Transient expression of Na+/H+ exchanger isoform NHE-2 in LLC-PK1 cells: inhibition of endogenous NHE-3 and regulation by hypertonicity.
Na+/H+ exchanger isoforms NHE-2 and NHE-3 demonstrate distinct tissue expression patterns in renal epithelial cells. NHE-2 is predominantly expressed in the inner medulla whereas NHE-3 is highly expressed in the proximal tubule cells. The purpose of the current experiments was to study the characteristics of NHE-2 upon its own expression in cultured proximal tubule cells, LLC-PK1. Toward this end, LLC-PK1 cells were subjected to six cycles of proton suicide. The mutant cells, when grown to confluence and assayed for Na+/H+ exchanger by 22Na+ influx, showed significant reduction in NHE activity as compared to the parent cells (10.4 nmole/mg prot/4 min in parent cells vs. 1.8 in mutant cells, P < 0.001, n = 4). This remaining exchanger activity was mostly mediated via NHE-3 as shown by inhibition of the Na influx following PKC stimulation (65% with PMA vs. 100% without PMA. P < 0.05, n = 4). The mutant cells were transiently transfected with a pCMV/NHE-2 expression vector using calcium phosphate precipitation method. Northern blot analysis showed the expression of a 3.4 kb transcript only in the transfected cells. The expression peaked at 48 hr and diminished by 96 hr. The exchanger activity at 48 hr after transfection was mostly due to NHE-3 (as shown by inhibition in the presence of PMA) but was significantly lower than in sham transfected cells (1.2 nmoles/mg prot. in NHE-2-transfected and 2.1 in sham-transfected, P < 0.05, n = 4). At 60 hr after transfection, the cells exhibited PMA-stimulated Na influx (>28%) indicating functional expression of NHE-2. Increasing the osmolality of the media to 510 mOsm/l stimulated the Na+/H+ exchanger in NHE-2 transfected cells but inhibited the exchanger activity in sham transfected cells. In conclusion, NHE-2 appears as a 3.4 kb transcript in transfected LLC-PK1 cells and functional expression of NHE-2 is preceded by inhibition of endogenous NHE-3 activity. The NHE-2 is stimulated by hypertonicity, indicating a likely role for this isoform in cell volume regulation.
Singh
G
G
Department of Medicine, Indiana University School of Medicine, and Veterans Affairs Medical Center, Indianapolis, IN, USA.
Orlowski
J
J
Soleimani
M
M
D000818:Animals; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000494:pharmacology; D006982:Hypertonic Solutions / Q000494:pharmacology*; D066298:In Vitro Techniques; D017136:Ion Transport; D007527:Isoenzymes / Q000096:biosynthesis*; D018374:LLC-PK1 Cells / Q000378:metabolism*; D009154:Mutation; D012964:Sodium / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000502:physiology; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D018076:DNA, Complementary; D006982:Hypertonic Solutions; D007527:Isoenzymes; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D013755:Tetradecanoylphorbol Acetate