title: Characterization of Na(+)-phosphate cotransporters in renal cortical endosomes.
abstract: To determine the role of membrane recycling in proximal tubular P(i) transport, we studied the transport functions of simultaneously prepared rat renal cortical endosomal vesicles (EV) and brush-border membrane vesicles (BBMV). Initial P(i) uptake was Na+ gradient-dependent in both vesicles. Kinetic studies showed a lower apparent maximal rate (Vmax) for P(i) uptake in EV compared with BBMV (446 +/- 69 vs. 1493 +/- 117 pmol.mg protein-1 x 15 s-1, respectively; n = 4, P < 0.05), with no difference in the apparent Michaelis constant (Km). Endosomal Na(+)-P(i) cotransport was inhibited by phosphate analogues, phosphonoformic acid and arsenate, but not by the anion-exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Na(+)-P(i) cotransport was electroneutral in both vesicles, with a stoichiometry of 2 for BBMV and 1 for EV. The nonpermeant sulfhydryl reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), produced a marked inhibition of Na(+)-P(i) cotransport in EV but not in BBMV, suggesting accessible sulfhydryl groups, an "inside-out" orientation in EV, and "right-side-out" orientation in BBMV. The EV and the BBMV differed significantly in their phospholipid composition and lipid fluidity. The Na(+)-P(i) cotransporter protein (NaPi-2) abundance, determined by Western blots, was five times lower in EV than in BBMV (0.25 +/- 0.05 vs. 1.36 +/- 0.20 arbitrary units). Renal cortical endosomes contain Na(+)-P(i) cotransporters, albeit at a lower density, suggesting involvement of membrane recycling in the regulation of proximal tubular P(i) transport.
authors.affiliation: Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, USA.
authors.forename: M
authors.initials: M
authors.lastname: Loghman-Adham
authors.forename: G T
authors.initials: GT
authors.lastname: Motock
authors.forename: P
authors.initials: P
authors.lastname: Wilson
authors.forename: M
authors.initials: M
authors.lastname: Levi
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D004563:Electrochemistry; D011992:Endosomes / Q000378:metabolism* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000502:physiology; D010710:Phosphates / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics; D010743:Phospholipids / Q000378:metabolism; D017494:Proton Pumps / Q000378:metabolism; D051381:Rats; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
chemical_list: D002352:Carrier Proteins; D010710:Phosphates; D010743:Phospholipids; D017494:Proton Pumps; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters