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 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.
Guerra
L
L
Institute of General Physiology, Bari, Italy.
Di Sole
F
F
Valenti
G
G
Ronco
P M
PM
Perlino
E
E
Casavola
V
V
Reshkin
S J
SJ
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D002927:Cimetidine / Q000494:pharmacology; D003000:Clonidine / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology
D017931:DNA Primers; D006146:Guanidines; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C069139:growth factor-activatable Na-H exchanger NHE-1; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; C039465:5-dimethylamiloride; D000584:Amiloride; D002927:Cimetidine; D011494:Protein Kinases; D003000:Clonidine