Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB099

Poster Communications

Functional activity of plasma membrane NHE isoforms expressed in the self-differentiating intestinal epithelial cell line Caco-2Bbe

K. Zhou1, Y. Yu1, A. Seidler1, H. Xu2, U. E. Seidler1, K. Nikolovska1

1. Medical School Hannover, Hannover, Germany. 2. University of Arizona, Tucson, Arizona, USA Minor Outlying Islands.


The absorption of NaCl, HCO3- and fluid in the gastrointestinal tract is strongly dependent on apical Na+/H+ exchange. Na+/H+ exchanger NHE2, NHE3, and NHE8 are presumably expressed in the enterocyte apical membrane. Unlike the genetic deletion of NHE3, which results in diarrhea in mice, deletion of NHE2 or NHE8 does not cause a diarrheal phenotype. For further elucidation of the function and localization of enterocyte NHE2 and NHE8, we utilized fully differentiated Caco-2Bbe cells, whose genetic expression profile closely resembles mature enterocytes. Using specific inhibitors and shRNA knockdown of NHE2, the activity of the different NHEs was analyzed by fluorometric pHi-metry in a perfusion chamber with separate apical and basolateral perfusion and their expression was quantified by qPCR. The specificity and inhibitory potential of inhibitors for NHE1,2,3 and NHE8 were determined by overexpression in the NHE-deficient PS120 fibroblast cell line and in NHE1,2 and 3 knockout mouse intestine. Differentiated Caco-2Bbe cells express endogenous NHE1, NHE2, NHE3 and NHE8, with NHE2 having the highest and NHE1 the lowest mRNA expression levels. The acid-activated basolateral Na+/H+ exchange rates (0.127±0.018 △pH/min) were 3-fold higher compared to the apical rates (0.038±0.0062 △pH/min). 96,97 ± 2,4 % of the total acid-activated basolateral Na+/H+ exchange showed a NHE1-typical inhibitor profile. On the apical membrane, 50.9 ± 3.1 % of the acid-activated Na+/H+ exchange rates displayed a NHE2-typical inhibitor profile, 30.8 ± 5.6 % an NHE3-typical inhibitor profile and 25,6 ± 11,75 % an NHE8-typical inhibitor profile. shRNA downregulation of NHE2 expression in Caco-2Bbe cells (~30 % remaining NHE2 expression) resulted in increased expression of NHE8 compared to mock transfected cells, whereas NHE1 and NHE3 expression levels did not change significantly. NHE2 silencing did not significantly influence the total apical acid-activated Na+/H+ exchange activity. However, the percentage of apical Na+/H+ activity with a NHE2-typical inhibitor profile was reduced, while that with a NHE8 typical inhibitor profile has increased to 37±8,5 %. Apical NHE3 activity and basolateral NHE1 activity remained constant. Caco-2Bbe cells highly express NHE2 mRNA, and NHE2 activity was functionally identified in the apical membrane, but its transport rate during pHi-recovery on the apical membrane was modest compared to the basolateral NHE1 activity, proposing a high NHE2 turnover rate, an unknown NHE2 activation mode, or both. NHE8 activity is also found in the apical membrane and partly compensates for the loss of NHE2, but the majority of NHE8 appears localized in organelle localization and does not participate in cytoplasmic pHi-regulation.

Where applicable, experiments conform with Society ethical requirements