title: Na-K-2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues.
abstract: The effects of several sulfamoyl benzoic acid derivatives on Na-K-Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC50 values) and order of potency of these derivatives were benzmetanide, 5 X 10(-8) M greater than bumetanide 3 X 10(-7) M greater than piretanide 3 X 10(-6) M greater than furosemide 7 X 10(-6) M greater than amino piretanide 1 X 10(-5) 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [3H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values for Kd and number of binding sites (n) were: bovine kidney, Kd = 1.6 X 10(-7), n = 10.5 pmol/mg protein; winter flounder intestine, Kd 1.2 X 10(-7), n = 7.3 pmol/mg protein, and brush-border membranes from winter flounder, Kd = 5.3 X 10(-7), n = 20.4 pmol/mg protein. The estimated Kd for bumetanide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8 X 10(-7) M. The similarity in magnitudes of IC50 and Kd for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5 mM concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D002034:Bumetanide / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002712:Chlorides / Q000378:metabolism*; D004232:Diuretics / Q000378:metabolism*; D005432:Flounder; D005665:Furosemide / Q000031:analogs & derivatives* / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007679:Kidney Medulla / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D004232:Diuretics; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D005665:Furosemide; D012964:Sodium; D011188:Potassium