Search

In polarized intestinal epithelial cells, DRA is an apical Cl-/HCO3 - exchanger that is part of neutral NaCl absorption under baseline conditions but in cAMP driven diarrheas it is stimulated and contributes to increased anion secretion. To further understand regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and ATP. FSK and ATP stimulated DRA in a concentration dependent manner, ATP acting via P2Y1 receptors. FSK at 1µM and ATP at 0.25 µM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP increased Ca2+ in a concentration dependent manner, while FSK (1 µM), that by itself did not significantly alter Ca2+, followed by 0.25 µM ATP produced a large increase in Ca2+ that was ~equal to the elevation caused by 1 µM ATP. BAPTA-AM pretreatment prevented the ATP and FSK/ATP synergistic increased DRA activity and the increase in Ca2+i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. Conclusion: In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca2+) synergistically increased Ca2+i and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, while separation of DRA from NHE3 contributes to reduced NaCl absorption. more...

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action—they form binary complexes with FKBP through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mTOR and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles that are named rapafucins using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin., Graphical abstract more...

There is increasing evidence that the study of normal human enteroids duplicates many known aspects of human intestinal physiology. However, this epithelial cell-only model lacks the many nonepithelial intestinal cells present in the gastrointestinal tract and exposure to the mechanical forces to which the intestine is exposed. We tested the hypothesis that physical shear forces produced by luminal and blood flow would provide an intestinal model more closely resembling normal human jejunum. Jejunal enteroid monolayers were studied in the Emulate, Inc. Intestine-Chip under conditions of constant luminal and basolateral flow that was designed to mimic normal intestinal fluid flow, with human umbilical vein endothelial cells (HUVECs) on the basolateral surface and with Wnt3A, R-spondin, and Noggin only on the luminal surface. The jejunal enteroids formed monolayers that remained confluent for 6–8 days, began differentiating at least as early as day 2 post plating, and demonstrated continuing differentiation over the entire time of the study, as shown by quantitative real-time polymerase chain reaction and Western blot analysis. Differentiation impacted villus genes and proteins differently with early expression of regenerating family member 1α (REG1A), early reduction to a low but constant level of expression of Na(+)-K(+)-2Cl(−) cotransporter 1 (NKCC1), and increasing expression of sucrase-isomaltase (SI) and downregulated in adenoma (DRA). These results were consistent with continual differentiation, as was shown to occur in mouse villus enterocytes. Compared with differentiated enteroid monolayers grown on Transwell inserts, enteroids exposed to flow were more differentiated but exhibited increased apoptosis and reduced carbohydrate metabolism, as shown by proteomic analysis. This study of human jejunal enteroids-on-chip suggests that luminal and basolateral flow produce a model of continual differentiation over time and NaCl absorption that mimics normal intestine and should provide new insights in intestinal physiology. NEW & NOTEWORTHY This study showed that polarized enteroid models in which there is no basolateral Wnt3a, are differentiated, regardless of the Wnt3a status of the apical media. The study supports the concept that in the human intestine villus differentiation is not an all or none phenomenon, demonstrating that at different days after lack of basolateral Wnt exposure, clusters of genes and proteins exist geographically along the villus with different domains having different functions. more...

Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion. more...

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler’s diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+concentration [Ca2+]i). The ST elevation of [Ca2+]iwas from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms. more...

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler’s diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca(2+) concentration [Ca(2+)](i)). The ST elevation of [Ca(2+)](i) was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms. more...

Calcium-activated chloride channels (CaCCs) mediate numerous physiological functions and are best known for the transport of electrolytes and water in epithelia. In the intestine, CaCC currents are considered necessary for the secretion of fluid to protect the intestinal epithelium. Although genetic ablation of ANO1/TMEM16A, a gene encoding a CaCC, reduces the carbachol-induced secretion of intestinal fluid, its mechanism of action is still unknown. Here, we confirm that ANO1 is essential for the secretion of intestinal fluid. Carbachol-induced transepithelial currents were reduced in the proximal colon of Ano1-deficient mice. Surprisingly, cholera toxin-induced and cAMP-induced fluid secretion, believed to be mediated by CFTR, were also significantly reduced in the intestine of Ano1-deficient mice. ANO1 is largely expressed in the apical membranes of intestines, as predicted for CaCCs. The Ano1-deficient colons became edematous under basal conditions and had a greater susceptibility to dextran sodium sulfate-induced colitis. However, Ano1 depletion failed to affect tumor development in a model of colorectal cancer. We thus conclude that ANO1 is necessary for cAMP- and carbachol-induced Cl− secretion in the intestine, which is essential for the protection of the intestinal epithelium from colitis., Colitis: Intestinal membrane protein implicated in defective fluid secretion An ion channel, a membrane protein allowing ion transport, that controls the flow of chloride is needed for proper secretion of protective fluids in the intestine. Uhtaek Oh from the Korea Institute of Science & Technology in Seoul, South Korea, and colleagues showed that cells lining the intestinal surface express a calcium-activated chloride channel called anoctamin-1 (ANO1) that regulates fluid secretion in the gut. Compared to control animals, ANO1-deficient mice released less fluid into their intestines following exposure to a diarrhea-inducing toxin or to a chloride transport–stimulating signaling molecule. This fluid secretion was previously thought to be mediated via a different ion channel. The ANO1-deficient mice accumulated fluid within colonic tissues, which increased their susceptibility to colitis. The findings point to ANO1 activation as a potential therapeutic strategy for treating colitis. more...

CK2 regulates NHE3 by phosphorylating a single C-terminal amino acid, which, when mutated, reduces basal NHE3 activity and its acute stimulation and inhibition. It also is necessary for binding of proteins throughout the C-terminus, which means that it determines the C-terminal structure., Casein kinase 2 (CK2) binds to the NHE3 C-terminus and constitutively phosphorylates a downstream site (S719) that accounts for 40% of basal NHE3 activity. The role of CK2 in regulation of NHE3 activity in polarized Caco-2/bbe cells was further examined by mutation of NHE3-S719 to A (not phosphorylated) or D (phosphomimetic). NHE3-S719A but not -S719D had multiple changes in NHE3 activity: 1) reduced basal NHE3 activity—specifically, inhibition of the PI3K/AKT-dependent component; 2) reduced acute stimulation of NHE3 activity by LPA/LPA5R stimulation; and 3) reduced acute inhibition of NHE3 activity—specifically, elevated Ca2+ related (carbachol/Ca2+ ionophore), but there was normal inhibition by forskolin and hyperosmolarity. The S719A mutant had reduced NHE3 complex size, reduced expression in lipid rafts, increased BB mobile fraction, and reduced binding to multiple proteins that bind throughout the NHE3 intracellular C-terminus, including calcineurin homologous protein, the NHERF family and SNX27 (related PDZ domains). These studies show that phosphorylation of the NHE3 at a single amino acid in the distal part of the C-terminus affects multiple aspects of NHE3 complex formation and changes the NHE3 lipid raft distribution, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity. more...

Equilibrative nucleoside transporters (ENTs) play a crucial role in the transport of nucleoside and nucleoside analogues, which are important for nucleotide synthesis and chemotherapy. In addition, ENTs regulate extracellular adenosine levels in the vicinity of its receptors and hence influence adenosine-related functions. The clinical applications of ENT inhibitors in the treatment of cardiovascular diseases and cancer therapy have been explored in numerous studies. However, all ENT inhibitors to date are selective for ENT1 but not ENT2. In the present study, we investigated the novel compound 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) as an inhibitor of ENT1 and ENT2. Nucleoside transporter-deficient PK15NTD cells stably expressing ENT1 and ENT2 showed that FPMINT inhibited [3H]uridine and [3H]adenosine transport through both ENT1 and ENT2 in a concentration-dependent manner. The IC50 value of FPMINT for ENT2 was 5-10-fold less than for ENT1, and FPMINT could not be displaced with excess washing. Kinetic studies revealed that FPMINT reduced Vmax of [3H]uridine transport in ENT1 and ENT2 without affecting KM. Therefore, we conclude that FPMINT inhibits ENTs in an irreversible and non-competitive manner. Although already selective for ENT2 over ENT1, further modification of the chemical structure of FPMINT may lead to even better ENT2-selective inhibitors of potential clinical, physiological and pharmacological importance. more...

Background & AimsDRA (SLC26A3) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute cAMP-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates or does not affect its activity.MethodsThis study reevaluated cAMP regulation of DRA using new “tools” including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on non-specific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2.ResultsDRA is an apical protein in human proximal colon, differentiated colonoid monolayers and Caco-2 cells. It is glycosylated and appears as two bands. cAMp(forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with IC50 of 0.5 μmol/L and 0.2 µmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked CFTR. When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172.ConclusionsDRA is acutely stimulated by cAMP by a process that is CFTR-dependent but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity. more...

Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE. more...

Background: Vasopressin induced trafficking of aquaporin-2 (AQP2) containing vesicles has been studied in kidney cell lines using conventional fluorescent proteins as tags. However, trafficking of fluorescent tagged AQP2, which resembles the vectorial translocation of native AQP2 from cytoplasm to apical membrane has not been demonstrated at real time. Using a photoconvertible fluorescent protein tag on AQP2 might allow the simultaneous tracking of two separate populations of AQP2 vesicle after subcellular local photoconversion. Methods: A spacer was used to link a photoconvertible fluorescent protein (mEos2) to the amino-terminus of AQP2. The DNA constructs were expressed in mpkCCD cells. The trafficking of chimeric protein was visualized with high speed confocal microscopy in 4 dimensions. Results: Chimeric AQP2 expressed in mpkCCD cell conferred osmotic water permeability to the cells. Subcellular photoconversion with a 405 nm laser pulse converted green chimeras to red chimeras locally. Forskolin stimulation triggered chimeric AQP2 to translocate from acidic organelles to apical plasma membrane. By serendipity, the rate of apical accumulation was found to increase when mEos2 was tagged to the carboxyl-terminus in at least one of the AQP2 molecules within the tetramer. Conclusion: Functional photoconvertible chimeric AQP2 was successfully expressed in mpkCCD cells, in which forskolin induced apical trafficking and accumulation of chimeric AQP2. The proof-of-concept to monitor two populations of AQP2 vesicle simultaneously was demonstrated. more...

Background & Aims: Human enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation. Methods: Human duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s). Results: Differentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids. Conclusions: Human enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion. Keywords: Chloride Secretion, Bicarbonate Secretion, DRA, Ion Transport more...

Basal activity of the BB Na+/H+ exchanger NHE3 requires multiprotein complexes that form on its C terminus. One complex stimulates basal NHE3 activity and contains ezrin and phosphoinositides as major components; how it stimulates NHE3 activity is not known. This study tested the hypothesis that ezrin dynamically associates with this complex, which sets ezrin binding. NHE3 activity was reduced by an Akti. This effect was eliminated if ezrin binding to NHE3 was inhibited by a point mutant. Recombinant AKT phosphorylated NHE3 C terminus in the domain ezrin directly binds. This domain (amino acids 475–589) is predicted to be α-helical and contains a conserved cluster of three serines (Ser515, Ser522, and Ser526). Point mutations of two of these (S515A, S515D, or S526A) reduced basal NHE3 activity and surface expression and had no Akti inhibition. S526D had NHE3 activity equal to wild type with normal Akti inhibition. Ezrin binding to NHE3 was regulated by Akt, being eliminated by Akti. NHE3-S515A and -S526D did not bind ezrin; NHE3-S515D had reduced ezrin binding; NHE3-S526D bound ezrin normally. NHE3-Ser526 is predicted to be a GSK-3 kinase phosphorylation site. A GSK-3 inhibitor reduced basal NHE3 activity as well as ezrin-NHE3 binding, and this effect was eliminated in NHE3-S526A and -S526D mutants. The conclusions were: 1) NHE3 basal activity is regulated by a signaling complex that is controlled by sequential effects of two kinases, Akt and GSK-3, which act on a Ser cluster in the same NHE3 C-terminal domain that binds ezrin; and 2) these kinases regulate the dynamic association of ezrin with NHE3 to affect basal NHE3 activity. more...

Na+/H+exchanger NHE3 mediates the majority of intestinal and renal electroneutral sodium absorption. Dysfunction of NHE3 is associated with a variety of diarrheal diseases. We previously reported that the NHE3 gene ( SLC9A3) has more than 400 single-nucleotide polymorphisms (SNPs) but few nonsynonymous polymorphisms. Among the latter, one polymorphism (rs2247114-G>A), which causes a substitution from arginine to cysteine at amino acid position 799 (p.R799C), is common in Asian populations. To improve our understanding of the population distribution and potential clinical significance of the NHE3-799C variant, we investigated the frequency of this polymorphism in different ethnic groups using bioinformatics analyses and in a cohort of Japanese patients with cardiovascular or renal disease. We also characterized the function of human NHE3-799C and its sensitivity to regulatory ligands in an in vitro model. NHE3-799C had an allele frequency of 29.5–57.6% in Asian populations, 11.1–23.6% in European populations, and 10.2–22.7% in African populations. PS120/FLAG-NHERF2 fibroblasts stably expressing NHE3-799C had lower total protein expression but a higher percentage of surface expression than those expressing NHE3-799R. NHE3-799C had similar basal activity to NHE3-799R and was similarly stimulated or inhibited, by serum or forskolin, respectively. Tenapanor, a small-molecule NHE3 inhibitor, dose-dependently inhibited NHE3-799R and NHE3-799C activities. The IC50values of tenapanor for NHE3-799C and NHE3-799R were significantly different, but both were in the nanomolar range. These results suggest that NHE3-799C is a common variant enriched in Asian populations, is not associated with compromised function or abnormal regulation, and is unlikely to contribute to clinical disease.NEW & NOTEWORTHY This study reports results on the functional significance of human NHE3-799C under basal conditions and in response to regulatory ligands, including a novel NHE3 inhibitor called tenapanor. We demonstrate that NHE3-799C is a common variant of NHE3 that is enriched in Asian populations; however, in contrast to our previous studies using rabbit NHE3, its presence seems to have limited clinical significance in humans and is not associated with compromised function or abnormal transport regulation. more...

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression. more...

An increasing amount of data showing the beneficial use of zinc (Zn) in treating diarrhea continues to emerge from epidemiological and clinical trials. However, without a thorough understanding of physiological mechanisms of Zn, it does not support policy recommendation to advocate the therapeutic use of Zn. Our data demonstrate that Zn is a potential antidiarrheal agent that provides substantial benefit by stimulating sodium absorption and inhibiting chloride secretion in intestinal epithelial cells. Thus, inclusion of Zn in oral rehydration solution (ORS) has the potential to markedly augment the effectiveness of ORS in the treatment of diarrhea. more...

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with Kmvalues of 2.5 ± 0.7 μM for [3H]adenine, 6.4 ± 0.5 μM for [3H]guanine, 1.1 ± 0.1 mM for [3H]guanosine, and 4.2 ± 0.5 mM [3H]adenosine. The uptake of [3H]guanine (0.05 μM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5–1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine >>> thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2′ and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [3H]guanine: 2-chloro-2′-deoxyadenosine > 2-chloroadenosine > 2′3′-dideoxyadenosine = 2′-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2′-deoxyadenosine and 2-chloroadenosine inhibited [3H]guanine uptake with IC50values of 68 ± 5 and 99 ± 10 μM, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-β-d-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 (IC50of ∼1 μM), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells. more...

NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na+-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in V-max of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)(i) of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number. more...

The etiology of the atherosclerosis that occurs in diabetes mellitus is unclear. Adenosine has been shown to inhibit growth of rat aortic smooth muscle cells. Nucleoside transporters play an integral role in adenosine function by regulating adenosine levels in the vicinity of adenosine receptors. Therefore, we studied the effect of 25 mM d-glucose, which mimics hyperglycemia of diabetes, on adenosine transport in cultured human aortic smooth muscle cells (HASMCs). Although RT-PCR demonstrated the presence of equilibrative nucleoside transporter-1 (ENT-1) and ENT-2 mRNA, functional studies revealed that adenosine transport in HASMCs was predominantly mediated by ENT-1 and inhibited by nitrobenzylmercaptopurine riboside (NBMPR, IC50 = 0.69 ± 0.05 nM). Adenosine transport in HASMCs was increased by >30% after treatment for 48 h with 25 mM d-glucose, but not with equimolar d-mannitol and l-glucose. Kinetic studies showed that d-glucose increased Vmax of adenosine transport without affecting Km. Similarly, d-glucose increased Bmax of high-affinity [3H]NBMPR binding, while the dissociation constant ( Kd) was not changed. Consistent with these observations, 25 mM d-glucose increased mRNA and protein expression of ENT-1. Treatment of serum-starved cells with the selective inhibitors of MAPK/ERK, PD-98059 (40 μM) and U-0126 (10 μM), abolished the effect of d-glucose on ENT-1. We conclude that d-glucose upregulates the protein and message expression and functional activity of ENT-1 in HASMCs, possibly via MAPK/ERK-dependent pathways. Pathologically, the increase in ENT-1 activity in diabetes may affect the availability of adenosine in the vicinity of adenosine receptors and, thus, alter vascular functions in diabetes. more...

Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca 2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca 2+ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca 2+ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca 2+ stores, caused dramatic increase in cation entry measured by Mn 2+ quenching of fura-2 and by Ca 2+ transients. OAG-induced responses were ≈700-fold more resistant to La 3+ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca 2+ or inhibition of store-operated Ca 2+ entry with La 3+ and SK&F-96365 reversed the elevated basal [Ca 2+ ] i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension. more...

Protein glycosylation is important for nucleoside transport, and this has been demonstrated for the human equilibrative nucleoside transporter-1 (hENT1). It is not known whether glycosylation affects the functions of hENT2 or where hENT2 is glycosylated. We address these questions using N-glycosylation mutants (N48D, N57D, and N48/57D) and demonstrate that hENT2 is glycosylated at Asn48 and Asn57. Our results show that although the apparent affinities for [ 3 H ]uridine and [ 3 H ]cytidine of the mutants were indistinguishable from those of the wild-type protein, N-glycosylation was required for efficient targeting of hENT2 to the plasma membrane. All mutants had a two- to threefold increase in IC50 for dipyridamole. N57D and N48/57D, but not N48D, also had a twofold increase in IC50 for NBMPR. We conclude that the relative insensitivity of hENT2 to inhibitors is primarily due to its primary structure and not to glycosylation. Glycosylation modulates hENT1 function, but is not required for hENT2. more...

The luminal fluid microenvironment of the uterus is important for sperm capacitation and embryo development. In an attempt to understand the possible role of Na(+)/H(+) exchangers (NHEs) in uterine function, the mRNAs of different NHE isoforms as well as their subcellular localization (apical versus basolateral) and functional activity were investigated in mouse endometrial epithelial cells using reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and intracellular pH (pH(i)) measurement techniques. The presence of NHE1, NHE2, and NHE4, but not NHE3 mRNAs were revealed by RT-PCR. Immunostaining showed that NHE1, NHE2, and NHE4 were present in both apical and basolateral membranes. The pH(i) recovery from intracellular acidification was Na(+)-dependent; however, the rate of pH(i) recovery depending on basolateral Na(+) was 12.4 times faster than that depending on apical Na(+). The Na(+)-dependent rate of pH(i) recovery was also inhibited by amiloride, indicating H(+) extrusion through NHEs; however, the amiloride sensitivity of the apical membrane was less than that of the basolateral membrane, suggesting the involvement of different types of NHEs in the two membranes. The results indicate that the basolaterally located NHE1, NHE2, and NHE4, in addition to participating in the homeostatic control of intracellular pH, may play a role in H(+) extrusion in order to achieve transepithelial HCO(3)(-) secretion. The apically located NHEs may be involved in mediating Na(+) absorption as alternatives of or complementary to epithelial Na(+) channels. more...

The effect of nucleoside on Na+ reabsorption via Na+/nucleoside cotransporter in cultured rat epididymal epithelia was studied by short-circuit current (Isc) technique. Guanosine added apically stimulated Isc in a dose-dependent manner, with a median effective concentration (EC50) of 7 +/- 2 microM (mean +/- SEM). Removal of Na+ from the apical bathing solution or pretreatment with a nonspecific Na+/nucleoside cotransporter inhibitor, phloridzin, completely blocked the Isc response to guanosine. Moreover, the guanosine response was abolished by pretreatment of the tissue with ouabain, a Na+/K+-ATPase inhibitor, suggesting the involvement of Na+/nucleoside cotransporter on the apical side and Na+/K+-ATPase on the basolateral side in Na+ reabsorption. In contrast, the Isc response to guanosine was not affected after desensitization of purinoceptors by ATP. Addition of the Na+/K+/2Cl- symport inhibitor bumetanide to the basolateral side or the nonspecific Cl- channel blocker diphenylamine-2-carboxylate to the apical side showed no effect on the Isc response to guanosine, excluding stimulation of Cl- secretion by guanosine as the cause of the guanosine-induced Isc. The Isc response to purine nucleoside (guanosine and inosine) was much higher than that to pyrimidine nucleoside (thymidine and cytidine). Consistent with substrate specificity, results of reverse transcription-polymerase chain reaction revealed mRNA for concentrative nucleoside transporter (CNT2), which is a purine nucleoside-selective Na+/nucleoside cotransporter in the epididymis, but not for CNT1. It is suggested that the Na+/nucleoside cotransporter (i.e., CNT2) may be one of the elements involved in Na+ and fluid reabsorption in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa. more...

Although earlier work has pointed to the presence of Na+/H+ exchangers (NHEs) in the rat epididymis, little is known about the regional distribution of various NHE isoforms and their functions. In the present work, expression of different isoforms of NHE in cultured epithelia of the efferent duct and cauda epdidymidis were studied. Reverse transcription-polymerase chain reaction revealed the presence of NHE1, NHE2, and NHE3, but not NHE4, message in both cultured epithelia. Western blot analysis detected the presence of NHE1 and NHE2 proteins in both cultured epithelia, but NHE3 protein was only detected in the cultured epithelial cells from the efferent duct. Immunohistochemical studies demonstrated that NHE2 was localized in the cytoplasm of the ciliated cells, whereas NHE3 was localized at the apical membrane of the principal cells of the efferent duct. The NHE activities in both cultured epithelia were inhibited by 10 μM HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine methanesulphona... more...

Initiation of intestinal Na+-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na+/H+exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na+/H+exchange might also be required for Na+-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na+-glucose cotransport, inhibition of Na+/H+exchange with 200 μM 5-( N, N-dimethyl)- amiloride induced 36 ± 2% increases in transepithelial resistance (TER). Evaluation using multiple Na+/H+exchange inhibitors showed that inhibition of the Na+/H+exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH4Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na+-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 ± 5% after inhibition of Na+/H+exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na+-glucose cotransport. However, NHE3 inhibitors did not diminish Na+-glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na+-glucose cotransport-dependent tight junction regulation. more...

Previous studies from our laboratory have provided evidence that the rat epididymis utilizes the Na(+)/H(+) exchanger to transport acid and base. The present study was undertaken to use immunohistochemistry for investigating the localization (apical versus basolateral) and distribution of NHE1 and NHE2 proteins along intact rat epididymis. Both proteins were found to be exclusively localized within the epithelium. Immunoreactivity for NHE1 was detected on the basolateral surface, whereas NHE2 immunoreactivity was detected on the apical side of the epithelium. Interestingly, NHE1 was found along the entire length of the epididymal tubule whereas NHE2 was absent in the initial segment but present in the caput, corpus, and cauda regions. These results, when interpreted along with those of previous functional studies, may suggest that the apical NHE2 is involved in Na(+) reabsorption and the basolateral NHE1 in HCO(3)(-) secretion in the rat epididymis. more...

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed. more...

When expressed either in polarized epithelial cells or in fibroblasts, two Na(+)/H(+) exchanger isoforms, NHE1 and NHE3, have different subcellular distributions. Using a quantitative cell surface biotinylation technique, we found PS120 cells target approximately 90% of mature NHE1 but only 14% of NHE3 to the cell surface, and this pattern occurs irrespective of NHE protein expression levels. In this study, we examined surface fractions of NHE3 C-terminal truncation mutants to identify domains involved in the targeting of NHE3. Removing the C-terminal 76 amino acids doubled surface fractions to 30% of total and doubled the V(max) from 1300 to 2432 microM H(+)/s. Removal of another 66 amino acids increased surface levels to 55% of total with an increase in the V(max) to 5794 microM H(+)/s. Surface fractions did not change with a further 105 amino acid truncation. We postulated that inhibition of the basal recycling of NHE3 could result in the surface accumulation of the NHE3 truncations. Accordingly, we found that, unlike wild-type NHE3, the truncations were shown to internalize poorly and were not affected by PI3 kinase inhibition. However, while the truncations demonstrated reduced basal recycling, they retained the same serum response as full-length NHE3, with a mobilization of approximately 10% of total NHE to the surface. We conclude that basal recycling of NHE3 is controlled by endocytic determinants contained within its C-terminal 142 amino acids and that serum-mediated exocytosis is independently regulated through a different part of the protein. more...

Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 μg/ml, respectively) and also increased K′[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+activity by 51%. Further investigation into the mechanism of squalamine’s effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator. more...

Redistribution of apical Na+/H+exchangers (NHE) in the proximal tubules as a plausible mechanism of pressure natriuresis was investigated with confocal immunofluorescence microscopy in Sprague-Dawley rats (SD), spontaneously hypertensive rats (SHR), and two-kidney, one-clip Goldblatt hypertensive rats (GH). NHE isoform NHE3 was localized in the brush border of proximal tubules in SD. Twenty minutes of induced acute hypertension (20–40 mmHg) resulted in a pronounced redistribution of isoform NHE3 from the brush border into the base of microvilli, where clathrin-coated pits were localized. Prehypertensive young SHR (5 wk old, mean blood pressure 105 ± 3 mmHg, n = 11) produced similar findings. However, NHE3 was found to concentrate in the base of microvilli in adult SHR (12 wk old, mean blood pressure 134 ± 6 mmHg, n = 12) and nonclipped kidneys of GH (mean blood pressure 131 ± 6 mmHg, n = 6). In clipped kidneys of GH, which were not exposed to the hypertension because of the arterial clips, NHE3 was localized on the brush border as in normal SD. No further redistribution of NHE3 was detected in adult SHR or GH when acute hypertension was induced. Since both acute and chronic increase of arterial pressure can provoke the redistribution of apical NHE in proximal tubules, the pressure-induced NHE redistribution could be a physiological response and an integral part of pressure natriuresis. more...

We previously reported that mammalian small intestinal and colonic brush borders (BBs) contained both epithelial Na+/H+exchangers NHE2 and NHE3. We now show that, in the avian (chicken) colon, NHE2 is the major functional isoform under basal conditions and when stimulated by a low-NaCl diet. Hubbard chickens were maintained for 2 wk on a high- or low-NaCl diet. After the chickens were killed, the ileum and colon were removed, and BBs were prepared by Mg2+precipitation and22Na andd-[14C]glucose uptake determined in the BB vesicles. NHE2 and NHE3 were separated by differential sensitivity to HOE-694 (NHE2 defined as Na+/H+exchange inhibited by 50 μM HOE-694). Chickens on a low-Na+diet have increased plasma aldosterone (10 vs. 207 pg/ml). On the high-NaCl diet, both NHE2 and NHE3 contributed to ileal and colonic apical Na+/H+exchange, contributing equally in ileum, but NHE2 being the major component in colon (86%). Low-NaCl diet significantly increased ileal and colonic BB Na+/H+exchange; the increase in BB Na+/H+exchange in both ileum and colon was entirely due to an increase in NHE2 with no change in NHE3 activity. In contrast, low-NaCl diet decreased ileal and colonic Na+-dependentd-glucose uptake. Western analysis showed that low-Na+diet increased the amount of NHE2 in the ileal and colonic BB and decreased the amount of ileal Na+-dependent glucose transporter SGLT1. Both NHE2 and NHE3 were present in the apical but not basolateral membranes (BLM) of ileal and colonic epithelial cells. In summary, 1) NHE2 and NHE3 are both present in the BB and not BLM of chicken ileum and colon; 2) NHE2 is the major physiological colonic BB Na+/H+exchanger under basal conditions; 3) low-NaCl diet, which increases plasma aldosterone, increases ileal and colonic BB Na+/H+exchange and decreases Na+-dependentd-glucose uptake; 4) the stimulation of colonic BB Na+/H+exchange is due to increased activity and amount of NHE2; and 5) the inhibition of ileald-glucose uptake is associated with a decrease in SGLT1 amount. NHE2 is the major chicken colonic BB Na+/H+exchanger. more...

Intestinal neutral NaCl absorption, which is made up of brush-border (BB) Na+/H+exchange linked to BB Cl−/[Formula: see text]exchange, is up- and downregulated as part of digestion and diarrheal diseases. Glucocorticoids stimulate ileal NaCl absorption and BB Na+/H+exchange. Intestinal BB contains two Na+/H+exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbit ileal BB Na+/H+exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BB Na+/H+exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ∼50% to ileal Na+/H+exchange. Glucocorticoids (methylprednisolone) increase BB Na+/H+exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times), without an effect on NHE2 activity. Thus ileal BB Na+/H+exchange in animals treated with glucocorticoids is 69% via NHE3. A quantitative Western analysis for NHE3 was developed, using as an internal standard a fusion protein of the COOH-terminal 85 amino acids of NHE3 and maltose binding protein. Glucocorticoid treatment increased the amount of BB NHE3. The quantitative Western analysis showed that NHE3 makes up 0.018% of ileal BB protein in control rabbits and 0.042% (2.3 times as much) in methylprednisolone-treated rabbits. Methylprednisolone treatment did not alter the amount of ileal BB NHE2 protein. NHE3 turnover number was estimated to be 458 cycles/s under basal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thus methylprednisolone stimulates ileal BB Na+/H+exchange activity only by an effect on NHE3 and not on NHE2; it does so primarily by increasing the amount of BB NHE3, although it also increases the NHE3 turnover number. more...

There are no reported, convenient in vitro models for studying polarized functions in salivary epithelial cells. Accordingly, we examined three often-used salivary cell lines for their ability to form a polarized monolayer on permeable, collagen-coated polycarbonate filters. Only the SMIE line, derived from rat submandibular gland, had this ability. The SMIE cell monolayer exhibited junctional complexes, with a tight-junction-associated protein, ZO-1, localized to cell-cell contact areas. The Na+/K+-ATPase alpha1-subunit was detected predominantly in the basolateral membranes, while the Na+/H+ exchanger isoform 2 appeared primarily in the apical membranes. Using adenovirus-mediated cDNA transfer, SMIE cells were shown to be capable of routing marker proteins (beta-galactosidase +/- a nuclear targeting signal, alpha1-antitrypsin, aquaporin-1) to appropriate locations. Furthermore, this salivary cell monolayer provided a convenient tool for studying aquaporin-1-mediated, osmotically directed, transepithelial fluid movement in vitro. Thus, SMIE cells appear to be a useful experimental model with which to study some polarized functions in a salivary epithelial cell line. more...

Apical Na+/H+ exchangers (NHE) in the proximal tubule and medullary thick ascending limb (MTAL) display similar functions and regulation, suggesting that similar NHE isoforms are present. In the rat proximal tubule, NHE2 and NHE3 are present in the apical membrane, however, in the MTAL, NHE3, but not NHE2, mRNA has been found. In this study, the expression and subcellular localization of NHE2 in both rat and mouse MTAL were studied. To detect NHE2 mRNA, reverse transcription-polymerase chain reaction (RT-PCR) was performed in microdissected MTAL tubules using primers specific for NHE2. Analysis of PCR products with and without digestion by restriction enzymes chosen from the published NHE2 sequence gave predicted sizes. Subcloning and sequencing of the PCR product from mouse MTAL revealed 91% and 75% identity to the published NHE2 nucleotide sequence of comparable regions in rat and rabbit, respectively. Thus, NHE2 mRNA is expressed in the MTAL of mouse and rat. The subcellular localization of NHE2 was determined by immunochemistry using a specific NHE2 antibody. Immunofluorescence staining was observed in the apical, but not basolateral, membrane of MTAL of both species. In addition, anti-NHE2 antibody recognized an 85 kD protein in plasma membranes prepared from mouse and rat renal outer medulla and a MTAL cell line by Western analysis, which further support that NHE2 protein is expressed in the MTAL of both species. We conclude that NHE2 is expressed in the apical membrane of MTAL in both mouse and rat. more...

NHE3 is most likely the isoform involved in renal reabsorption of HCO3- and Na+. The functional properties of the "cloned" NHE3 isoform, including its transport regulation by extra- and intracellular stimuli, have so far been studied using non-epithelial expression systems. In the present report we stably transfected NHE3 cDNA (rabbit isoform) into Madin-Darby canine kidney cells (MDCK) cells and compared the sensitivity to inhibitors and the regulation of the Na+/H+-exchanger by vasotocin in NHE3 transfectants to that of the intrinsic basolateral Na+/H+-exchanger in untransfected and control transfected MDCK cells. By Southern blot analysis we documented that the NHE3 transcript is expressed in NHE3 transfectants. Na+/H+-exchange activity, measured as sodium-dependent recovery of intracellular pH from an acid load using 2', 7'-bis(carboxymethyl)-5(6)-carboxy-fluorescein (BCECF), was equally present at the basolateral cell surface of all cell lines; however, NHE3 transfectants demonstrated transport activity in the apical membrane that was significantly higher than that in untransfected or control transfected MDCK cells. Studies with ethylisopropylamiloride (EIPA) have shown that there is a similar sensitivity to inhibitors of the apical and/or basolateral Na+/H+-exchanger in transfected and untransfected MDCK cell lines. In contrast, the apical Na+/H+-exchanger (as compared to the basolateral Na+/H+-exchanger) of NHE3 transfectants was found to be relatively insensitive to the inhibitor HOE 694. Vasotocin decreased the activity of the apical Na+/H+-exchanger in NHE3 transfectants and stimulated the activity of the basolateral Na+/H+-exchanger in transfected (with NHE3 or pMAMneo) and untransfected MDCK cells. Phorbol ester, as expected, increased the activity of the Na+/H+-exchanger in the basolateral membrane of all cell lines; also, it stimulated transport activity at the apical cell surface of NHE3 transfectants. No change of Na+/H+-exchange activities was seen in studies with 8-bromo-cAMP. The PKC inhibitor calphostin C completely suppressed regulation of the apical and/or basolateral Na+/H+-exchanger by vasotocin, it partially blocked activation of the apical Na+/H+-exchanger in NHE3 transfectants by phorbol 12-myristate 13-acetate (PMA), and completely blocked stimulation of basolateral Na+/H+-exchanger by PMA. Consistent with a V1 receptor action, the effects of vasotocin in NHE3 transfectants and in MDCK cells were blocked by the V1 receptor antagonist, d(CH2)5Tyr(Me)-AVP, but were not reproduced by the V2 receptor agonist desmopressin. It is concluded that NHE3 in the apical membrane of NHE3-transfected MDCK cells contributes to the differential regulation of the apical and basolateral Na+/H+-exchanger by vasotocin; NHE3 is inhibited and endogenous Na+/H+-exchange activity is stimulated by vasotocin via V1 receptor activation of the protein kinase C pathway. more...

NHE3 is the Na + /H + exchanger located on the intestinal and renal brush border membrane, where it functions in transepithelial Na + absorption. The brush border Na + absorptive process is acutely inhibited by activation of cAMP-dependent protein kinase, but the molecular mechanism of this inhibitory effect is poorly understood. We have identified two regulatory proteins, E3KARP and NHERF, that interact with NHE3 to enable cAMP to inhibit NHE3. The two regulatory proteins are structurally related, sharing ≈50% identity in amino acid sequences. It has been previously shown that when NHE3 is transfected into PS120 fibroblasts or Caco-2 cells, cAMP failed to inhibit NHE3 activity. Northern blot analysis showed that both PS120 and Caco-2 cells lacked the expression of both E3KARP and NHERF. In contrast, other cell lines in which cAMP inhibits NHE3, including OK, CHO, and LLC-PK 1 cells, expressed NHERF-related regulatory proteins. To determine their functions in cAMP-dependent inhibition of NHE3, E3KARP and NHERF were transfected into PS120/NHE3 fibroblasts. Transfection in PS120/NHE3 fibroblasts with either NHERF or E3KARP reconstituted cAMP-induced inhibition of NHE3, resulting in 25–30% inhibition in these cells. more...

Rabbit NHE2 and NHE3 are two epithelial isoform Na+/H+ exchangers (NHE), the messages for which are found predominantly and entirely, respectively, in renal, intestinal, and gastric mucosa. The current studies used Western analysis and immunohistochemistry to identify and characterize the apical vs. basolateral membrane distribution of NHE2 and NHE3 in intestinal epithelial cells. Based on Western analysis, NHE2 and NHE3 both are present in brush-border but not basolateral membranes of small intestine. Both NHE2 and NHE3 are 85-kDa proteins. Consistent with Western analysis, NHE2 and NHE3 are immunolocalired to the brush-border but not basolateral membranes of villus epithelial cells, but not goblet cells, in human jejunum and ileum and in surface epithelial cells in the ascending and descending colon and rectum. In addition, NHE2 and NHE3 are present in small amounts in the crypt cell brush border of human jejunum, ileum, ascending and descending colon, and rectum. In rabbit jejunum, ileum, and ascending colon, NHE2 and NHE3 are present in the brush border of epithelial and not goblet cells, again much more in the villus (small intestine)/ surface cells (colon) than the crypt. NHE2 but not NHE3 is present in the brush border of rabbit descending colon surface cells and in small amounts in crypt cells. NHE2 and NHE3 are both human and rabbit small intestinal and colonic epithelial cell brush-border Na+/H+ exchanger isoforms that colocalize in all intestinal segments except rabbit descending colon, which lacks NHE3. more...

All cloned members of the mammalian Na+/H+ exchanger gene family encode proteins that consist of two functionally distinct domains: a membrane-bound N terminus and a cytoplasmic C terminus, which are required for ion transport and regulation of transport, respectively. Despite their similarity in structure, three members of this family, designated NHE1, NHE2, and NHE3, exhibit different kinetic mechanisms in response to growth factors and protein kinases. For instance, growth factors stimulate NHE1 by a change in the affinity constant for intracellular H+, K'(Hi+), and regulate NHE2 and NHE3 by a change in Vmax. We have constructed chimeric Na+/H+ exchangers by exchanging the N and C termini among three cloned rabbit Na+/H+ exchangers (NHE1 to NHE3) to determine which domain is responsible for the above Vmax-vs.-K'(H(i)+) effect of the Na+/H+ isoforms. All of the chimeras had functional exchange activity and basal kinetic properties similar to those of wild-type exchangers. Studies with serum showed that the N terminus is responsible for the Vmax-vs.-K'(H(i)+) stimulation of the Na+/H+ exchanger isoforms. Moreover, phorbol 12-myristate 13-acetate and fibroblast growth factor altered Na+/H+ exchange only in chimeras that had an epithelial N-terminal domain matched with an epithelial C-terminal domain. Therefore, the protein kinase-induced regulation of Na+/H+ exchangers is mediated through a specific interaction between the N- and C-termini, whcih is restricted so that epithelial N- and epithelial N-and C-terminal portions of the exchangers are required for regulation. more...