IDENTIFICATION OF PTH-RESPONSIVE NA/H-EXCHANGER ISOFORMS IN A RABBIT PROXIMAL TUBULE CELL-LINE (RKPC-2)

Renal epithelial cells may express apical and basolateral Na/H exchangers which are different in their physiological regulation and different in their sensitivities to the inhibitor amiloride. In the present study RKPC-2 cells [a Simian virus 40 (SV-40) transformed cell line of rabbit S2 proximal tubular origin] were examined for localization (apical vs basolateral) and regulation of Na/H-exchange activity(ies) by parathyroid hormone (PTH). In addition, using specific cDNA probes we determined the expression of multiple isoforms of Na/H exchangers in RKPC-2 cells. By the use of BCECF [2',7',bis(2-carboxyethyl)-5,6-carboxyfluorescein intracellular pH (pH(i)) indicator] and single cell fluorescence microscopy, Na/H-exchange activities (defined as initial rate of Na-dependent pH(i) recovery) were found on the apical and basolateral membrane of RKPC-2 cells; apical and basolateral transport activities differed in sensitivity to dimethylamiloride, the basolateral being more sensitive. Northern blot analysis demonstrated the presence of a 5.2-kb transcript, related to Na/H-exchanger activity NHE-1, and a 3.2-kb transcript, related to Na/H-exchanger activity NHE-2. PTH (10(-8) M) inhibited apically and basolaterally located Na/H-exchanger activities. The inhibitory effect of PTH was mimicked by 8-bromo-adenosine 3'5'-cyclic monophosphate (cAMP); it was blunted in the presence of H-89 (inhibitor of protein kinase A) and was unaffected by calphostin C (inhibitor of protein kinase C). In contrast to 8-bromo-cAMP (and PTH), exposure of RKPC-2 cells to phorbol 12-myristate 13-acetate (TPA) caused a significant stimulation of both Na/H-exchange activities. Examination of the intracellular Ca2+ concentration ([Ca2+]i; using fura-2) and cAMP content (using a cAMP binding protein assay) revealed only PTH-dependent stimulation of adenylate cyclase. These results suggest that PTH (mediated by protein kinase A) inhibits in RKPC-2 cells structurally distinct Na/H-exchange activities (NHE-2 and NHE-1); we assume apical location of NHE-2 ('amiloride-resistant') and basolateral location of NHE-1 ('amiloride-sensitive') isoforms of Na/H-exchange activities.