[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

¹ Department of Pathophysiology, Karol Marcinkowski's University Medical School, Poznan, Poland
² Department of Physiological Sciences, The Medical School, Framlington Place, The University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH

Cultured renal MDCK cells possess a Na⁺ + K⁺ + Cl^- co-transport system which is inhibited with high affinity by loop diuretics (K_0·5 for bumetanide inhibition = 0·28 µM). 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 µM comprises a non-saturable linear component, assessed by the inclusion of 100 µM-unlabelled bumetanide and a saturable component (K_0·5 = 0·19 µM and B_max = 0·55 pmol/10⁶ cells). Though the magnitude of the linear non-specific component was little diffe; ent 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/10⁶ cells measured at 10 µM-[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 µM-[³H]bumetanide were measured in the same cell batch. At 37 °C this was 113 K⁺ ions site^-1 s^-1.

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