Effects of inhibitors on anion exchangers in rabbit renal brush border membrane vesicles.
Cl-formate, Cl-oxalate, and SO4-CO3 exchange participate in Cl and organic anion transport across the brush border membrane of the rabbit proximal tubule. To determine the functional similarity of these transporters to each other and to band 3, we characterized, in isolated membrane vesicles, the inhibition of these transporters by compounds known to inhibit erythrocyte band 3. 4,4'-Dinitro-2,2'-disulfonic stilbene (DNDS), diphenylamine-2-carboxylate (DPC), flufenamate, and 4-aceto-4'-isothiocyano-2,2'-disulfonic stilbene (SITS) were effective inhibitors of Cl-oxalate and SO4-CO3 exchange, suggesting at least some common structural motifs between these exchangers and band 3. Cl-formate exchange was relatively insensitive to DNDS and DPC but sensitive to flufenamate (IC50 = 43 microM). Sensitivity to DNDS but not 4-amino-4'-amino-2,2'-disulfonic stilbene, a feature of band 3, was seen only for the SO4-CO3 exchanger. None of the exchangers had significant affinity for dipyridamole, furosemide, or probenecid. Finally, the presence of DPC or flufenamate increased the IC50 for reversible inhibition by DNDS, consistent with at least a partial overlap between the disulfonic stilbene and diphenylamine carboxylate binding sites of the Cl-oxalate exchanger. We next examined the effect of irreversible SITS binding. The Cl-oxalate exchanger was inhibited 90%, consistent with its high affinity for reversible inhibition by stilbenes. SITS pretreatment caused 50% inhibition of the Cl-formate exchanger, consistent with the reduced affinity of this exchanger for reversible binding of stilbenes. Despite the high sensitivity of the SO4-CO3 exchanger to reversible inhibition by stilbenes, SITS pretreatment caused < 20% irreversible inhibition of this exchanger. Finally, we characterized the stilbene inhibition of the Cl-oxalate exchanger in more detail. The presence of oxalate increased the IC50 for reversible inhibition by DNDS or SITS, implying that oxalate can directly compete at the reversible stilbene binding site of the Cl-oxalate exchanger. However, oxalate could not protect against covalent inactivation of the Cl-oxalate exchanger by SITS, indicating the presence of a separate site for irreversible binding of disulfonic stilbenes. These results suggest a dissociation between the sensitivities of proximal tubule anion exchangers to reversible and irreversible inhibition by disulfonic stilbenes. In contrast to band 3, the Cl-oxalate exchanger must possess separate sites for reversible and irreversible interaction with stilbenes, with only the former overlapping the substrate binding site.
McConnell
K R
KR
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Aronson
P S
PS
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001692:Biological Transport; D002254:Carbonates / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005439:Flufenamic Acid / Q000494:pharmacology; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D010070:Oxalates / Q000378:metabolism; D011817:Rabbits; D013267:Stilbenes / Q000494:pharmacology; D013431:Sulfates / Q000378:metabolism
D017920:Antiporters; D002254:Carbonates; D002712:Chlorides; D010070:Oxalates; D013267:Stilbenes; D013431:Sulfates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D005439:Flufenamic Acid; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid