Characterization of na+-phosphate cotransporters in renal cortical
endosomes.
Loghman-Adham, Mahmoud, George T. Motock, Paul Wilson, and Moshe Levi.
Division of Nephrology and Hypertension, Department of Pediatrics,
University of Utah School of Medicine and Veterans Affairs Medical
Center, Salt Lake City, Utah 84132, University of Texas, Southwestern
Medical Center, and Veterans Affairs Medical Center, Dallas, Texas
75216
APStracts 2:0022F, 1995.
To determine the role of membrane recycling in proximal tubular
phosphate (Pi) transport, we studied the transport functions of
simultaneously prepared rat renal cortical endosomes (EV) and brush
border membrane vesicles (BBMV). Initial Pi uptake was Na+ gradient
-dependent in both vesicles. Kinetic studies showed a lower apparent
Vmax for Pi uptake in EV compared with BBMV (446 +/- 69 vs 1493 +/- 117
pmol/mg protein/15 sec, respectively, n=4, p<0.05), with no
difference in the apparent Km. Endosomal Na+-Pi cotransport was
inhibited by phosphate analogues, phosphonoformic acid and arsenate,
but not by the anion exchange inhibitor, 4,4'
-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Na+-Pi
cotransport was electroneutral in both vesicles with a stoichiometry
of 2 for BBMV and 1 for EV. The non-permeant sulfhydryl (SH) reagent,
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), produced a marked
inhibition of Na+- Pi cotransport in EV but not in BBMV, suggesting
accessible SH groups and 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+-Pi cotransporter protein (NaPi-2) abundance, determined by
Western blots, was 5 times lower in EV than in BBMV (0.25 +/- 0.05 vs
1.36 +/- 0.20 arbitrary units). Renal cortical endosomes contain Na+-Pi
cotransporters, albeit at a lower density, suggesting involvement of
membrane recycling in the regulation of proximal tubular Pi
transport.
Received 19 December 1994; accepted in final form 14 February
1995.
APS Manuscript Number F447-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 February 1995.