Expression, Localization and Functional Activity of the Major Na⁺/H⁺ Exchange Isoforms Expressed in the Intestinal Cell Line Caco-2BBe.

BACKGROUND/AIMS: Enterocytes express a number of NHE isoforms with presumed localization in the apical (NHE2, 3 and 8) or basolateral (NHE1) membrane. Functional activity and localization of enterocyte NHE isoforms were assessed using fully differentiated Caco-2BBe cells, whose genetic expression profile closely resembles mature enterocytes. METHODS: The activity of the different NHEs was analyzed by fluorometric pHi-metry in a perfusion chamber with separate apical and basolateral perfusion, using specific inhibitors and shRNA knockdown of NHE2. The expression of the NHEs and of other relevant acid extrusion transporters was quantified by qPCR. RESULTS: Quantitative comparison of the mRNA expression levels of the different NHE isoforms in 14 day-differentiated Caco-2BBe cells showed the following order: NHE2>NHE8>NHE3>NHE1. Acid-activated NHE exchange rates in the basolateral membrane were >6-fold higher than in the apical membrane. 79 ± 3 % of the acid-activated basolateral Na⁺/H⁺ exchange rate displayed a NHE1-typical inhibitor profile, and no NHE2/3/8 typical activity could be observed. Analysis of the apical Na⁺/H⁺ exchange rates revealed that approximately 51 ± 3 % of the total apical activity displayed a NHE2/8-typical inhibitor profile and 31 ± 6 % a NHE3-typical inhibitor profile. Because no selective NHE2 inhibitor is available, a stable NHE2 knockdown cell line (C2NHE2KD) was generated. C2NHE2KD displayed a reduced NHE2-typical apical Na⁺/H⁺ exchange rate and maintained a lower steady-state pHi, despite high expression levels of other acid extruders, in particular NBCn1 (Slc4a7). CONCLUSION: Differentiated Caco-2BBe cells display particularly high mRNA expression levels of NHE2, which can be functionally identified in the apical membrane. Although at low intracellular pH, NHE2 transport rate was far lower than that of NHE1. NHE2 activity was nevertheless essential for the maintenance of the steady-state pHi of these cells.

Sodium/bicarbonate cotransporter NBCn1/slc4a7 increases cytotoxicity in magnesium depletion in primary cultures of hippocampal neurons.

Growing evidence suggests that pharmacological inhibition of Na/H exchange and Na/HCO(3) transport provides protection against damage or injury in cardiac ischemia. In this study, we examined the contribution of the sodium/bicarbonate cotransporter NBCn1 (slc4a7) to cytotoxicity in cultured hippocampal neurons of rats. In neurons exposed to extracellular pH (pH(o)) ranging from 6.2 to 8.3, NBCn1 protein expression increased by fivefold at pH < 6.5 compared to the expression at pH(o) 7.4. At pH(o) 6.5, the intracellular pH of neurons was approximately 1 unit lower than that at pH 7.4. Immunochemistry showed a marked increase in NBCn1 immunofluorescence in plasma membranes and cytosol of the soma as well as in dendrites, at pH(o) 6.5. NBCn1 expression also increased by 40% in a prolonged Mg(2+)-free incubation at normal pH(o). Knockdown of NBCn1 in neurons had negligible effect on cell viability. The effect of NBCn1 knockdown on cytotoxicity was then determined by exposing neurons to 0.5 mm glutamate for 10 min and measuring lactate dehydrogenase (LDH) release from neurons. Compared to normal incubation (pH(o) 7.2 for 6 h) after glutamate exposure, acidic incubation (pH(o) 6.3 for 6 h) reduced cytotoxicity by 75% for control neurons and 78% for NBCn1-knockdown neurons. Thus, both controls and knockdown neurons showed acidic protection from cytotoxicity. However, in Mg(2+)-free incubation after glutamate exposure, NBCn1 knockdown progressively attenuated cytotoxicity. This attenuation was unaffected by acidic preincubation before glutamate exposure. We conclude that NBCn1 has a dynamic upregulation in low pH(o) and Mg(2+) depletion. NBCn1 is not required for acidic protection, but increases cytotoxicity in Mg(2+)-free conditions.

Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds. Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5.

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney (OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.