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

Pflugers Arch. 2001 Oct;443(1):123-31. doi: 10.1007/s004240100629.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ammonium Chloride / pharmacology
  • Animals
  • Binding, Competitive
  • Cell Line
  • Cell Membrane / metabolism
  • Cell Size / physiology
  • Colforsin / pharmacology
  • Diuretics / pharmacology
  • Electric Conductivity
  • Epithelial Cells / metabolism
  • Fluoresceins / administration & dosage
  • Furosemide / pharmacology
  • Gene Expression*
  • Hydrogen-Ion Concentration
  • Kidney Medulla / metabolism*
  • Kidney Tubules, Collecting / metabolism*
  • Kinetics
  • Mice
  • Osmolar Concentration
  • Potassium / metabolism
  • Quaternary Ammonium Compounds / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Rubidium Radioisotopes / metabolism
  • Sodium-Potassium-Chloride Symporters / genetics*
  • Sodium-Potassium-Chloride Symporters / physiology*
  • Solute Carrier Family 12, Member 2

Substances

  • Diuretics
  • Fluoresceins
  • Quaternary Ammonium Compounds
  • Rubidium Radioisotopes
  • Slc12a2 protein, mouse
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 2
  • Ammonium Chloride
  • Colforsin
  • Furosemide
  • 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
  • Potassium