Expression, Localization and Functional Activity of the Major Na+/H+ Exchange Isoforms Expressed in the Intestinal Cell Line Caco-2BBe
Cellular Physiology and Biochemistry
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 pH i -metry in a perfusion chamber with separate apical and basolateral perfusion,
... olateral 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 pH i , despite high expression levels of other acid extruders, in particular NBCn1 (Slc4a7). Caco-2BBe cells were transfected with full length human NHE3 tagged with VSVG (with the amino acid sequence YTDIEMNRLGK derived from the Vesicular Stomatitis viral glycoprotein) at C-terminus (a kind gift from Chris Yun, Emory University, Atlanta, USA with the permission of use from Ming Tse, John Hopkins University, Baltimore) and stable cell lines were established using 800 µg/mL G418 (Promo cell GmbH, Heidelberg, Germany) in the medium. The cells were grown at 37°C in a humidified atmosphere containing 5% CO 2 , in Dulbecco's Modified Eagle Medium with 4.5 g/l D-glucose and sodium pyruvate (Life Technologies GmbH, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin, 100 µg/mL streptomycin and 1% non-essential amino acids. The cells were maintained with 800 µg/mL G418 antibiotic in the medium. Then the cells were subjected to repeated acid selection for 5 cycles as described previously  . After 5 acid suicide selection cycles, the expression of NHE3-VSVG protein expression using anti-VSVG antibody and a robust NHE3 protein expression was detected ( Supplementary Fig. 2) . Acid extrusion mechanisms that may explain the non-NHE inhibitor sensitive Na + -dependent basolateral pH i recovery. Inhibition of NHE1 at the basolateral membrane uncovered existence of Na + -transporters that are not sensitive to high concentrations of HOE642. One obvious candidate is NHE4. Although rat intestinal NHE4 expression is reportedly low to nonexistent [33, 34] , and NHE4-deficient mice displayed no intestinal phenotype [35, 36] , its insensitivity to specific NHE inhibitors (except high concentrations of dimethyl-amiloride which inhibits a number of non-NHE transporters in these high concentration) makes it difficult to study its function. However, expression and functional activity has been reported in human enterocytes by some [37, 38] , while reported absent by others [39, 40] . For the low residual Na + -dependent Fig. 9 . Acid-activated apical Na + /H + exchange rates in differentiated C2NHE2KD cells. The experimental design, solutions and inhibitors were the same as in Fig. 6. (A) pH i -recovery curve without inhibitors, (B) in the presence of 60 μM HOE642, (C) in the presence of 1 μM tenapanor, (D) in the presence of 60 μM HOE642 and 1 μM tenapanor during apical Na + re-addition in C2NHE2KD cells. (E) Comparisons of acid-activated apical Na + /H + exchange rates under control, 60 μM HOE642, 1 μM tenapanor, and 60 μM HOE642 plus 1 μM tenapanor. The total apical Na + /H + exchange rate of C2NHE2KD cells was 0.050 ± 0.005 (△pH/min). 60 μM HOE642 reduced acid-activated apical Na + /H + exchange rate to 0.031 ±0.004 (△pH/min), 1 μM tenapanor to 0.036 ± 0.003 (△pH/min) and 60 μM HOE642 plus 1μM tenapanor to 0.015 ± 0.004 (△pH/min). (F) Comparisons of initial pH i at the beginning of acid-induced pH i recovery among different groups. (n=5, mean ± SEM, one-way ANOVA with Tukey's multiple comparison tests, *p< 0.05, **p< 0.001, ***p< 0.0001).