POLARIZED DISTRIBUTION OF NA+ H+ EXCHANGER ISOFORMS IN RABBIT COLLECTING DUCT CELLS/

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 measurement s of intracellular pH (pH(i)) in monolayers mounted in a cuvette conta ining two fluid compartments, making it possible to independently meas ure Na+/H+ exchange activity on either the apical or basolateral surfa ce. RC.SV3 monolayers express Na+/H+ exchange activities in both the a pical and basolateral membrane domains. The two exchangers have half-s aturation constants (K-m) for external sodium and sensitivities to dim ethylamiloride, to HOE-694 and to cimetidine and clonidine consistant with the NHE-1 isoform on the basolateral cell surface and the NHE-2 i soform on the apical surface. Protein I;kinase A inhibition of basolat eral exchanger activity was significantly higher than that of the apic al exchanger. Protein kinase C significantly stimulated both exchanger s equally. RT-PCR analysis found RNA for only NHE-1 and NHE-2, and imm unofluoresence with an antibody against NHE-1 demonstrated a basolater al location for this isoform. The results suggest that RC.SV3 cells ha ve two Na+/H+ exchange activities separated spatially to the two cellu lar membranes, with the NHE-1 and the NHE-2 isoforms located on the ba solateral and the apical membranes, respectively.