Polarized distribution of Na+/H+ exchanger isoforms in rabbit collecting duct cells

Abstract

The present study describes two Na+/H+ exchanger (NHE) isoforms in an immortalized rabbit renal cortical collecting tubule cell line (RC.SV3). Na+/H+ exchange activity was assayed using fluorescence measurements of intracellular pH (pHi) in monolayers mounted in a cuvette containing two fluid compartments, making it possible to independently measure Na+/H+ exchange activity on either the apical or basolateral surface. RC.SV3 monolayers express Na+/H+ exchange activities in both the apical and basolateral membrane domains. The two exchangers have half-saturation constants (Km) for external sodium and sensitivities to dimethylamiloride, to HOE-694 and to cimetidine and clonidine consistant with the NHE-1 isoform on the basolateral cell surface and the NHE-2 isoform on the apical surface. Protein kinase A inhibition of basolateral exchanger activity was significantly higher than that of the apical exchanger. Protein kinase C significantly stimulated both exchangers equally. RT-PCR analysis found RNA for only NHE-1 and NHE-2, and immunofluorescence with an antibody against NHE-1 demonstrated a basolateral location for this isoform. The results suggest that RC.SV3 cells have two Na+/H+ exchange activities separated spatially to the two cellular membranes, with the NHE-1 and the NHE-2 isoforms located on the basolateral and the apical membranes, respectively.