Effect of the preparation method on Na+-H+ exchange and ion permeabilities in rat renal brush-border membranes

Biochim Biophys Acta. 1984 May 16;772(2):140-8. doi: 10.1016/0005-2736(84)90037-3.

Abstract

The delta pH-dependent quenching of Acridine orange was used to characterize Na+-H+ exchange and K+ and H+ conductances in brush-border membrane vesicles isolated by precipitation with either CaCl2 or MgCl2 from rat kidney cortex. A transmembrane pH difference of 2.5 units (inside acidic) was imposed and the initial rate of its dissipation was followed after injecting a puls of tetramethylammonium gluconate (control) or sodium or potassium gluconate. In membranes isolated by CaCl2, the Na+-H+ exchange was partially electroneutral (45% to 77% of the total exchange) and the rest was due to electrically coupled Na+ and H+ movements through conductive pathways in the membranes. In membranes prepared by MgCl2, the rate of total Na+-H+ exchange was about twice as high as that in membranes obtained by CaCl2 precipitation. However, total and electroneutral exchanges were equal indicating negligible electrically coupled Na+ and H+ movements in these membranes. K0.5 for Na+ in all preparations was in the same range, being in average 30 mM. Amiloride was a competitive inhibitor of Na+-H+ exchange in membranes obtained with both preparations; Ki values ranged between 0.1 and 0.58 mM. The rates of delta pH-dissipation with K+ gradients (+/- valinomycin) were by 50% to 150% higher in membranes prepared with CaCl2 than in membranes isolated with MgCl2 indicating much higher H+ and K+ conductances in membranes obtained with CaCl2. Therefore, the rate of Na+-H+ exchange as well as the conductances for various ions in the isolated brush-border membranes depend on membrane preparation.

MeSH terms

  • Amiloride / pharmacology
  • Animals
  • Carrier Proteins / metabolism*
  • Cell Fractionation
  • Cell Membrane Permeability* / drug effects
  • Electric Conductivity
  • Hydrogen-Ion Concentration
  • Kidney Cortex / metabolism*
  • Kidney Cortex / ultrastructure
  • Kinetics
  • Microvilli / drug effects
  • Microvilli / metabolism*
  • Microvilli / ultrastructure
  • Potassium / metabolism
  • Rats
  • Sodium / metabolism*
  • Sodium-Hydrogen Exchangers

Substances

  • Carrier Proteins
  • Sodium-Hydrogen Exchangers
  • Amiloride
  • Sodium
  • Potassium