[3H]bumetanide binding and inhibition of Na+ + K+ + Cl- co-transport: demonstration of specificity by the use of MDCK cells deficient in co-transport activity.
Cultured renal MDCK cells possess a Na+ + K+ + Cl- co-transport system which is inhibited with high affinity by loop diuretics (K0.5 for bumetanide inhibition = 0.28 microM). By the use of 'mutant' cell lines deficient in co-transport flux the specific interaction of [3H]bumetanide with the co-transporter has been identified. [3H]Bumetanide uptake in parental MDCK-N cells in the range 0-1 microM comprises a non-saturable linear component, assessed by the inclusion of 100 microM-unlabelled bumetanide and a saturable component (K0.5 = 0.19 microM and Bmax = 0.55 pmol/10(6) cells). Though the magnitude of the linear non-specific component was little different between the parental MDCK-N cell line and two co-transport-deficient mutant cell lines (LKC1 and LKA3), the magnitude of the saturable component was markedly reduced in both co-transport-deficient mutants. In addition to the saturable component associated with flux inhibition a lower-affinity uptake displaceable by excess unlabelled bumetanide was evident in MDCK-N cells comprising 8 pmol/10(6) cells measured at 10 microM-[3H]bumetanide. This lower-affinity uptake was present in both co-transport-deficient mutant cell lines confirming its lack of association with inhibition of co-transport flux. In MDCK cells possessing the co-transporter, an estimate of the turnover number was made when co-transport flux and specific bumetanide uptake at 0.5 microM-[3H]bumetanide were measured in the same cell batch. At 37 degrees C this was 113 K+ ions site-1 s-1.
Popowicz
P
P
Department of Physiological Sciences, Medical School, University of Newcastle upon Tyne.
Simmons
N L
NL
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002034:Bumetanide / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D004232:Diuretics / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
D002712:Chlorides; D004232:Diuretics; D002034:Bumetanide; D012964:Sodium; D011188:Potassium