Functional characteristics of a cloned epithelial Na+/H+ exchanger (NHE3): resistance to amiloride and inhibition by protein kinase C.
We previously cloned an isoform Na+/H+ exchanger (NHE3), which was expressed only in intestine, kidney, and stomach. We show here the functional characteristics of NHE3 as a Na+/H+ exchanger by stably transfecting NHE3 cDNA into PS120 cells, a fibroblast cell line that lacks endogenous Na+/H+ exchangers. NHE3 was 39- and 160-fold more resistant to inhibition by amiloride and ethylisopropyl amiloride, respectively, than NHE1, the housekeeping Na+/H+ exchanger isoform. Although both exchangers were stimulated by serum, NHE3 was inhibited by phorbol 12-myristate 13-acetate (PMA), which stimulated NHE1. Mechanistically, serum and PMA stimulated NHE1 by an increase in the apparent affinity of the exchanger for intracellular H+. In contrast, serum stimulated and PMA inhibited NHE3 by a Vmax change. When NHE3 was stably expressed in Caco-2 cells, an intestinal epithelial cell line, NHE3 was functionally expressed in the apical membrane. Thus, NHE3 is a good candidate to be an epithelial brush border Na+/H+ exchanger. Furthermore, Na+/H+ exchangers can be rapidly regulated by mechanisms that change either the Vmax or the affinity for intracellular H+, depending on the Na+/H+ exchanger subtype.
Tse
C M
CM
Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, MD 21205.
Levine
S A
SA
Yun
C H
CH
Brant
S R
SR
Pouyssegur
J
J
Montrose
M H
MH
Donowitz
M
M
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate