Vazhaikkurichi Rajendran - Academia.edu (original) (raw)
Papers by Vazhaikkurichi Rajendran
Biomaterials, 2018
Lineage specification is an essential process in stem cell fate, tissue homeostasis and developme... more Lineage specification is an essential process in stem cell fate, tissue homeostasis and development. Microenvironmental cues provide direct and selective extrinsic signals to regulate lineage specification of stem cells. Microenvironmental milieu consists of two essential components, one being extracellular matrix (ECM) as the substratum, while the other being cell secreted exosomes and growth factors. ECM of differentiated cells modulates phenotypic expression of stem cells, while their exosomes contain phenotype specific instructive factors (miRNA, RNA and proteins) that control stem cell differentiation. This study demonstrates that osteoblasts-derived (Os-Exo) and adipocytes-derived (Ad-Exo) exosomes contain instructive factors that regulate the lineage specification of human mesenchymal stem cells (hMSCs). Analyses of exosomes revealed the presence of transcription factors in the form of RNA and protein for osteoblasts (RUNX2 and OSX) and adipocytes (C/EBPα and PPARγ). In addit...
The Faseb Journal, Apr 1, 2014
Advances in Comparative and Environmental Physiology, 1993
American Journal of Physiology Gastrointestinal and Liver Physiology, Jul 1, 1985
The characteristics of [14C]glycyl-L-proline transport have been studied using brush-border membr... more The characteristics of [14C]glycyl-L-proline transport have been studied using brush-border membrane vesicles from mouse small intestine in order to investigate the transport of nonhydrolyzable peptide across the brush-border membrane. Uptake curves for the peptide did not exhibit overshoot phenomena and were similar under Na+ or K+ gradient conditions (extravesicular greater than intravesicular). However, L-proline was transported by Na+ gradient-dependent system. Analysis of the incubation medium and the intravesicular contents showed that there was negligible hydrolysis of the peptide. Transport of glycyl-L-proline was saturable, conforming to Michaelis-Menten kinetics with a Km of 30.8 +/- 1.9 mM and a Vmax of 5.96 +/- 0.17 nmol.mg prot-1.0.4 min-1. Uptake of glycyl-L-proline was not significantly inhibited by free amino acids nor by most of the peptides containing D amino acids but was strongly inhibited (up to 64%) by various di- and tripeptides of L amino acids. These results clearly show that glycyl-L-proline was transported by a Na+-independent, carrier-mediated process. Our results suggest that the nonhydrolyzable peptides are transported mostly by carrier-mediated processes in contrast to hydrolyzable peptides.
American Journal of Physiology Gastrointestinal and Liver Physiology, 1999
Na depletion inhibits electroneutral Na-Cl absorption in intact tissues and Na/H exchange in apic... more Na depletion inhibits electroneutral Na-Cl absorption in intact tissues and Na/H exchange in apical membrane vesicles (AMV) of rat distal colon. Two anion (Cl/HCO3 and Cl/OH) exchanges have been identified in AMV from surface cells of rat distal colon. To determine whether Cl/HCO3 and/or Cl/OH exchange is responsible for vectorial Cl movement, this study examined the spatial distribution and the effect of Na depletion on anion-dependent 36Cl uptake by AMV in rat distal colon. These studies demonstrate that HCO3 concentration gradient-driven 36Cl uptake (i.e., Cl/HCO3 exchange) is 1) primarily present in AMV from surface cells and 2) markedly reduced by Na depletion. In contrast, OH concentration gradient-driven 36Cl uptake (i.e., Cl/OH exchange) present in both surface and crypt cells is not affected by Na depletion. In Na-depleted animals HCO3 also stimulates 36Cl via Cl/OH exchange with low affinity. These results suggest that Cl/HCO3 exchange is responsible for vectorial Cl absorption, whereas Cl/OH exchange is involved in cell volume and/or cell pH homeostasis.
American Journal of Physiology Gastrointestinal and Liver Physiology, Mar 1, 1987
A proton-peptide symport mechanism has been postulated for transport of dipeptides in rabbit inte... more A proton-peptide symport mechanism has been postulated for transport of dipeptides in rabbit intestinal and renal brush-border membrane vesicles (BBMV). We have investigated the effects of a transmembrane potential (in mouse) and an inwardly directed proton gradient (in mouse, rabbit, and human) on the transport of glycyl-L-proline in intestinal BBMV. Membrane potential alterations, induced by permeant anions or generated by a K+-diffusion potential in the presence of valinomycin, did not accelerate the uptake of glycyl-L-proline. In contrast, in parallel experiments the uptake of D-glucose, whose cotransport system is electrogenic, was markedly increased by an interior negative membrane potential. Thus the transport of glycyl-L-proline in mouse intestinal BBMV is not electrogenic. Further studies on the effect of a proton gradient (extravesicular pH 5.5; intravesicular pH 7.5) on transport of glycyl-L-proline revealed an absence of stimulation of glycyl-L-proline transport and lower uptake rates in the presence of a proton gradient. The simultaneous presence of an interior negative membrane potential and an inwardly directed proton gradient did not accelerate the transport of glycyl-L-proline. These results demonstrate that the transport of glycyl-L-proline in mouse intestinal BBMV is neither electrogenic nor energized by an inwardly directed proton gradient. Likewise, pH gradients do not stimulate glycyl-L-proline uptake in either rabbit or human BBMV.
American Journal of Physiology Gastrointestinal and Liver Physiology, 1999
A novel Na/H exchange activity that requires Cl was recently identified in the apical membrane of... more A novel Na/H exchange activity that requires Cl was recently identified in the apical membrane of crypt cells of the rat distal colon. This study explores the nature of the coupling of Cl and Na/H exchange. A concentration of 100 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl channel blocker, inhibited the Cl dependence of both proton gradient-driven 22Na uptake from crypt cell apical membrane vesicles and Na-dependent intracellular pH recovery from an acid load during microperfusion of the crypt lumen. Cl-dependent proton gradient-driven 22Na uptake was inhibited by 94% by 500 microM DIDS but only by 1% by 10 microM DIDS, an anion exchange inhibitor at low concentrations but a Cl channel blocker at high concentrations. In addition, a polyclonal antibody to the cystic fibrosis transmembrane conductance regulator (CFTR) inhibited Cl-dependent proton gradient-driven 22Na uptake by 38%. These results indicate that the Cl dependence of Na/H exchange in the colonic crypt apical membrane involves a Cl channel and not a Cl/anion exchange and permit the speculation that this Cl channel activity represents both CFTR and the outward rectifying Cl conductance.
Gastroenterology, 2000
Preterm infants frequently suffer from intestinal disorders probably related to immature digestiv... more Preterm infants frequently suffer from intestinal disorders probably related to immature digestive enzyme function. The aims of this study were to determine the effects of glucagon-like peptide 2 (GLP-2) on brush border enzymes activities and expression in the neonatal pig small intestine and to establish whether these effects were altered by preterm delivery. Piglets were delivered by cesarean section either preterm (n = 15; day 107 of gestation) or at term (n = 15; day 115) and maintained in incubators with a continuous infusion of total parenteral nutrition. GLP-2 (25nmol kgld", i.v.) or saline were administered daily and the piglets were sacrificed after 6 days (n = 7-8 in each group). Maltase activity was significantly increased by GLP-2 regardless of age at delivery whereas lactase activity was not affected . Maltase-glucoamylase and lactase-phloridzin hydrolase mRNA levels measured by reverse transcription PeR and northern blot techniques, respectively, followed the same trend as the activities. Aminopeptidase N and A and dipeptidyl peptidase (DPP) IV activities were significantly increased in the preterm delivered piglets but not in the term-delivered piglets( . We conclude that brush border enzymes are differentially regulated by GLP-2 in the neonate pig intestine. In addition, certain enzymes in the premature delivered neonate (i.e. peptidases) appear more sensitive to postnatal GLP-2 stimulation than the corresponding enzymes in the term-delivered neonate.
American Journal of Physiology Gastrointestinal and Liver Physiology, Feb 1, 1987
This study characterizes the transport of radiolabeled acidic amino acids into brush-border membr... more This study characterizes the transport of radiolabeled acidic amino acids into brush-border membrane vesicles prepared from human jejunum. The uptakes of L-glutamic, L-aspartic, and D-aspartic acids were stimulated by a Na+ gradient (extravesicular greater than intravesicular). Concentrative uptake (resulting in an "overshoot" phenomenon) of these dicarboxylic amino acids occurred when there was an outward K+ gradient (intravesicular greater than extravesicular). In addition, increasing K+ gradients (0-100 mM) resulted in enhanced uptake of L-glutamic acid. This K+ requirement is somewhat specific as Rb+ and Cs+ could enhance uptake to a limited extent, whereas Li+ and choline+ showed no enhancement. The presence of a K+ gradient did not affect the affinity of the carrier system for L-glutamic acid but it did increase the Vmax. The presence of extravesicular anions having differing membrane permeabilities did not alter L-glutamic acid uptake indicating an absence of an effect of membrane potential on the transport process. Finally, the human transport system for L-glutamic acid appears to be specific for acidic amino acids as demonstrated by inhibition studies. Our studies demonstrate a transport system in human jejunum specific for acidic amino acids that is energized by an inward Na+ gradient and an outward K+ gradient.
Gastroenterology, 2000
Univ, New Haven, CT.
American Journal of Physiology Gastrointestinal and Liver Physiology, Mar 1, 2001
A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cell... more A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cells of rat distal colon. The presence of Cl is required for both outward proton gradient-driven Na uptake in apical membrane vesicles (AMV) and Na-dependent intracellular pH recovery from an acid load in the crypt gland. The present study establishes that Cl-dependent outward proton gradient-driven (22)Na uptake 1) is saturated with increasing extravesicular Na concentration with a Michaelis constant (K(m)) for Na of approximately 24.2 mM; 2) is saturated with increasing outward H concentration gradient with a hyperbolic curve and a K(m) for H of approximately 1.5 microM; 3) is inhibited by the Na/H exchange (NHE) inhibitors amiloride, ethylisopropylamiloride, and HOE-694 with an inhibitory constant (K(i)) of approximately 480.2, 1.1, and 9.5 microM, respectively; 4) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor at low concentration and a Cl channel blocker at high dose, and by 5-nitro-2(3-phenylpropylamino)benzoic acid, a Cl channel blocker, with a K(i) of approximately 280.6 and 18.3 microM, respectively; and 5) substantially stimulated Cl-NHE activity by dietary Na depletion, which increases plasma aldosterone and inhibits NHE in surface cell AMV. These properties of Cl-NHE are distinct from those of NHE1, NHE2, and NHE3 isoforms that are present in colonic epithelial cells; thus these results suggest that the colonic crypt cell Cl-NHE is a novel NHE isoform.
World Journal of Gastroenterology Wjg, Sep 1, 2009
Inflammatory bowel disease (IBD) is a common and lifelong disabling gastrointestinal disease. Eme... more Inflammatory bowel disease (IBD) is a common and lifelong disabling gastrointestinal disease. Emerging treatments are being developed to target inflammatory cytokines which initiate and perpetuate the immune response. Adenosine is an important modulator of inflammation and its anti-inflammatory effects have been well established in humans as well as in animal models. High extracellular adenosine suppresses and resolves chronic inflammation in IBD models. High extracellular adenosine levels could be achieved by enhanced adenosine absorption and increased de novo synthesis. Increased adenosine concentration leads to activation of the A2a receptor on the cell surface of immune and epithelial cells that would be a potential therapeutic target for chronic intestinal inflammation.
American Journal of Physiology Cell Physiology, Feb 1, 1999
Gastroenterology, 2012
Background: Decreased Na-Cl absorption and increased K + and Clsecretion contribute to diarrhea i... more Background: Decreased Na-Cl absorption and increased K + and Clsecretion contribute to diarrhea in patients with chronic ulcerative colitis (CUC). In general, diarrhea occurs as a result of inhibition of electroneutral Na-Cl absorption and/or active Clsecretion. A recent study has shown active K + secretion as an alternate mechanism for secretory diarrhea in patients with colonic pseudo-obstruction. Although numerous studies have investigated the role and mechanism of inhibition of Na-Cl absorption, the role and mechanism of K + secretion in CUC has not been investigated. Both large conductance K + (BK) channels (also known as Kcnma1 and K Ca 1.1) and intermediate conductance K + (IK) channels (also known as Kcnn4, and K Ca 3.1) are present on the apical membranes of mammalian colon. Aim: The present study was initiated to identify whether active K + secretion mediated via BK and/or IK channels is induced in CUC. Methods: CUC was induced in rats by giving 5% dextransulfate-sodium (DSS) ad libitum for five alternate weeks. Mucosal to serosal (m-s), serosal to mucosal (s-m) and net 86 Rb (a K + surrogate) fluxes were measured in colonic mucosa mounted under voltage clamp conditions. Gene specific mRNA abundance and protein expression were measured using QRT-PCR and western blot analyses, respectively. Mucosal cytokine levels were measured using a rat cytokine/Chemokine Milliplex Map Kit, while nitric oxide (NO) level was determined using a total NO detection kit. Results: Similar to the earlier observations, net K + absorption is present in normal rat distal colon. In contrast, net K + secretion (normal vs CUC: 1.6 ± 0.2 vs -0.9 ± 0.1 μEq/h.cm 2 ) occurred as a result of both reduced m-s (normal vs CUC: 2.1 ± 0.2 vs 0.4 ± 0.03, μEq/h.cm 2 ) and enhanced s-m (normal vs CUC: 0.5 ± 0.1 vs 1.2 ± 0.1 μEq/h.cm 2 ) fluxes in CUC distal colon. The active K+ secretion was blocked 92% by mucosal iberiotoxin (IbTX, a BK channel blocker) and charibdotoxin (CTX, a common BK and IK channel blocker) but not by TRAM-34 (an IK channel blocker). In the presence of mucosal VO 4 (a P-type ATPase inhibitor), serosal addition of NONOate (a NO donor) induced IbTX-sensitive net K+ secretion in the normal rat distal colon (basal vs NONOate: 0.02 ± 0.04 vs -0.32 ± 0.06 μEq/h.cm 2 ). BK channel specific protein expression and mRNA abundance were increased by 5-fold and 3-fold in CUC, respectively. Mucosal proinflammatory cytokines (TNF-α, IFN-γ, IL-1β and IL-6) and NO levels were significantly increased in CUC. Conclusions: 1) BK, but not IK channel mediated, K + secretion is induced in CUC; 2) cytosolic nitrosylation of tyrosine and/or cysteine residue(s) may induce BK channels; and 3) enhanced BK channel expression may occur at the transcriptional level. Speculation: We speculate that BK channel mediated K + secretion might, in part, provide the driving force for diarrhea in CUC.
The American journal of physiology, 1996
The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly ... more The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly homologous to the recently cloned nongastric isoforms of H-K-adenosinetriphosphatase H-K-ATPase). We have generated polyclonal antibodies against the protein encoded by ATP1AL1 and used them to monitor the protein's expression and distribution in transfection studies. The protein was retained in the endplasmic reticulum when it was transiently expressed alone in COS cells. In COS cells cotransfected with ATP1AL1 plus gastric H-K-ATPase beta-subunit cDNAs (ATP1AL1-gH-K beta), both proteins reached the surface. Stably transfected lines of HEK 293 cells expressing both of these proteins demonstrate a 86Rb+ uptake activity sensitive to both 2-methyl,8-(phenylmeoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH-28080) and ouabain (inhibitory constants of approximately 131 and 42 microM, respectively). Outward proton fluxes were measured in the same cells as the spontaneous intracellular pH (p...
Gastroenterology, 1993
The mechanism of short-chain fatty acid (SCFA) absorption by the colon is not known. The aim of t... more The mechanism of short-chain fatty acid (SCFA) absorption by the colon is not known. The aim of these experiments was to identify the transport mechanisms present in the basolateral membrane to develop an overall model of colonic SCFA absorption. These studies determined the uptake of [14C]butyrate (used as a model SCFA) by basolateral membrane vesicles prepared from rat distal colonic mucosa. Significantly higher [14C]butyrate uptake under an acidic environment (extravesicular pH [pHo] = intravesicular pH [pHi] = 5.5) than that under alkaline environment (pHo = pHi = 7.5) indicates the presence of nonionic diffusion. In the absence of a pH gradient (pHo/pHi = 7.5/7.5), outward gradients of bicarbonate significantly stimulated [14C]butyrate uptake. Additional presence of a pH gradient (pHo/pHi = 6.0/7.5) further enhanced the bicarbonate gradient-stimulated [14C]butyrate uptake that was not inhibited by voltage clamping but was inhibited substantially by an anion exchange inhibitor 4...
Gastroenterology, 1991
In this study, the presence of a bicarbonate gradient-dependent, carrier-mediated anion exchange ... more In this study, the presence of a bicarbonate gradient-dependent, carrier-mediated anion exchange process for butyrate (a representative short-chain fatty acid) uptake in apical membrane vesicles isolated from rat distal colon is described. An outward gradient of both butyrate- and bicarbonate-stimulated [14C]butyrate uptake and resulted in transient accumulation (an "overshoot" phenomenon). Butyrate gradient-stimulated [14C]butyrate uptake was not altered either by an imposed pH gradient or at different pH values. In contrast, bicarbonate gradient-stimulated [14C]butyrate uptake was stimulated severalfold by an additional imposition of an outward pH gradient (pHi = 7.5; pH0 = 6.0). This bicarbonate- and pH gradient-stimulated butyrate uptake was not inhibited by either voltage clamping, with equimolar intravesicular and extravesicular K+ and valinomycin, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an anion-exchange inhibitor. Increasing butyrate c...
Gastroenterology, 1992
To determine the effect of mucosal sodium and mucosal ouabain on active Rb+(K+) absorption, unidi... more To determine the effect of mucosal sodium and mucosal ouabain on active Rb+(K+) absorption, unidirectional and net 86Rb+ fluxes were measured under voltage-clamp conditions in the distal colon of normal and sodium-depleted rats. The role of mucosal sodium (independent of serosal sodium) was evaluated in a model of Rb+(K+) absorption in which serosal ouabain markedly enhanced active Rb+(K+) absorption. In normal rats, mucosal sodium was a competitive inhibitor of Rb+(K+) absorption, and Rb+(K+) absorption consisted of a mucosal sodium-sensitive component and a mucosal sodium-insensitive component. Further, mucosal ouabain almost completely inhibited the mucosal sodium-insensitive component but did not affect the mucosal sodium-sensitive component. In sodium-depleted rats, both mucosal sodium-sensitive and mucosal sodium-insensitive fractions of Rb+(K+) absorption were also identified. Aldosterone markedly stimulated the mucosal sodium-sensitive component (1.68 +/- 0.15 vs. 0.60 +/- 0...
Biomaterials, 2018
Lineage specification is an essential process in stem cell fate, tissue homeostasis and developme... more Lineage specification is an essential process in stem cell fate, tissue homeostasis and development. Microenvironmental cues provide direct and selective extrinsic signals to regulate lineage specification of stem cells. Microenvironmental milieu consists of two essential components, one being extracellular matrix (ECM) as the substratum, while the other being cell secreted exosomes and growth factors. ECM of differentiated cells modulates phenotypic expression of stem cells, while their exosomes contain phenotype specific instructive factors (miRNA, RNA and proteins) that control stem cell differentiation. This study demonstrates that osteoblasts-derived (Os-Exo) and adipocytes-derived (Ad-Exo) exosomes contain instructive factors that regulate the lineage specification of human mesenchymal stem cells (hMSCs). Analyses of exosomes revealed the presence of transcription factors in the form of RNA and protein for osteoblasts (RUNX2 and OSX) and adipocytes (C/EBPα and PPARγ). In addit...
The Faseb Journal, Apr 1, 2014
Advances in Comparative and Environmental Physiology, 1993
American Journal of Physiology Gastrointestinal and Liver Physiology, Jul 1, 1985
The characteristics of [14C]glycyl-L-proline transport have been studied using brush-border membr... more The characteristics of [14C]glycyl-L-proline transport have been studied using brush-border membrane vesicles from mouse small intestine in order to investigate the transport of nonhydrolyzable peptide across the brush-border membrane. Uptake curves for the peptide did not exhibit overshoot phenomena and were similar under Na+ or K+ gradient conditions (extravesicular greater than intravesicular). However, L-proline was transported by Na+ gradient-dependent system. Analysis of the incubation medium and the intravesicular contents showed that there was negligible hydrolysis of the peptide. Transport of glycyl-L-proline was saturable, conforming to Michaelis-Menten kinetics with a Km of 30.8 +/- 1.9 mM and a Vmax of 5.96 +/- 0.17 nmol.mg prot-1.0.4 min-1. Uptake of glycyl-L-proline was not significantly inhibited by free amino acids nor by most of the peptides containing D amino acids but was strongly inhibited (up to 64%) by various di- and tripeptides of L amino acids. These results clearly show that glycyl-L-proline was transported by a Na+-independent, carrier-mediated process. Our results suggest that the nonhydrolyzable peptides are transported mostly by carrier-mediated processes in contrast to hydrolyzable peptides.
American Journal of Physiology Gastrointestinal and Liver Physiology, 1999
Na depletion inhibits electroneutral Na-Cl absorption in intact tissues and Na/H exchange in apic... more Na depletion inhibits electroneutral Na-Cl absorption in intact tissues and Na/H exchange in apical membrane vesicles (AMV) of rat distal colon. Two anion (Cl/HCO3 and Cl/OH) exchanges have been identified in AMV from surface cells of rat distal colon. To determine whether Cl/HCO3 and/or Cl/OH exchange is responsible for vectorial Cl movement, this study examined the spatial distribution and the effect of Na depletion on anion-dependent 36Cl uptake by AMV in rat distal colon. These studies demonstrate that HCO3 concentration gradient-driven 36Cl uptake (i.e., Cl/HCO3 exchange) is 1) primarily present in AMV from surface cells and 2) markedly reduced by Na depletion. In contrast, OH concentration gradient-driven 36Cl uptake (i.e., Cl/OH exchange) present in both surface and crypt cells is not affected by Na depletion. In Na-depleted animals HCO3 also stimulates 36Cl via Cl/OH exchange with low affinity. These results suggest that Cl/HCO3 exchange is responsible for vectorial Cl absorption, whereas Cl/OH exchange is involved in cell volume and/or cell pH homeostasis.
American Journal of Physiology Gastrointestinal and Liver Physiology, Mar 1, 1987
A proton-peptide symport mechanism has been postulated for transport of dipeptides in rabbit inte... more A proton-peptide symport mechanism has been postulated for transport of dipeptides in rabbit intestinal and renal brush-border membrane vesicles (BBMV). We have investigated the effects of a transmembrane potential (in mouse) and an inwardly directed proton gradient (in mouse, rabbit, and human) on the transport of glycyl-L-proline in intestinal BBMV. Membrane potential alterations, induced by permeant anions or generated by a K+-diffusion potential in the presence of valinomycin, did not accelerate the uptake of glycyl-L-proline. In contrast, in parallel experiments the uptake of D-glucose, whose cotransport system is electrogenic, was markedly increased by an interior negative membrane potential. Thus the transport of glycyl-L-proline in mouse intestinal BBMV is not electrogenic. Further studies on the effect of a proton gradient (extravesicular pH 5.5; intravesicular pH 7.5) on transport of glycyl-L-proline revealed an absence of stimulation of glycyl-L-proline transport and lower uptake rates in the presence of a proton gradient. The simultaneous presence of an interior negative membrane potential and an inwardly directed proton gradient did not accelerate the transport of glycyl-L-proline. These results demonstrate that the transport of glycyl-L-proline in mouse intestinal BBMV is neither electrogenic nor energized by an inwardly directed proton gradient. Likewise, pH gradients do not stimulate glycyl-L-proline uptake in either rabbit or human BBMV.
American Journal of Physiology Gastrointestinal and Liver Physiology, 1999
A novel Na/H exchange activity that requires Cl was recently identified in the apical membrane of... more A novel Na/H exchange activity that requires Cl was recently identified in the apical membrane of crypt cells of the rat distal colon. This study explores the nature of the coupling of Cl and Na/H exchange. A concentration of 100 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl channel blocker, inhibited the Cl dependence of both proton gradient-driven 22Na uptake from crypt cell apical membrane vesicles and Na-dependent intracellular pH recovery from an acid load during microperfusion of the crypt lumen. Cl-dependent proton gradient-driven 22Na uptake was inhibited by 94% by 500 microM DIDS but only by 1% by 10 microM DIDS, an anion exchange inhibitor at low concentrations but a Cl channel blocker at high concentrations. In addition, a polyclonal antibody to the cystic fibrosis transmembrane conductance regulator (CFTR) inhibited Cl-dependent proton gradient-driven 22Na uptake by 38%. These results indicate that the Cl dependence of Na/H exchange in the colonic crypt apical membrane involves a Cl channel and not a Cl/anion exchange and permit the speculation that this Cl channel activity represents both CFTR and the outward rectifying Cl conductance.
Gastroenterology, 2000
Preterm infants frequently suffer from intestinal disorders probably related to immature digestiv... more Preterm infants frequently suffer from intestinal disorders probably related to immature digestive enzyme function. The aims of this study were to determine the effects of glucagon-like peptide 2 (GLP-2) on brush border enzymes activities and expression in the neonatal pig small intestine and to establish whether these effects were altered by preterm delivery. Piglets were delivered by cesarean section either preterm (n = 15; day 107 of gestation) or at term (n = 15; day 115) and maintained in incubators with a continuous infusion of total parenteral nutrition. GLP-2 (25nmol kgld", i.v.) or saline were administered daily and the piglets were sacrificed after 6 days (n = 7-8 in each group). Maltase activity was significantly increased by GLP-2 regardless of age at delivery whereas lactase activity was not affected . Maltase-glucoamylase and lactase-phloridzin hydrolase mRNA levels measured by reverse transcription PeR and northern blot techniques, respectively, followed the same trend as the activities. Aminopeptidase N and A and dipeptidyl peptidase (DPP) IV activities were significantly increased in the preterm delivered piglets but not in the term-delivered piglets( . We conclude that brush border enzymes are differentially regulated by GLP-2 in the neonate pig intestine. In addition, certain enzymes in the premature delivered neonate (i.e. peptidases) appear more sensitive to postnatal GLP-2 stimulation than the corresponding enzymes in the term-delivered neonate.
American Journal of Physiology Gastrointestinal and Liver Physiology, Feb 1, 1987
This study characterizes the transport of radiolabeled acidic amino acids into brush-border membr... more This study characterizes the transport of radiolabeled acidic amino acids into brush-border membrane vesicles prepared from human jejunum. The uptakes of L-glutamic, L-aspartic, and D-aspartic acids were stimulated by a Na+ gradient (extravesicular greater than intravesicular). Concentrative uptake (resulting in an "overshoot" phenomenon) of these dicarboxylic amino acids occurred when there was an outward K+ gradient (intravesicular greater than extravesicular). In addition, increasing K+ gradients (0-100 mM) resulted in enhanced uptake of L-glutamic acid. This K+ requirement is somewhat specific as Rb+ and Cs+ could enhance uptake to a limited extent, whereas Li+ and choline+ showed no enhancement. The presence of a K+ gradient did not affect the affinity of the carrier system for L-glutamic acid but it did increase the Vmax. The presence of extravesicular anions having differing membrane permeabilities did not alter L-glutamic acid uptake indicating an absence of an effect of membrane potential on the transport process. Finally, the human transport system for L-glutamic acid appears to be specific for acidic amino acids as demonstrated by inhibition studies. Our studies demonstrate a transport system in human jejunum specific for acidic amino acids that is energized by an inward Na+ gradient and an outward K+ gradient.
Gastroenterology, 2000
Univ, New Haven, CT.
American Journal of Physiology Gastrointestinal and Liver Physiology, Mar 1, 2001
A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cell... more A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cells of rat distal colon. The presence of Cl is required for both outward proton gradient-driven Na uptake in apical membrane vesicles (AMV) and Na-dependent intracellular pH recovery from an acid load in the crypt gland. The present study establishes that Cl-dependent outward proton gradient-driven (22)Na uptake 1) is saturated with increasing extravesicular Na concentration with a Michaelis constant (K(m)) for Na of approximately 24.2 mM; 2) is saturated with increasing outward H concentration gradient with a hyperbolic curve and a K(m) for H of approximately 1.5 microM; 3) is inhibited by the Na/H exchange (NHE) inhibitors amiloride, ethylisopropylamiloride, and HOE-694 with an inhibitory constant (K(i)) of approximately 480.2, 1.1, and 9.5 microM, respectively; 4) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor at low concentration and a Cl channel blocker at high dose, and by 5-nitro-2(3-phenylpropylamino)benzoic acid, a Cl channel blocker, with a K(i) of approximately 280.6 and 18.3 microM, respectively; and 5) substantially stimulated Cl-NHE activity by dietary Na depletion, which increases plasma aldosterone and inhibits NHE in surface cell AMV. These properties of Cl-NHE are distinct from those of NHE1, NHE2, and NHE3 isoforms that are present in colonic epithelial cells; thus these results suggest that the colonic crypt cell Cl-NHE is a novel NHE isoform.
World Journal of Gastroenterology Wjg, Sep 1, 2009
Inflammatory bowel disease (IBD) is a common and lifelong disabling gastrointestinal disease. Eme... more Inflammatory bowel disease (IBD) is a common and lifelong disabling gastrointestinal disease. Emerging treatments are being developed to target inflammatory cytokines which initiate and perpetuate the immune response. Adenosine is an important modulator of inflammation and its anti-inflammatory effects have been well established in humans as well as in animal models. High extracellular adenosine suppresses and resolves chronic inflammation in IBD models. High extracellular adenosine levels could be achieved by enhanced adenosine absorption and increased de novo synthesis. Increased adenosine concentration leads to activation of the A2a receptor on the cell surface of immune and epithelial cells that would be a potential therapeutic target for chronic intestinal inflammation.
American Journal of Physiology Cell Physiology, Feb 1, 1999
Gastroenterology, 2012
Background: Decreased Na-Cl absorption and increased K + and Clsecretion contribute to diarrhea i... more Background: Decreased Na-Cl absorption and increased K + and Clsecretion contribute to diarrhea in patients with chronic ulcerative colitis (CUC). In general, diarrhea occurs as a result of inhibition of electroneutral Na-Cl absorption and/or active Clsecretion. A recent study has shown active K + secretion as an alternate mechanism for secretory diarrhea in patients with colonic pseudo-obstruction. Although numerous studies have investigated the role and mechanism of inhibition of Na-Cl absorption, the role and mechanism of K + secretion in CUC has not been investigated. Both large conductance K + (BK) channels (also known as Kcnma1 and K Ca 1.1) and intermediate conductance K + (IK) channels (also known as Kcnn4, and K Ca 3.1) are present on the apical membranes of mammalian colon. Aim: The present study was initiated to identify whether active K + secretion mediated via BK and/or IK channels is induced in CUC. Methods: CUC was induced in rats by giving 5% dextransulfate-sodium (DSS) ad libitum for five alternate weeks. Mucosal to serosal (m-s), serosal to mucosal (s-m) and net 86 Rb (a K + surrogate) fluxes were measured in colonic mucosa mounted under voltage clamp conditions. Gene specific mRNA abundance and protein expression were measured using QRT-PCR and western blot analyses, respectively. Mucosal cytokine levels were measured using a rat cytokine/Chemokine Milliplex Map Kit, while nitric oxide (NO) level was determined using a total NO detection kit. Results: Similar to the earlier observations, net K + absorption is present in normal rat distal colon. In contrast, net K + secretion (normal vs CUC: 1.6 ± 0.2 vs -0.9 ± 0.1 μEq/h.cm 2 ) occurred as a result of both reduced m-s (normal vs CUC: 2.1 ± 0.2 vs 0.4 ± 0.03, μEq/h.cm 2 ) and enhanced s-m (normal vs CUC: 0.5 ± 0.1 vs 1.2 ± 0.1 μEq/h.cm 2 ) fluxes in CUC distal colon. The active K+ secretion was blocked 92% by mucosal iberiotoxin (IbTX, a BK channel blocker) and charibdotoxin (CTX, a common BK and IK channel blocker) but not by TRAM-34 (an IK channel blocker). In the presence of mucosal VO 4 (a P-type ATPase inhibitor), serosal addition of NONOate (a NO donor) induced IbTX-sensitive net K+ secretion in the normal rat distal colon (basal vs NONOate: 0.02 ± 0.04 vs -0.32 ± 0.06 μEq/h.cm 2 ). BK channel specific protein expression and mRNA abundance were increased by 5-fold and 3-fold in CUC, respectively. Mucosal proinflammatory cytokines (TNF-α, IFN-γ, IL-1β and IL-6) and NO levels were significantly increased in CUC. Conclusions: 1) BK, but not IK channel mediated, K + secretion is induced in CUC; 2) cytosolic nitrosylation of tyrosine and/or cysteine residue(s) may induce BK channels; and 3) enhanced BK channel expression may occur at the transcriptional level. Speculation: We speculate that BK channel mediated K + secretion might, in part, provide the driving force for diarrhea in CUC.
The American journal of physiology, 1996
The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly ... more The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly homologous to the recently cloned nongastric isoforms of H-K-adenosinetriphosphatase H-K-ATPase). We have generated polyclonal antibodies against the protein encoded by ATP1AL1 and used them to monitor the protein's expression and distribution in transfection studies. The protein was retained in the endplasmic reticulum when it was transiently expressed alone in COS cells. In COS cells cotransfected with ATP1AL1 plus gastric H-K-ATPase beta-subunit cDNAs (ATP1AL1-gH-K beta), both proteins reached the surface. Stably transfected lines of HEK 293 cells expressing both of these proteins demonstrate a 86Rb+ uptake activity sensitive to both 2-methyl,8-(phenylmeoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH-28080) and ouabain (inhibitory constants of approximately 131 and 42 microM, respectively). Outward proton fluxes were measured in the same cells as the spontaneous intracellular pH (p...
Gastroenterology, 1993
The mechanism of short-chain fatty acid (SCFA) absorption by the colon is not known. The aim of t... more The mechanism of short-chain fatty acid (SCFA) absorption by the colon is not known. The aim of these experiments was to identify the transport mechanisms present in the basolateral membrane to develop an overall model of colonic SCFA absorption. These studies determined the uptake of [14C]butyrate (used as a model SCFA) by basolateral membrane vesicles prepared from rat distal colonic mucosa. Significantly higher [14C]butyrate uptake under an acidic environment (extravesicular pH [pHo] = intravesicular pH [pHi] = 5.5) than that under alkaline environment (pHo = pHi = 7.5) indicates the presence of nonionic diffusion. In the absence of a pH gradient (pHo/pHi = 7.5/7.5), outward gradients of bicarbonate significantly stimulated [14C]butyrate uptake. Additional presence of a pH gradient (pHo/pHi = 6.0/7.5) further enhanced the bicarbonate gradient-stimulated [14C]butyrate uptake that was not inhibited by voltage clamping but was inhibited substantially by an anion exchange inhibitor 4...
Gastroenterology, 1991
In this study, the presence of a bicarbonate gradient-dependent, carrier-mediated anion exchange ... more In this study, the presence of a bicarbonate gradient-dependent, carrier-mediated anion exchange process for butyrate (a representative short-chain fatty acid) uptake in apical membrane vesicles isolated from rat distal colon is described. An outward gradient of both butyrate- and bicarbonate-stimulated [14C]butyrate uptake and resulted in transient accumulation (an "overshoot" phenomenon). Butyrate gradient-stimulated [14C]butyrate uptake was not altered either by an imposed pH gradient or at different pH values. In contrast, bicarbonate gradient-stimulated [14C]butyrate uptake was stimulated severalfold by an additional imposition of an outward pH gradient (pHi = 7.5; pH0 = 6.0). This bicarbonate- and pH gradient-stimulated butyrate uptake was not inhibited by either voltage clamping, with equimolar intravesicular and extravesicular K+ and valinomycin, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an anion-exchange inhibitor. Increasing butyrate c...
Gastroenterology, 1992
To determine the effect of mucosal sodium and mucosal ouabain on active Rb+(K+) absorption, unidi... more To determine the effect of mucosal sodium and mucosal ouabain on active Rb+(K+) absorption, unidirectional and net 86Rb+ fluxes were measured under voltage-clamp conditions in the distal colon of normal and sodium-depleted rats. The role of mucosal sodium (independent of serosal sodium) was evaluated in a model of Rb+(K+) absorption in which serosal ouabain markedly enhanced active Rb+(K+) absorption. In normal rats, mucosal sodium was a competitive inhibitor of Rb+(K+) absorption, and Rb+(K+) absorption consisted of a mucosal sodium-sensitive component and a mucosal sodium-insensitive component. Further, mucosal ouabain almost completely inhibited the mucosal sodium-insensitive component but did not affect the mucosal sodium-sensitive component. In sodium-depleted rats, both mucosal sodium-sensitive and mucosal sodium-insensitive fractions of Rb+(K+) absorption were also identified. Aldosterone markedly stimulated the mucosal sodium-sensitive component (1.68 +/- 0.15 vs. 0.60 +/- 0...