Renal brush-border chloride transport mechanisms characterized using a fluorescent indicator.
Cl transport mechanisms in brush-border membrane vesicles (BBMV) isolated from rabbit renal cortex were characterized using the Cl-sensitive fluorescent indicator 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ). In control experiments using 36Cl, SPQ did not itself alter rates for Cl transport. Cl fluxes (JCl) in nanomoles per second per milligram vesicle protein in response to gradients of Cl and other ions were calculated from the SPQ fluorescence time course, fluorescence vs. [Cl] calibration, and BBMV glucose space (microliter/mg protein). For a 50 mM inwardly directed Cl gradient in BBMV (voltage and pH clamped) JCl was 0.26 +/- 0.01 nmol.s-1.mg-1 (mean +/- SD, 4 vesicle preparations). JCl was not altered by a 50 mM inwardly directed Na gradient (0.25 +/- 0.01 nmol.s-1.mg-1) but increased to 0.52 +/- 0.04 nmol.s-1.mg-1 when driven by a 1.5 pH unit inwardly directed proton gradient. The pH driven increase in JCl was blocked by H2DIDS (100 microM, JCl = 0.38 +/- 0.02 nmol.s-1.mg-1). In the absence of a gradient, JCl increased by 64 +/- 4% in the presence of 1 mM formate on both sides of the membrane; the increase was blocked by H2DIDS. JCl increased to 0.38 +/- 0.02 nmol.s-1.mg-1 for a 60-mV K valinomycin diffusion potential (internal positive); the increase was inhibited by the Cl channel blocker diphenylamine-2-carboxylate (DPC, 100 microM, JCl = 0.26 +/- 0.03 nmol.s-1.mg-1). Br and I transport was also blocked by DPC.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
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Division of Nephrology and Cardiovascular Research Institute, University of California, San Francisco 94143.
Illsley
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Verkman
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000465:Algorithms; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000648:ultrastructure; D008871:Microvilli / Q000378:metabolism*; D011806:Quinolinium Compounds; D011817:Rabbits
D002712:Chlorides; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid