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  • Title: The effect of lethal acid stress on Na+/H+ exchanger isoforms in cultured inner medullary collecting duct cells: deletion of NHE-2 and over expression of NHE-1.
    Author: Singh G, McAteer JA, Soleimani M.
    Journal: Biochim Biophys Acta; 1995 Oct 04; 1239(1):74-80. PubMed ID: 7548147.
    Abstract:
    Cultured inner medullary collecting duct (mIMCD-3) cells express Na+/H+ exchanger isoforms NHE-2 and NHE-1 (Soleimani et al. (1994) J. Biol. Chem. 269, 27973-27978). In the present studies we examined the effect of lethal acid stress on Na+/H+ exchanger activity and isoform expression in mIMCD-3 cells. mIMCD-3 cells were incubated for 10 min with 20 mM ammonium, and exposed to an ammonium-free acidic solution (pH 6.0) for 120 min. Thereafter, cells were recovered and grown in normal culture media. The surviving clones were isolated and subjected to two additional cycles of acid stress. A mutant clone was isolated and characterized for Na+/H+ exchange activity and isoform expression. The mutant mIMCD-3 clone demonstrated significant over-expression of Na+/H+ exchange activity as assessed by acid-stimulated 22Na influx (11.56 nmol/mg protein in mutant vs. 4.06 nmol/mg in parent cells, P < 0.001, n = 4) and sodium-dependent pHi recovery from an acid load (0.55 pH/min in mutant vs. 0.28 pH/min in parent cells, P < 0.01, n = 6). A dose-response inhibition of the exchanger showed that the mutant cells were very sensitive to dimethylamiloride (IC50 158 nM in mutant vs. 889 nM in parent mIMCD-3 cells, P < 0.001). To compare the Na+/H+ exchanger isoforms in mutant and parent mIMCD-3 cells, poly(A)+ RNA was isolated from each group and probed with radiolabeled NHE-1 or NHE-2 cDNA. The expression of NHE-1 mRNA was increased by approximately 100% in mutant cells. The NHE-2 mRNA, on the other hand, was found to be absent in mutant mIMCD-3 cells. Examination of the regulatory mechanisms of the Na+/H+ exchanger isoforms in parent mIMCD-3 cells, which express NHE-2 and NHE-1, and mutant mIMCD-3 cells, which only express NHE-1, would be helpful in elucidating the roles of NHE-2 and NHE-1 in inner medullary collecting duct cells.
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