Expression and role of sodium, potassium, chloride cotransport (NKCC1) in mouse inner medullary collecting duct (mIMCD-K2) epithelial cells

Loop-diuretic-sensitive Rb-86(+)(K+) transmembrane fluxes were determined i n cells of a mouse inner medullary collecting duct cell line (mIMCD-K2). Th e furosemide-sensitive (0.1 mM) influx was a substantial fraction of the to tal influx (0.39 +/-0.04 or 0.42 +/-0.03, n=5 in the presence or absence of ouabain, respectively). Furosemide also reduced Rb-86(+)(K+) efflux by a s imilar fraction (0.46). RT-PCR analysis revealed expression of mRNA for the Na+-K+-2Cl(-) cortransporter-1 (NKCC1), but not NKCC2. Loop-diuretic-sensi tive Rb-86(+)(K+) influx was confined to the basolateral membrane, confirmi ng its localisation there. The physiological properties of NKCC1 expressed in mIMCD-K2 cells, including the dependence upon medium Na+, K+ and Cl- and the relative sensitivity to loop diuretics as assessed by the concentratio n required for half-maximal inhibition (IC50) (bumetanide 3.3 +/-1.4x10(-7) M > piretanide 2.5 +/-0.15x10(-6) M > furosemide 2.3 +/-1.2x10(-5) M) were typical for NKCC1. Possible functions of NKCC1 were tested; furosemide did not inhibit the majority of forskolin-stimulated secretory short-circuit c urrent (I-SC) (83.5 +/-5.3% of the maintained response at 5 min). Secondly, total Rb-86(+)(K+) influx was stimulated markedly when external osmolarity was increased to 600 mosmol/l by mannitol due to an increase via NKCC1 fro m 55 +/- 11 to 191 +/-2 nmol/10(6) cells per 15 min, (both n=4, P <0.01). I n contrast, 10(-5) M forskolin did not stimulate total Rb-86(+)(K+) influx. Finally, the ability of both K+ and NH4+ to compete for ouabain-insensitiv e Rb-86(+)(K+) influx via NKCC1 was confirmed with similar concentrations f or half-maximal influx reduction (K-0.5). Apical exposure to NH4+ elicited rapid cytosolic alkalinisation in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluor escein (BCECF)loaded epithelial layers, consistent with selective permeabil ity of the apical membrane to NH3. Conversely, NH4+ (5 mM) at the basal cel l surface resulted in progressive acidification, the initial rate being red uced by 43% by furosemide. We conclude that NKCC1 participates in selective uptake of NH4+ at the basal surface, and that IMCD may function in direct NH4+ deposition to urine.