REVERSIBLE AND IRREVERSIBLE INHIBITION, BY STILBENEDISULPHONATES, OF LACTATE TRANSPORT INTO RAT ERYTHROCYTES - IDENTIFICATION OF SOME NEW HIGH-AFFINITY INHIBITORS

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'-diisothiocyanostilbene-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 K(i) values at 0.5 mM-L-lactate were approx. 36, 53 and 130-mu-M respectively. 3. DIDS and DBDS were competitive inhibitors with respect to L-lactate, with K(i) values of approx. 40-mu-M and 22-mu-M 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-mu-M. SITS was much less potent as an irreversible inhibitor of L-lactate transport, approx. 20% inhibition being obtained at 1000-mu-M. 5. The reversible inhibitor DBDS (1 mM) afforded protection against irreversible inhibition by DIDS and H2DIDS (100-mu-M); 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).