title: Inhibition of binding of [3H]metolazone to rat kidney membrane by stilbene disulfonates.
abstract: Thiazide diuretics inhibit an electroneutral chloride and sodium reabsorptive transport pathway in the renal distal convoluted tubule. [3H]Metolazone binds with high affinity to the drug recognition site on the thiazide-sensitive ion transporter. The molecular nature of this transporter is currently unknown. This report examines whether stilbene disulfonates [4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)], agents that have been used to identify other anion-transporting proteins, could be useful in the identification of the thiazide receptor. We found that high concentrations of DIDS or SITS were required to inhibit binding of [3H]metolazone to rat kidney membranes, with apparent IC50 values of 380 and 940 microM, respectively. The inhibition was due to a decrease in the number of binding sites without alteration in the affinity of the binding. The inhibition was not reversible, as judged by the inability of the inhibition to be reversed by removal of the DIDS or SITS from the membranes prior to equilibration with [3H]metolazone. Addition of 100 mM NaCl to the incubation medium did not protect the [3H]metolazone binding site from inhibition by DIDS. We infer that DIDS and SITS irreversibly inhibit binding of [3H]metolazone by reaction with the thiazide receptor at a site other than the anion-transporting site.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla 92093-0623.
authors.forename: H
authors.initials: H
authors.lastname: Luo
authors.forename: D D
authors.initials: DD
authors.lastname: Fanestil
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008297:Male; D008566:Membranes / Q000378:metabolism; D008788:Metolazone / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011955:Receptors, Drug / Q000037:antagonists & inhibitors; D012965:Sodium Chloride / Q000494:pharmacology; D049992:Sodium Chloride Symporters; D028021:Sodium-Potassium-Chloride Symporters; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters
chemical_list: D002352:Carrier Proteins; D011955:Receptors, Drug; C578876:Slc12a3 protein, rat; D049992:Sodium Chloride Symporters; D028021:Sodium-Potassium-Chloride Symporters; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; C064415:thiazide receptor; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D012965:Sodium Chloride; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D008788:Metolazone