Binding of 14C-furosemide to isolated human erythrocytes.

The incubation of 14C-furosemide at high specific activity with intact red blood cells at 37 degrees C, pH 7.4, has enabled the furosemide binding sites to be characterized with respect to time course, affinity and specificity. The binding reaction was rapid, reversible and close to thermodynamic equilibrium. Binding was dependent on cell and furosemide concentration and was saturable. At equilibrium, pharmacological doses of furosemide competitively inhibited 14C-furosemide binding with 50% inhibition at 3 x 10(-5) M. The Na+/K+ pump inhibitor ouabain had no effect on the 14C-furosemide binding. Bumetanide, which is more potent than furosemide as inhibitor of Na+/K+ co-transport system and equally effective in inhibiting anion transport, was less effective than furosemide in displacing 14C-furosemide from its binding sites, suggesting a different mechanism of action for the two drugs in the red blood cell. The preincubation of erythrocytes with 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS), the potent and specific inhibitor of anion permeability, reduced specific furosemide binding by more than 80% at a furosemide concentration of 0.1 microM, while it had little effect on the non-specific furosemide binding. Taken together, these data suggest that furosemide interacts with specific binding sites in the human red blood cell, whose nature has not been clarified, but whose location is probably on (or near) the protein in band 3, i.e. the membrane macromolecule-mediating anion transport.