Ming Tse

Structure/function studies of the epithelial isoforms of the mammalian Na+/H+ exchanger gene family

Na +/H + exchangers or antiporters are plasma membrane transport proteins, which in eukaryotes exchange extracellular Na + for intracellular H + with a stoichiometry of 1 : 1 [31,56]. In intact cells, Na + enters down the Na-K-ATPase generated electrochemical Na + gradient. All eukaryotic cells studied have plasma membrane Na +/H + exchangers, including yeast, Caenorhabditis elegans and crustaceans [1, 37, 49]. Prokaryotes have functionally similar Na +/H + exchanger proteins which regulate the intracellular Na + ion concentration and pH [38, 60]. In contrast to eukaryotic Na+/H + exchangers, prokaryotic Na+/H + exchangers are electrogenic, exchanging two intracellular Na + for 1 H + ; usually utilizing the intracellular H + ion electromotive force. In eukaryotic cells, the plasma membrane Na+/H + exchangers have multiple functions, including pH homeostasis, volume regulation, cell proliferation, and transcellular Na + absorption [reviewed in 31]. In no cell is it the only mechanism for any one of these functions. For instance, multiple mechanisms of pH homeostasis are present in most eukaryotic cells including a C1-/HCO3 exchanger, a NaHCO3 co-transporter, a Na+-dependent CI-/HCO3 exchanger and multiple mechanisms of H + extrusion [reviewed in 41], including the H-KATPase pump. In this review, we will focus on recent advances

Lysophosphatidic Acid Stimulates the Intestinal Brush Border Na+/H+ Exchanger 3 and Fluid Absorption via LPA5 and NHERF2

BACKGROUND & AIMS Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na(+)/H(+) exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na(+)/H(+) exchanger activity and Na(+)-dependent fluid absorption in the intestine. METHODS We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na(+)/H(+) exchanger regulatory factor 2 (NHERF2; Nherf2(-/-)) or LPA(2) (Lpa(2)(-/-)). Roles of LPA(5) and NHERF2 were determined by analysis of heterologous expression. RESULTS Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA(5) was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA(5) signaling required coexpression of NHERF2, which interacted with LPA(5). LPA-mediated intestinal fluid absorption was impaired in Nherf2(-/-) mice, demonstrating the requirement for NHERF2 in LPA(5) activity. However, fluid absorption was unaltered in Lpa(2)(-/-) mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. CONCLUSIONS LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA(5). Regulation by LPA(5) depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.

Membrane-limited expression and regulation of Na+-H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct

1 Cell‐specific reverse transcriptase‐polymerase chain reaction (RT‐PCR), immunolocalization and microspectrofluorometry were used to identify and localize the Na+‐H+ exchanger (NHE) isoforms expressed in the submandibular gland (SMG) acinar and duct cells and their regulation by basolateral and luminal P2 receptors in the duct. 2 The molecular and immunofluorescence analysis showed that SMG acinar and duct cells expressed NHE1 in the basolateral membrane (BLM). Duct cells also expressed NHE2 and NHE3 in the luminal membrane (LM). 3 Expression of NHE3 was unequivocally established by the absence of staining in SMG from NHE3 knockout mice. NHE3 was expressed in the LM and in subluminal regions of the duct. 4 Measurement of the inhibition of NHE activity by the amiloride analogue HOE 694 (HOE) suggested expression of NHE1‐like activity in the BLM and NHE2‐like activity in the LM of the SMG duct. Several acute and chronic treatments tested failed to activate NHE activity with low affinity for HOE as expected for NHE3. Hence, the physiological function and role of NHE3 in the SMG duct is not clear at present. 5 Activation of P2 receptors resulted in activation of an NHE‐independent, luminal H+ transport pathway that markedly and rapidly acidified the cells. This pathway could be blocked by luminal but not basolateral Ba2+. 6 Stimulation of P2U receptors expressed in the BLM activated largely NHE1‐like activity, and stimulation of P2Z receptors expressed in the LM activated largely NHE2‐like activity. 7 The interrelation between basolateral and luminal NHE activities and their respective regulation by P2U and P2Z receptors can be used to co‐ordinate membrane transport events in the LM and BLM during active Na+ reabsorption by the SMG duct.

NHE3 mobility in brush borders increases upon NHERF2-dependent stimulation by lyophosphatidic acid

The epithelial brush border (BB) Na+/H+ exchanger NHE3 is associated with the actin cytoskeleton by binding both directly and indirectly to ezrin; indirect binding is via attachment to NHERF family proteins. NHE3 mobility in polarized epithelial cell BBs is restricted by the actin cytoskeleton and NHERF binding such that only ~30% of NHE3 in the apical domain of an OK cell line stably expressing NHERF2 is mobile, as judged by FRAP analysis. Given that levels of NHE3 are partially regulated by changes in trafficking, we investigated whether the cytoskeleton association of NHE3 was dynamic and changed as part of acute regulation to allow NHE3 trafficking. The agonist studied was lysophosphatidic acid (LPA), an inflammatory mediator, which acutely stimulates NHE3 activity by increasing the amount of NHE3 on the BBs by stimulated exocytosis. LPA acutely stimulated NHE3 activity in OK cells stably expressing NHERF2. Two conditions that totally prevented LPA stimulation of NHE3 activity only partially prevented stimulation of NHE3 mobility: the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the NHE3F1 double mutant which has minimal direct binding of NHE3 to ezrin. These results show that LPA stimulation of NHE3 mobility occurs in two parts: (1) PI3K-dependent exocytic trafficking to the BB and (2) an increase in surface mobility of NHE3 in BBs under basal conditions. Moreover, the LPA stimulatory effect on NHE3 mobility required NHERF2. Although NHE3 and NHERF2 co-precipitated under basal conditions, they failed to co-precipitate 30 minutes after addition of LPA, whereas the physical association was re-established by 50-60 minutes. This dynamic interaction between NHERF2 and NHE3 was confirmed by acceptor photobleaching Förster Resonance energy Transfer (FRET). The restricted mobility of NHE3 in BBs under basal conditions as a result of cytoskeleton association is therefore dynamic and is reversed as part of acute LPA stimulation of NHE3. We suggest that this acute but transient increase in NHE3 mobility induced by LPA occurs via two processes: addition of NHE3 to the BB by exocytosis, a process which precedes binding of NHE3 to the actin cytoskeleton via NHERF2-ezrin, and by release of NHERF2 from the NHE3 already localized in the apical membrane, enabling NHE3 to distribute throughout the microvilli. These fractions of NHE3 make up a newly identified pool of NHE3 called the ‘transit pool’. Moreover, our results show that there are two aspects of LPA signaling involved in stimulation of NHE3 activity: PI3K-dependent stimulated NHE3 exocytosis and the newly described, PI3K-independent dissociation of microvillar NHE3 from NHERF2.

Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins

Secretory diarrheas caused by bacterial enterotoxins, including cholera and travelers diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido‐pyrrolooxazinedione (R)‐BPO‐27 inhibits CFTR chloride conductance with low‐nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)‐BPO‐27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)‐BPO‐27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat‐stable enterotoxin of E. coli (STa toxin), with IC50 down to ~5 nM. (R)‐BPO‐27 prevented cholera toxin and STa toxin‐induced fluid accumulation in small intestinal loops, with IC50 down to 0.1 μg/kg. (R)‐BPO‐27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7‐d administration at 5 μg/kg/d. As evidence to support efficacy in human diarrheas, (R)‐BPO‐27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)‐BPO‐27 for therapy of CFTR‐mediated secretory diarrheas.—Cil, O., Phuan, P.‐W., Gillespie, A.M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N.C., Lin, R., Donowitz, M., Verkman, A.S. Benzopyrimido‐pyrrolo‐oxazine‐dione CFTR inhibitor (R)‐BPO‐27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins. FASEB J. 31, 751–760 (2017). http://www.fasebj.org

Chapter 12 Molecular physiology of mammalian epithelial Na+/H+ exchangers NHE2 and NHE3

Publisher Summary This chapter discusses the molecular physiology of mammalian epithelial Na + /H + exchangers (NHE)—NHE2 and NHE3. These two exchangers contribute to small intestinal, colonic, gallbladder, renal, and salivary gland Na + and HCO 3 – absorption and, along with ENaC and Na + -linked substrate cotransporters, carry out most of the renal and intestinal Na + absorption. The chapter provides an overview of NHEs, including the identified members of the Na + /H + exchanger gene family and the structure/function relationships of the NHE N terminus; cellular and subcellular locations; physiological functions of apical Membrane NHEs and those attributed to NHE2 and/or NHE3; the regulation of transport activity; and diseases in which the epithelial NHEs play a pathogenic role. NHE2 and NHE3 can alter intracellular pH and intracellular volume. The cloned NHE isoforms are all separate genes and, with one exception, are present on different chromosomes. The related NHE2 and NHE4 are on the same chromosome. Pseudogenes are identified only for NHE3. All members of the NHE gene family have similar membrane topology based on similarity in hydrophobicity predicted by multiple algorithms. The chapter also describes the method used to assess the topology of NHE3 and NHE2.

Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3.

In polarized epithelial cells, SNX27 regulates PDZ domain–directed trafficking of NHE3 from endosomes to the plasma membrane and increases the stability of brush border NHE3. This establishes SNX27 as an important regulator of polarized sorting in epithelial cells.

6-mercaptopurine transport in human lymphocytes: Correlation with drug-induced cytotoxicity

OBJECTIVE:  6‐mercaptopurine (6‐MP) is efficacious in the treatment of inflammatory bowel disease (IBD). However, about one‐third of patients respond poorly to therapy. This study aimed to characterize the inherent differences in 6‐MP transport that may cotribute to the differences in treatment responses.

110 Missense Mutations of SLC9A3 in Patients With Congenital Sodium Diarrhea Are Associated With Reduced Na+/H+ Exchanger 3 (NHE3) Activity: Identification of the Cause of the Phenotype

Myo5b null mice display microvillus inclusions. Ezrin and p120 immunostaining were assessed in Myo5b null and wild type littermate mice. In a wildtype littermate, ezrin (left panel and red-right panel) was only expressed on the apical surface of the enterocytes. In all Myo5b null mice examined to date, ezrin was found in inclusions within the enterocytes. p120 localization (green-right panel) was unchanged in null mice. Heterozygote mice were identical to wild type mice and did not show microvillus inclusions. Scale bars = 50 μm.

886 Do Mutations of NHE3 At the Site of Poorly Functioning NHE3 Polymorphisms Contribute to Diarrhea of Congenital Na Diarrhea

of NHE3 from the membrane and diarrhea. Aim: We investigated the expression, brush border membrane (BBM) localization, and transport function of the major intestinal sodium absorptive transporter, the Na+/H+ exchanger NHE3, as well as the expression of its regulatory PDZ-adapter proteins NHERF1 and PDZK1, TNF-alpha, actin, and a variety of housekeeping genes, in the small and large intestine of mice after induction of a CD45RBhigh transfer colitis in the acute phase of diarrhea. Results: After colitis induction, TNF-alpha levels were increased both in the inflamed colon and microscopically normal-appearing small intestine. NHE3 mRNA expression was up regulated in the colon and unaltered in the small intestine, NHE3 protein expression and localization in the brush border membrane (BBM) was not altered, but acid-activated NHE3 transport rates in small intestinal villous and colonic surface cells were severely decreased, and fluid absorption In Vivo was decreased in the small intestine. PDZK1 mRNA expression was down regulated, whereas that of NHERF1 was not altered. Glucocorticoid treatment of colitic mice for 4 days decreased colonic mucosal cytokine expression and increased PDZK1 expression as well as fluid absorption. In order to investigate whether the down regulation of PDZK1 and the disturbed NHE3 transport activity is causally related, we studied PDZK1 protein expression and NHE3 transport activity in colonic surface enterocytes in the intestine of PDZK1 +/mice, and found PDZK1 content reduced by 60% and NHE3 transport activity significantly decreased. Conclusion: We suggest that during immune-mediated intestinal inflammation, NHE3 BBM protein abundance is normal, but the regulation of its transport activity is disturbed. PDZK1 down regulation during inflammation may be one factor responsible for inflammation-associated NHE3 dysfunction.