Attribution of [3H]bumetanide binding to the Na+K+Cl 'co-transporter' in rabbit renal cortical plasma membranes: a caveat.

The 3H-labelled loop diuretic bumetanide has been used to investigate loop diuretic binding to purified plasma membranes from rabbit kidney cortex (and outer medulla). Bumetanide binding to partially purified cortical plasma membranes in the range 0-10 microM, in a buffer containing principally Na, K and Cl ions, consists of a linear non-saturable component as assessed by 100 microM unlabelled bumetanide, and a saturable component consisting of high- and low-affinity binding sites, half-maximal binding being observed at 1.3 and 220 microM, respectively. The high-affinity site was found to be present in a fraction enriched in basolateral membrane markers when plasma membranes were further purified on a continuous Percoll gradient, whilst bumetanide binding to fractions enriched in brush-border or mitochondrial membrane markers was of lower affinity. Several features of bumetanide binding to basolateral membrane marker-enriched fractions are consistent with binding to the Na+K+Cl 'co-transporter' inhibited by loop diuretics: half-maximal binding was observed at 1.8 microM, with a finite maximal binding capacity of 78 pmol/mg. The relative efficacy of several loop diuretics for displacement of [3H]bumetanide was bumetanide greater than piretanide greater than furosemide = ethacrynic acid. Binding of loop diuretic was found to be dependent upon the medium ionic composition, Na, K and Cl being required to give maximal binding. The ability of probenecid and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) to compete with [3H]bumetanide was tested since these compounds are known inhibitors of anion secretion in the proximal nephron. Both DIDS and probenecid were able to effectively compete with [3H]bumetanide binding. A test of the ability of these compounds to inhibit 'co-transport' flux was made in intact MDCK cells using the ouabain-insensitive 86Rb (K) influx. Probenecid, at the concentrations seen to displace [3H]bumetanide binding to renal plasma membranes, was an effective inhibitor of 'co-transport' whereas DIDS was not. The adequacy of present criteria as to the identification of the 'co-transporter' in renal membranes using [3H]bumetanide binding are discussed in the light of this evidence.