeled dextran and altered ion selectivity. This led to ion and water loss into the intestinal lumen. Immunofluorescence studies revealed redistribution of claudin-2 and -4 to an intracellular location and accumulation of these proteins in the cytoplasm following infection. Zn2+ ameliorated this perturbed barrier by redistribution of claudin-2 and -4 back to the plasma membrane and by modulating the phosphorylation state of TJ proteins t hough extracellular signal-regulated kinase (ERK)1/2 dependency. Zn2+ prevents elevation of IL-6 and IL-8. Mice challenged with Shigella showed that oral Zn2+supplementation diminished diverse pathophysiological symptoms of shigellosis. Claudin-2 and -4 were susceptible to Shigella infection, resulting in altered barrier function and increased levels of IL-6 and IL-8. Zn2+ supplementation ameliorated this barrier dysfunction, and the inflammatory response involving ERK-mediated change of phosphorylation status for claudin-2 and -4. Thus, Zn2+ may have potential therapeutic value in inflammatory diarrhea and shigellosis. NEW & NOTEWORTHY Our study addresses whether Zn2+ could be an alternative strategy to reduce Shigella-induced inflammatory response and epithelial barrier dysfunction. We have defined a mechanism in terms of intracellular signaling pathways and tight-junction protein expression by Zn2+. Claudin-2 and -4 are susceptible to Shigella infection, whereas in the presence of Zn2+ they are resistant to infection-related barrier dysfunction involving ERK-mediated change of phosphorylation status of claudins.

Dextran sulfate sodium-induced chronic colitis attenuates Ca2+-activated Cl- secretion in murine colon by downregulating TMEM16A.

Attenuated Ca2+-activated Cl- secretion has previously been observed in the model of dextran sulfate sodium (DSS)-induced colitis. Prior studies have implicated dysfunctional muscarinic signaling from basolateral membranes as the potential perpetrator leading to decreased Ca2+-activated Cl- secretion. However, in our chronic model of DSS-colitis, cholinergic receptor muscarinic 3 ( Chrm3) transcript (1.028 ± 0.12 vs. 1.029 ± 0.27, P > 0.05) and CHRM3 protein expression (1.021 ± 0.24 vs. 0.928 ± 0.09, P > 0.05) were unchanged. Therefore, we hypothesized that decreased carbachol (CCH)-stimulated Cl- secretion in DSS-induced colitis could be attributed to a loss of Ca2+-activated Cl- channels (CaCC) in apical membranes of colonic epithelium. To establish this chemically-induced colitis, Balb/C mice were exposed to 4% DSS for five alternating weeks to stimulate a more moderate, chronic colitis. Upon completion of the protocol, whole thickness sections of colon were mounted in an Ussing chamber under voltage-clamp conditions. DSS-induced colitis demonstrated a complete inhibition of basolateral administration of CCH-stimulated Cl- secretion that actually displayed a reversal in polarity (15.40 ± 2.22 µA/cm2 vs. -2.47 ± 0.25 µA/cm2). Western blotting of potential CaCCs, quantified by densitometric analysis, demonstrated no change in bestrophin-2 and cystic fibrosis transmembrane regulator, whereas anoctamin-1 [ANO1, transmembrane protein 16A (TMEM16A)] was significantly downregulated (1.001 ± 0.13 vs. 0.510 ± 0.12, P < 0.05). Our findings indicate that decreased expression of TMEM16A in DSS-induced colitis contributes to the decreased Ca2+-activated Cl- secretion in murine colon.

Calcium-Activated Cl- Channel: Insights on the Molecular Identity in Epithelial Tissues.

Calcium-activated chloride secretion in epithelial tissues has been described for many years. However, the molecular identity of the channel responsible for the Ca2+-activated Cl− secretion in epithelial tissues has remained a mystery. More recently, TMEM16A has been identified as a new putative Ca2+-activated Cl− channel (CaCC). The primary goal of this article will be to review the characterization of TMEM16A, as it relates to the physical structure of the channel, as well as important residues that confer voltage and Ca2+-sensitivity of the channel. This review will also discuss the role of TMEM16A in epithelial physiology and potential associated-pathophysiology. This will include discussion of developed knockout models that have provided much needed insight on the functional localization of TMEM16A in several epithelial tissues. Finally, this review will examine the implications of the identification of TMEM16A as it pertains to potential novel therapies in several pathologies.

Na-H Exchanger Isoform-2 (NHE2) Mediates Butyrate-dependent Na+ Absorption in Dextran Sulfate Sodium (DSS)-induced Colitis.

Diarrhea associated with ulcerative colitis (UC) occurs primarily as a result of reduced Na(+) absorption. Although colonic Na(+) absorption is mediated by both epithelial Na(+) channels (ENaC) and Na-H exchangers (NHE), inhibition of NHE-mediated Na(+) absorption is the primary cause of diarrhea in UC. As there are conflicting observations reported on NHE expression in human UC, the present study was initiated to identify whether NHE isoforms (NHE2 and NHE3) expression is altered and how Na(+) absorption is regulated in DSS-induced inflammation in rat colon, a model that has been used to study UC. Western blot analyses indicate that neither NHE2 nor NHE3 expression is altered in apical membranes of inflamed colon. Na(+) fluxes measured in vitro under voltage clamp conditions in controls demonstrate that both HCO3 (-)-dependent and butyrate-dependent Na(+) absorption are inhibited by S3226 (NHE3-inhibitor), but not by HOE694 (NHE2-inhibitor) in normal animals. In contrast, in DSS-induced inflammation, butyrate-, but not HCO3 (-)-dependent Na(+) absorption is present and is inhibited by HOE694, but not by S3226. These observations indicate that in normal colon NHE3 mediates both HCO3 (-)-dependent and butyrate-dependent Na(+) absorption, whereas DSS-induced inflammation activates NHE2, which mediates butyrate-dependent (but not HCO3 (-)-dependent) Na(+) absorption. In in vivo loop studies HCO3 (-)-Ringer and butyrate-Ringer exhibit similar rates of water absorption in normal rats, whereas in DSS-induced inflammation luminal butyrate-Ringer reversed water secretion observed with HCO3 (-)-Ringer to fluid absorption. Lumen butyrate-Ringer incubation activated NHE3-mediated Na(+) absorption in DSS-induced colitis. These observations suggest that the butyrate activation of NHE2 would be a potential target to control UC-associated diarrhea.

Segmental differences in upregulated apical potassium channels in mammalian colon during potassium adaptation.

Rat proximal and distal colon are net K+ secretory and net K+ absorptive epithelia, respectively. Chronic dietary K+ loading increases net K+ secretion in the proximal colon and transforms net K+ absorption to net K+ secretion in the distal colon, but changes in apical K+ channel expression are unclear. We evaluated expression/activity of apical K+ (BK) channels in surface colonocytes in proximal and distal colon of control and K+-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K+ secretory proximal colon (39% of patches) than in those from K+-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K+ loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K+ loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting (P < 0.001). Thus apical BK channels are normally more abundant in K+ secretory proximal colon than in K+ absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K+ secretory response to dietary K+ loading.

Glucose-dependent insulinotropic polypeptide-mediated signaling pathways enhance apical PepT1 expression in intestinal epithelial cells.

We have shown recently that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1) augments H(+) peptide cotransporter (PepT1)-mediated peptide absorption in murine jejunum. While we observed that inhibiting cAMP production decreased this augmentation of PepT1 activity by GIP, it was unclear whether PKA and/or other regulators of cAMP signaling pathway(s) were involved. This study utilized tritiated glycyl-sarcosine [(3)H-glycyl-sarcosine (Gly-Sar), a relatively nonhydrolyzable dipeptide] uptake to measure PepT1 activity in CDX2-transfected IEC-6 (IEC-6/CDX2) cells, an absorptive intestinal epithelial cell model. Similar to our earlier observations with mouse jejunum, GIP but not GLP-1 augmented Gly-Sar uptake (control vs. +GIP: 154 ± 22 vs. 454 ± 39 pmol/mg protein; P < 0.001) in IEC-6/CDX2 cells. Rp-cAMP (a PKA inhibitor) and wortmannin [phosophoinositide-3-kinase (PI3K) inhibitor] pretreatment completely blocked, whereas neither calphostin C (a potent PKC inhibitor) nor BAPTA (an intracellular Ca(2+) chelator) pretreatment affected the GIP-augmented Gly-Sar uptake in IEC-6/CDX2 cells. The downstream metabolites Epac (control vs. Epac agonist: 287 ± 22 vs. 711 ± 80 pmol/mg protein) and AKT (control vs. AKT inhibitor: 720 ± 50 vs. 75 ± 19 pmol/mg protein) were shown to be involved in GIP-augmented PepT1 activity as well. Western blot analyses revealed that both GIP and Epac agonist pretreatment enhance the PepT1 expression on the apical membranes, which is completely blocked by wortmannin in IEC-6/CDX2 cells. These observations demonstrate that both cAMP and PI3K signaling pathways augment GIP-induced peptide uptake through Epac and AKT-mediated pathways in intestinal epithelial cells, respectively. In addition, these observations also indicate that both Epac and AKT-mediated signaling pathways increase apical membrane expression of PepT1 in intestinal absorptive epithelial cells.

Calcium oxalate urolithiasis in mice lacking anion transporter Slc26a6.

Urolithiasis is one of the most common urologic diseases in industrialized societies. Calcium oxalate is the predominant component in 70-80% of kidney stones, and small changes in urinary oxalate concentration affect the risk of stone formation. SLC26A6 is an anion exchanger expressed on the apical membrane in many epithelial tissues, including kidney and intestine. Among its transport activities, SLC26A6 mediates Cl(-)-oxalate exchange. Here we show that mutant mice lacking Slc26a6 develop a high incidence of calcium oxalate urolithiasis. Slc26a6-null mice have significant hyperoxaluria and elevation in plasma oxalate concentration that is greatly attenuated by dietary oxalate restriction. In vitro flux studies indicated that mice lacking Slc26a6 have a defect in intestinal oxalate secretion resulting in enhanced net absorption of oxalate. We conclude that the anion exchanger SLC26A6 has a major constitutive role in limiting net intestinal absorption of oxalate, thereby preventing hyperoxaluria and calcium oxalate urolithiasis.

Enhanced K(+) secretion in dextran sulfate-induced colitis reflects upregulation of large conductance apical K(+) channels (BK; Kcnma1).

Defective colonic Na(+) and Cl(-) absorption is a feature of active ulcerative colitis (UC), but little is known about changes in colonic K(+) transport. We therefore investigated colonic K(+) transport in a rat model of dextran sulfate-induced colitis. Colitis was induced in rat distal colon using 5% dextran sulfate sodium (DSS). Short-circuit current (Isc, indicating electrogenic ion transport) and (86)Rb (K(+) surrogate) fluxes were measured in colonic mucosa mounted in Ussing chambers under voltage-clamp conditions in the presence of mucosal orthovanadate (a P-type ATPase inhibitor). Serum aldosterone was measured by immunoassay. Control animals exhibited zero net K(+) flux. By contrast, DSS-treated animals exhibited active K(+) secretion, which was inhibited by 98, 76, and 22% by Ba(2+) (nonspecific K(+) channel blocker), iberiotoxin (IbTX; BK channel blocker), and TRAM-34 (IK channel blocker), respectively. Apical BK channel α-subunit mRNA abundance and protein expression, and serum aldosterone levels in DSS-treated animals, were enhanced 6-, 3-, and 6-fold respectively, compared with controls. Increasing intracellular Ca(2+) with carbachol (CCH), or intracellular cAMP with forskolin (FSK), stimulated both active Cl(-) secretion and active K(+) secretion in controls but had no or little effect in DSS-treated animals. In DSS-induced colitis, active K(+) secretion involves upregulation of apical BK channel expression, which may be aldosterone-dependent, whereas Cl(-) secretion is diminished. Since similar ion transport abnormalities occur in patients with UC, diarrhea in this disease may reflect increased colonic K(+) secretion (rather than increased Cl(-) secretion), as well as defective Na(+) and Cl(-) absorption.

Glucose-dependent insulinotropic polypeptide regulates dipeptide absorption in mouse jejunum.

Glucose-dependent insulinotropic polypeptide (GIP) secreted from jejunal mucosal K cells augments insulin secretion and plays a critical role in the pathogenesis of obesity and Type 2 diabetes mellitus. In recent studies, we have shown GIP directly activates Na-glucose cotransporter-1 (SGLT1) and enhances glucose absorption in mouse jejunum. It is not known whether GIP would also regulate other intestinal nutrient absorptive processes. The present study investigated the effect of GIP on proton-peptide cotransporter-1 (PepT1) that mediates di- and tripeptide absorption as well as peptidomimetic drugs. Immunohistochemistry studies localized both GIP receptor (GIPR) and PepT1 proteins on the basolateral and apical membranes of normal mouse jejunum, respectively. Anti-GIPR antibody detected 50-, 55-, 65-, and 70-kDa proteins, whereas anti-PepT1 detected a 70-kDa proteins in mucosal homogenates of mouse jejunum. RT-PCR analyses established the expression of GIPR- and PepT1-specific mRNA in mucosal cells of mouse jejunum. Absorption of Gly-Sar (a nondigestible dipeptide) measured under voltage-clamp conditions revealed that the imposed mucosal H(+) gradient-enhanced Gly-Sar absorption as an evidence for the presence of PepT1-mediated H(+):Gly-Sar cotransport on the apical membranes of mouse jejunum. H(+):Gly-Sar absorption was completely inhibited by cephalexin (a competitive inhibitor of PepT1) and was activated by GIP. The GIP-activated Gly-Sar absorption was completely inhibited by RP-cAMP (a cAMP antagonist). In contrast to GIP, the ileal L cell secreting glucagon-like peptide-1 (GLP-1) did not affect the H(+):Gly-Sar absorption in mouse jejunum. We conclude from these observations that GIP, but not GLP-1, directly activates PepT1 activity by a cAMP-dependent signaling pathway in jejunum.

Inflammatory cytokine TNF-α inhibits Na(+)-glutamine cotransport in intestinal epithelial cells.

Glutamine (Gln), a preferred fuel source for enterocytes, is critical for intestinal epithelial cell integrity and barrier function. Chronic enteritis inhibits apical Na(+)-Gln cotransport. It is not known whether inflammatory cytokines that are secreted during inflammation inhibit Na(+)-Gln cotransport. Thus, this study aimed to examine whether TNF-α would affect apical Na(+)-Gln cotransport in intestinal epithelial cells. In this study, the presence of Na(+)-Gln cotransport was established by measuring Gln uptake in 10 days postconfluent IEC-6 cells grown on transwell plates. Cation, amino acid specificity, and siRNA transfection studies established that Na(+)-Gln cotransport is mediated via B(0)AT1. Immunoblotting and immunofluorescence studies established the apical membrane localization of B(0)AT1 in IEC-6 cells. Tumour necrosis factor α (TNF-α) inhibited Na(+)-Gln cotransport in a concentration- and time-dependent manner with an inhibitory concentration of 1.53 nmol·L(-1). Quantitative real-time PCR and Western blot analyses indicated that TNF-α did not alter B(0)AT1-specific transcripts or protein expression level. Kinetic studies revealed that TNF-α inhibited Na(+)-Gln cotransport by reducing the affinity of the cotransporters for Gln, and this effect was antagonized by genistein. Thus, we conclude that the TNF-α inhibition of Na(+)-Gln cotransport occurs at the post-translational level, and that the IEC-6 cell line is an excellent system to study the role of cytokines in Na(+)-Gln cotransport.

Cyclic AMP-induced K+ secretion occurs independently of Cl- secretion in rat distal colon.

cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.

Aldosterone induces active K⁺ secretion by enhancing mucosal expression of Kcnn4c and Kcnma1 channels in rat distal colon.

Although both Kcnn4c and Kcnma1 channels are present on colonic mucosal membranes, only Kcnma1 has been suggested to mediate K(+) secretion in the colon. Therefore, studies were initiated to investigate the relative roles of Kcnn4c and Kcnma1 in mediating aldosterone (Na-free diet)-induced K(+) secretion. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net (86)Rb(+) (K(+) surrogate) fluxes as well as short circuit currents (I(sc); measure of net ion movement) were measured under voltage clamp condition in rat distal colon. Active K(+) absorption, but not K(+) secretion, is present in normal, while aldosterone induces active K(+) secretion (1.04 ± 0.26 vs. -1.21 ± 0.15 µeq·h(-1)·cm(-2); P < 0.001) in rat distal colon. Mucosal VO(4) (a P-type ATPase inhibitor) inhibited the net K(+) absorption in normal, while it significantly enhanced net K(+) secretion in aldosterone animals. The aldosterone-induced K(+) secretion was inhibited by the mucosal addition of 1) either Ba(2+) (a nonspecific K(+) channel blocker) or charybdotoxin (CTX; a common Kcnn4 and Kcnma1 channel blocker) by 89%; 2) tetraethyl ammonium (TEA) or iberiotoxin (IbTX; a Kcnma1 channel blocker) by 64%; and 3) TRAM-34 (a Kcnn4 channel blocker) by 29%. TRAM-34, but not TEA, in the presence of IbTX further significantly inhibited the aldosterone-induced K(+) secretion. Thus the aldosterone-induced Ba(2+)/CTX-sensitive K(+) secretion consists of IbTX/TEA-sensitive (Kcnma1) and IbTX/TEA-insensitive fractions. TRAM-34 inhibition of the IbTX-insensitive fraction is consistent with the aldosterone-induced K(+) secretion being mediated partially via Kcnn4c. Western and quantitative PCR analyses indicated that aldosterone enhanced both Kcnn4c and Kcnma1α protein expression and mRNA abundance. In vitro exposure of isolated normal colonic mucosa to aldosterone also enhanced Kcnn4c and Kcnma1α mRNA levels, and this was prevented by exposure to actinomycin D (an RNA synthesis inhibitor). These observations indicate that aldosterone induces active K(+) secretion by enhancing mucosal Kcnn4c and Kcnma1 expression at the transcriptional level.

Regulation of Glucose Insulinotropic Peptide and Intestinal Glucose Transporters in the Diet-Induced Obese Mouse.

Our recent studies have shown that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1), augments Na-glucose transporter 1- (SGLT1-) mediated glucose absorption in mouse jejunum. Na-dependent glucose absorption sharply rose and peaked in 3 months of high-fat (i.e., obese) compared to normal (i.e., normal weight) diet fed animals. Previous studies have shown that GIP-augmented SGLT1 and PEPT1 (peptide transporter 1) are regulated by protein kinase A (PKA) signaling in mouse jejunum. Additional studies have indicated that cAMP and PI3 kinase signaling augment PEPT1 through EPAC and AKT activation pathways, respectively, through increased apical PEPT1 trafficking in intestinal epithelial cells. However, little is known about how the signaling glucose transport paradigm is altered over a long period. Early on, increased glucose absorption occurs through SGLT1, but as the obesity and diabetes progress, there is a dramatic shift towards a Na-independent mechanism. Surprisingly, at the peak of glucose absorption during the fifth month of the progression of obesity, the SGLT1 activity was severely depressed, while a Na-independent glucose absorptive process begins to appear. Since glucose transporter 2 (GLUT2) is expressed on the apical membrane of the small intestine in obese patients and animal models of obesity, it was hypothesized to be the new more efficient route. Western blot analyses and biotinylation of the apical membrane revealed that the GIP expression increases in the obese animals and its trafficking to the apical membrane increases with the GIP treatment.