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

Abstract

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.

The Role of Plasma Membrane Sodium/Hydrogen Exchangers in Gastrointestinal Functions: Proliferation and Differentiation, Fluid/Electrolyte Transport and Barrier Integrity

“…Since the inhibitory potential of the “NHE1-selective” inhibitors is also very high for NHE2 and NHE8 (not tested for the intracellular isoforms NHE6, 7 and 9), a selective inhibition of NHE1 by amiloride derivatives or by the “NHE1-selective” inhibitors HOE694 or HOE642 is not possible in vivo ( Table 1 ). However, these substances were useful to functionally localize the different NHEs to the apical vs. basolateral membranes in the Caco2BBe intestinal epithelial cell line ( Yu et al, 2019 ; Zhou et al, 2021 ). In the absence of external CO 2 and HCO 3 − , the NHE1 isoform mediates a very rapid pH i -recovery after an experimental intracellular acid load, evidenced by the virtually total inhibition of pH i recovery by 3 µM HOE642 in the basolateral bath solution of a perfusion chamber that can separately perfuse the apical and the basolateral membrane.…”

“…However, due to the overlapping inhibitory concentration for some of the NHE isoforms, and the difficulty to separate the basal from the apical membrane, the assessment of the activity of the different NHE isoforms is still a challenge ( Table 1 ). In two recent publications we studied the activity of the different NHE isoforms in Caco2BBe cell monolayers cultured on transwell filters with relatively high endogenous mRNA expression of NHE1,NHE2, NHE3 and NHE8, but not NHE4 ( Yu et al, 2019 ; Zhou et al, 2021 ). We were able to successfully separate the activity of the apical NHE isoforms 2,3 and 8 from the basolateral NHE1 using a fluorometric approach with a dual perfusion chamber and the selective addition of Na + and inhibitors to the apical and basolateral side of the cells.…”

“…In this model, lack of Na + in one perfusate will block the activity of all NHE isoforms in the respective membrane, but will allow the detection of NHE activity in the opposite membrane where Na + perfusion is ongoing. This approach enabled inhibition of the abundant NHE1 activity in the basolateral membrane without interfering with the modest NHE2, NHE3 and NHE8 activities in the apical membrane ( Yu et al, 2019 ; Zhou et al, 2021 ). In our studies, we have used HOE642, instead of HOE694 since it offers better separation between NHE1, NHE2, and NHE3 with IC 50 values of 0.05, 3, and 1,000 μM, respectively ( Scholz et al, 1995 ), and we were able to completely inhibit NHE2 activity in the apical membrane using 60 µM HOE642 without interfering with apical NHE3 activity ( Yu et al, 2019 ).…”

“…This approach enabled inhibition of the abundant NHE1 activity in the basolateral membrane without interfering with the modest NHE2, NHE3 and NHE8 activities in the apical membrane ( Yu et al, 2019 ; Zhou et al, 2021 ). In our studies, we have used HOE642, instead of HOE694 since it offers better separation between NHE1, NHE2, and NHE3 with IC 50 values of 0.05, 3, and 1,000 μM, respectively ( Scholz et al, 1995 ), and we were able to completely inhibit NHE2 activity in the apical membrane using 60 µM HOE642 without interfering with apical NHE3 activity ( Yu et al, 2019 ). Later, however, an inhibition of the NHE8 with 3 µM HOE642 was shown in the same culture model, indicating that the activity attributed to NHE2 in the studies by Yu et al (2019) , and Bachmann et al (2004) overlaps with the activity of NHE8 which remained masked.…”

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

“…Although the CFTR-mediated anion secretion is often dependent on protein kinase A (PKA), phosphoinositide 3-kinase (PI3K) or an increase in intracellular calcium ([Ca] i ) [ [9] , [10] , [11] ], it is unclear whether PTH directly alters PKA/PI3K phosphorylation or [Ca] i all of which are crucial for CFTR activity. Meanwhile, NHE1—known to be abundantly expressed in the basolateral membrane of enterocytes—is postulated to help maintaining normal pH i during anion secretion [ [12] , [13] , [14] ]. Nevertheless, whether prolonged PTH-induced HCO 3 − efflux eventually disturbs pH i balance and hence ends the entire transport process remains unknown.…”