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/

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 effec t of lethal acid stress on Na+/H+ exchanger activity and isoform expre ssion in mIMCD-3 cells. mIMCD-3 cells were incubated for 10 min with 2 0 mM ammonium, and exposed to an ammonium-free acidic solution (pH 6.0 ) for 120 min. Thereafter, cells were recovered and grown in normal cu lture media. The surviving clones were isolated and subjected to two a dditional cycles of acid stress. A mutant clone was isolated and chara cterized for Na+/H+ exchange activity and isoform expression. The muta nt mIMCD-3 clone demonstrated significant over-expression of Na+/H+ Na -22 influx (11.56 nmol/mg protein in mutant vs. 4.06 nmol/mg in parent cells, exchange activity as assessed by acid-stimulated P < 0.001, n = 4) and sodium-dependent pH(i) recovery from an acid load (0.55 pH/mi n in mutant vs. 0.28 pH/min in parent cells, P < 0.01, it = 6). A dose -response inhibition of the exchanger showed that the mutant cells wer e very sensitive to dimethylamiloride (IC50 158 nM in mutant vs. 889 n M 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 isola ted from each group and probed with radiolabeled NHE-1 or NHE-2 cDNA, The expression of NHE-1 mRNA was increased by similar to 100% in mutan t 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 medull ary collecting duct cells.