Characterization of basolateral Na/H exchange (Na/H-1) in MDCK cells

MDCK cells were grown to confluent monolayers on permeant filter supports; pH was analysed by using the pH-sensitive fluorescent probe 2′7′-biscarboxyethyl-5,6-carboxyfluorescein and a routine spectrofluorometer equipped with a perfusion cuvette [Krayer-Pawlowska et al. (1990) J Membr Biol 120:173–183]. Superfusion of the basolateral (but not apical) cell surface with Na+-containing solutions led to immediate recovery of pHi from an acid load (NH4 prepulse). This pHi recovery was reversibly inhibited by ethylisopropylamiloride indicating Na/H exchange activity. Na/H exchange activity showed an apparent K m for Na+ of about 25 mM Na+ and an apparent K i for inhibition by dimethylamiloride of around 0.2 μM; inhibition by dimethylamiloride was competitive with Na+ interaction. Lowering pHi prior to analysis of Na/H exchange leads to sharp activation of Na/H exchange; the apparent V max for Na/H exchange is increased more than tenfold by lowering the pHi from 7.0 to 6.7 without an effect on apparent K m values for Na+ interaction. It is concluded that MDCK cells (strain I) grown on a permeant support contain only basolateral Na/H exchange activity, most likely Na/H-1 [for nomenclature see Igarashi et al. (1991) Kidney Int 40:S84–S89].