[3H]bumetanide binding to duck red cells. Correlation with inhibition of (Na + K + 2Cl) co-transport.
Bumetanide is a potent inhibitor of cation-chloride co-transport systems in many cell types, including duck red cells. We studied equilibrium binding of [3H]bumetanide to intact duck red cells under a number of conditions known to affect (Na + K + 2Cl) co-transport in these cells. Saturable [3H]bumetanide binding to duck red cells is markedly stimulated by addition of norepinephrine or cell shrinkage, conditions which similarly stimulate co-transport. In the presence of norepinephrine and saturating concentrations of extracellular sodium, potassium, and chloride for the co-transporter, we found approximately 1000 [3H]bumetanide-binding sites/red cell, and measurement of 24Na+ influx on the same cells yielded a turnover number of approximately 4000/s for the co-transporter. 24Na+ influx was negatively correlated with the amount of bound [3H]bumetanide, and both saturable binding and inhibition of influx were half-maximal at approximately 10(-7) M [3H]bumetanide. Binding of [3H]bumetanide to duck red cells is stimulated in a saturable manner by increasing extracellular sodium and potassium. Chloride has a biphasic effect on [3H]bumetanide binding; increasing [Cl-]o (by replacement of methylsulfate) from 0 to 32 mM markedly enhances binding, whereas further increasing [Cl-]o to 160 mM inhibits binding. This behavior is similar to that reported for bumetanide inhibition of duck red cell (Na + K + 2Cl) co-transport (Haas, M., and McManus, T. J. (1983) Am. J. Physiol. 245, C235-C240; Haas, M., and McManus, T. J. (1982) Biophys. J. 37, 214a) and [3H]bumetanide binding to membranes from dog kidney outer medulla (Forbush, B. III, and Palfrey, H. C. (1983) J. Biol. Chem. 258, 11787-11792).
D000085:Acetates / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002034:Bumetanide / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004232:Diuretics / Q000378:metabolism*; D004372:Ducks; D004912:Erythrocytes / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology; D009566:Nitrates / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
D000085:Acetates; D002712:Chlorides; D004232:Diuretics; D008698:Mesylates; D009566:Nitrates; D002034:Bumetanide; C045880:methanesulfonic acid; D012964:Sodium; D011188:Potassium; D009638:Norepinephrine