title: Reversible and irreversible inhibition, by stilbenedisulphonates, of lactate transport into rat erythrocytes. Identification of some new high-affinity inhibitors.
abstract: 1. Inhibition of L-lactate transport into rat erythrocytes by stilbenedisulphonates was studied under conditions which allowed the contribution of reversible and irreversible inhibition to be assessed. 2. At low temperatures (7 degrees C), 4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) and other stilbenedisulphonates were found to inhibit lactate transport instantaneously, in a manner which was fully reversible. The most potent reversible inhibitors were 4,4'-dibenzamidostilbene-2,2'-disulphonate (DBDS), DIDS and 4-acetamido-4'isothiocyanostilbene-2,2'-disulphonate (SITS), for which apparent Ki values at 0.5 mM-L-lactate were approx. 36, 53 and 130 microM respectively. 3. DIDS and DBDS were competitive inhibitors with respect to L-lactate, with Ki values of approx 40 microM and 22 microM respectively. 4. After incubation for 1 h at 37 degrees C with DIDS or its dihydro derivative (H2DIDS), which contain the amino-reactive isothiocyanate group, most of the inhibition observed was irreversible. Under these conditions the IC50 value (concn. causing 50% inhibition) for irreversible inhibition by both compounds was approx 100 microM. SITS was much less potent as an irreversible inhibitor of L-lactate transport, approx. 20% inhibition being obtained at 100 microM. 5. The reversible inhibitor DBDS (1 mM) afforded protection against irreversible inhibition by DIDS and H2DIDS (100 microM); protection was 60 and 65% respectively after a 60 min incubation. This indicates that specific binding of the irreversible inhibitors is required before covalent modification can take place. 6. These compounds may be useful high-affinity probes for lactate transport in other tissues and might act as affinity labels for the transport protein(s).
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, UK.
authors.forename: R C
authors.initials: RC
authors.lastname: Poole
authors.forename: A P
authors.initials: AP
authors.lastname: Halestrap
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; D002250:Carbon Radioisotopes; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D007773:Lactates / Q000097:blood*; D015394:Molecular Structure; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D002250:Carbon Radioisotopes; D007773:Lactates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid