Mingmin Chen

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.

The Role of the NHERF Family of PDZ Scaffolding Proteins in the Regulation of Salt and Water Transport

The four members of the NHERF (Na+/H+ exchanger regulatory factor) family of PDZ adapter proteins bind to a variety of membrane transporters and receptors and modulate membrane expression, mobility, interaction with other proteins, and the formation of signaling complexes. All four family members are expressed in the intestine. The CFTR (cystic fibrosis transmembrane regulator) anion channel and the Na+/H+ exchanger NHE3 (Na/H exchanger‐ isoform 3) are two prominent binding partners to this PDZ‐adapter family, which are also known key players in the regulation of intestinal electrolyte and fluid transport. Experiments in heterologous expression systems have provided a number of mechanistic models how NHERF protein interactions can affect the function of their targets at the molecular level. Recently, NHERF1, 2, and 3 knockout mice have become available, and this review summarizes the reports on electrolyte and fluid transport regulation in the native intestine of these mice.

Na/H Exchanger Regulatory Factor 1 Overexpression- dependent Increase of Cytoskeleton Organization Is Fundamental in the Rescue of F508del Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway CFBE41o- Cells

NHERF1 overexpression increases functional apical expression of F508del CFTR in CFBE41o- cells. Here, we show that this occurs via the formation of the multiprotein complex NHERF1-phosphoezrin-actin, which provides a regulated linkage between F508del CFTR and the actin cytoskeleton resulting in an increased F508del CFTR stability in the membrane.

NHE3 inhibition by cAMP and Ca2+ is abolished in PDZ-domain protein PDZK1-deficient murine enterocytes.

The PDZ‐binding protein PDZK1 (NHERF3/CAP70/PDZ‐dc‐1) in vitro binds to NHE3, but its role in the regulation of NHE3 activity in native enterocytes is unknown. This study was undertaken to understand the physiological role of PDZK1 in regulating NHE3 activity in native murine colonic enterocytes. NHE3 transport rates were assessed fluorometrically in BCECF‐loaded colonic crypts in the NHE3‐expressing cryptal openings by measuring acid‐activated, Na+‐dependent, Hoe 642‐insensitive proton efflux rates. NHE3 mRNA expression levels and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative PCR and Western analysis and immunohistochemistry. In pdzk1−/− colonic surface cells, acid‐activated NHE3 transport rates were strongly reduced, and the inhibitory effect of forskolin and ionomcyin was virtually abolished. Hyperosmolarity, on the other hand, still had an inhibitory effect. In addition, the NHE3‐selective inhibitor S1611 inhibited acid‐activated NHE3 activity in pdzk1−/− and +/+ mice, suggesting that functional NHE3 is present in pdzk1‐deficient colonocytes. NHE1 and NHE2 activity was not altered in pdzk1−/− colonic crypts. Immunohistochemistry revealed apical NHE3 staining in pdzk1−/− and +/+ proximal colon, and Western blot analysis revealed no difference in NHE3 abundance in colonic enterocyte homogenate as well as brush border membrane. Lack of the PDZ‐adaptor protein PDZK1 in murine proximal colonic enterocytes does not influence NHE3 abundance or targeting to the apical membrane, but abolishes NHE3 regulation by cAMPergic and Ca2+ ‐dependent pathways. It leaves NHE3 inhibition by hyperosmolarity intact, suggesting an important and selective role for PDZK1 in the agonist‐mediated regulation of intestinal NHE3 activity.

Defective jejunal and colonic salt absorption and alteredNa +/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

We investigated the role of the Na+/H+ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na+ absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

The switch of intestinal Slc26 exchangers from anion absorptive to HCO3- secretory mode is dependent on CFTR anion channel function

CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO(3)(-)) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PAT1) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO(3)(-) secretion may be an unmasking of Slc26a6- and Slc26a3-mediated Cl(-)/HCO(3)(-) exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S1611 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO(3)(-) secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO(3)(-) secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na(+)/H(+) exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl(-) absorptive mode and Cl(-) exit via CFTR to operate in the HCO(3)(-) secretory mode.

Gene ablation for PEPT1 in mice abolishes the effects of dipeptides on small intestinal fluid absorption, short-circuit current, and intracellular pH

PEPT1 function in mouse intestine has not been assessed by means of electrophysiology and methods to assess its role in intracellular pH and fluid homeostasis. Therefore, the effects of the dipeptide glycilsarcosin (Gly-Sar) on jejunal fluid absorption and villous enterocyte intracellular pH (pH(i)) in vivo, as well as on enterocyte[(14)C]Gly-Sar uptake, short-circuit current (I(sc)) response, and enterocyte pH(i) in vitro were determined in wild-type and PEPT1-deficient mice and in mice lacking PEPT1. Immunohistochemistry for PEPT1 failed to detect any protein in enterocyte apical membranes in Slc15a1(-/-) animals. Saturable Gly-Sar uptake in Slc15a1(-/-) everted sac preparations was no longer detectable. Similarly, Gly-Sar-induced jejunal I(sc) response in vitro was abolished. The dipeptide-induced increase in fluid absorption in vivo was also abolished in animals lacking PEPT1. Since PEPT1 acts as an acid loader in enterocytes, enterocyte pH(i) was measured in vivo by two-photon microscopy in SNARF-4-loaded villous enterocytes of exteriorized jejuni in anesthetized mice, as well as in BCECF-loaded enterocytes of microdissected jejunal villi. Gly-Sar-induced pH(i) decrease was no longer observed in the absence of PEPT1. A reversal of the proton gradient across the luminal membrane did not significantly diminish Gly-Sar-induced I(sc) response, whereas a depolarization of the apical membrane potential by high K(+) or via Na(+)-K(+)-ATPase inhibition strongly diminished Gly-Sar-induced I(sc) responses. This study demonstrates for the first time that proton-coupled electrogenic dipeptide uptake in the native small intestine, mediated by PEPT1, relies on the negative apical membrane potential as the major driving force and contributes significantly to intestinal fluid transport.

Loss of PDZ‐adaptor protein NHERF2 affects membrane localization and cGMP‐ and [Ca2+]‐ but not cAMP‐dependent regulation of Na+/H+ exchanger 3 in murine intestine

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal‐dependent and possibly segment‐specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger‐mediated and receptor‐mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid‐activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin‐induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore‐ or carbachol‐mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal‐ and segment‐specific fashion, and is therefore an important regulator of intestinal fluid transport.

Knockout mouse models for intestinal electrolyte transporters and regulatory PDZ adaptors: new insights into cystic fibrosis, secretory diarrhoea and fructose-induced hypertension.

Knockout mouse models have provided key insights into the physiological significance of many intestinal electrolyte transporters. This review has selected three examples to highlight the importance of knockout mouse technology in unravelling complex regulatory relationships important for the understanding of human diseases. Genetic ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) has created one of the most useful mouse models for understanding intestinal transport. Recent work has provided an understanding of the key role of the CFTR anion channel in the regulation of HCO3− secretion, and the important consequences that a defect in HCO3− output may have on the viscoelastic properties of mucus, on lipid absorption and on male and female reproductive function. The regulation of CFTR activity, and also that of the intestinal salt absorptive transporter NHE3, occurs via the formation of PSD95‐Drosophila homologue Discs‐large‐tight junction protein ZO‐1 (PDZ) adaptor protein‐mediated multiprotein complexes. The recent generation of knockout mice for three members of the sodium‐hydrogen regulatory factor (NHERF) family of PDZ adaptor proteins, namely NHERF1 (EBP50), NHERF2 (E3KARP) and NHERF3 (PDZK1), has helped to explain why NHERF1 is essential for both normal and mutant CFTR function. In addition, they have provided new insight into the molecular mechanisms of secretory diarrhoeas. Genetic ablation of members of the recently discovered Slc26 anion transporter gene family not only reproduced the phenotype of the genetic diseases that led to the discovery of the gene family, but also resulted in new insights into complex human diseases such as secretory diarrhoea, fructose‐induced hypertension and urolithiasis.

The electroneutral Na+:HCO3− cotransporter NBCn1 is a major pHi regulator in murine duodenum

•  The mucosa of the proximal duodenum is regularly exposed to the strong acid of gastric juice, and acid damage causes peptic ulceration and bleeding. •  The epithelial cells of the duodenum protect themselves against acid‐induced chemical damage by secreting bicarbonate into the lumen and by regulating their intracellular pH, but the molecular mechanisms of duodenal pHi control are incompletely understood. •  We have found a high expression of the electroneutral sodium bicarbonate transporter Slc4a7 (NBCn1) in the basolateral membrane of duodenal villous enterocytes. •  Genetic knockout of Slc4a7 resulted in a strong defect in duodenocyte pHi recovery from acid loads, and reduced bicarbonate secretory rates. •  This suggests that the electroneutral sodium bicarbonate cotransporter Slc4a7 is a major import mechanism for HCO3− from the blood into the duodenal epithelial cells. Defects in this transporter may severely endanger duodenal mucosal integrity