Expression and role of sodium, potassium, chloride cotransport (NKCC1) in mouse inner medullary collecting duct (mIMCD-K2) epithelial cells.
Loop-diuretic-sensitive 86Rb+(K+) transmembrane fluxes were determined in cells of a mouse inner medullary collecting duct cell line (mIMCD-K2). The furosemide-sensitive (0.1 mM) influx was a substantial fraction of the total influx (0.39+/-0.04 or 0.42+/-0.03, n=5 in the presence or absence of ouabain, respectively). Furosemide also reduced 86Rb+(K+) efflux by a similar fraction (0.46). RT-PCR analysis revealed expression of mRNA for the Na+-K+-2Cl- cortransporter-1 (NKCC1), but not NKCC2. Loop-diuretic-sensitive 86Rb+(K+) influx was confined to the basolateral membrane, confirming 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 concentration 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 current (Isc) (83.5+/-5.3% of the maintained response at 5 min). Secondly, total 86Rb+(K+) influx was stimulated markedly when external osmolarity was increased to 600 mosmol/l by mannitol due to an increase via NKCC1 from 55+/-11 to 191+/-2 nmol/106 cells per 15 min, (both n=4, P<0.01). In contrast, 10-5 M forskolin did not stimulate total 86Rb+(K+) influx. Finally, the ability of both K+ and NH4+ to compete for ouabain-insensitive 86Rb+(K+) influx via NKCC1 was confirmed with similar concentrations for half-maximal influx reduction (K0.5). Apical exposure to NH4+ elicited rapid cytosolic alkalinisation in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded epithelial layers, consistent with selective permeability of the apical membrane to NH3. Conversely, NH4+ (5 mM) at the basal cell surface resulted in progressive acidification, the initial rate being reduced 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.
Glanville
M
M
Department of Physiological Sciences, Medical School, Framlington Place, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK.
Kingscote
S
S
Thwaites
D T
DT
Simmons
N L
NL
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D048429:Cell Size / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D004232:Diuretics / Q000494:pharmacology; D004553:Electric Conductivity; D004847:Epithelial Cells / Q000378:metabolism; D005452:Fluoresceins / Q000008:administration & dosage; D005665:Furosemide / Q000494:pharmacology; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007679:Kidney Medulla / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012414:Rubidium Radioisotopes / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics* / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2
D004232:Diuretics; D005452:Fluoresceins; D000644:Quaternary Ammonium Compounds; D012414:Rubidium Radioisotopes; C578878:Slc12a2 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D000643:Ammonium Chloride; D005576:Colforsin; D005665:Furosemide; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D011188:Potassium