Inhibition of renal Na(+)-Pi cotransporter by mercuric chloride: role of sulfhydryl groups.
We studied the role of sulfhydryl groups in Na(+)-Pi cotransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na(+)-dependent 32Pi transport in a dose-dependent manner (IC50 = 54 microM). Na(+)-independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium-exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D-glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (greater than 100 microM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 +/- 0.13 to 0.75 +/- 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected against inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na(+)-Pi cotransporter of renal BBM.
Loghman-Adham
M
M
Department of Pediatrics, University of Utah School of Medicine, Salt Lake City 84132.
D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008627:Mercuric Chloride / Q000494:pharmacology*; D008871:Microvilli / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D013347:Subcellular Fractions / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters; D013997:Time Factors
D002352:Carrier Proteins; D010710:Phosphates; D050608:Sodium-Phosphate Cotransporter Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; D008627:Mercuric Chloride; D012964:Sodium