Techniques for isolation of brush-border and basolateral membrane vesicles from dog kidney cortex

1989 ◽  
Vol 983 (1) ◽  
pp. 77-81 ◽  
Author(s):  
Shirley A. Hilden ◽  
Conrado A. Johns ◽  
William B. Guggino ◽  
Nicolaos E. Madias
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Kidney Cortex ◽  
Basolateral Membrane Vesicles ◽  
Dog Kidney

Na+ gradient-dependent glycine uptake in basolateral membrane vesicles from the dog kidney

1985 ◽  
Vol 249 (3) ◽  
pp. F338-F345
Author(s):  
S. J. Schwab ◽  
M. R. Hammerman
Keyword(s):  
Amino Acids ◽  
Membrane Potential ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Transport Processes ◽  
Basolateral Membrane Vesicles ◽  
Glycine Uptake ◽  
Dog Kidney ◽  
Dependent Transport

The imposition of a Na+ gradient (extravesicular greater than intravesicular) stimulated the uptake of [3H]glycine measured over time in basolateral membrane vesicles from dog kidney over that measured in the presence of a choline+ gradient or measured under Na+-equilibrated conditions. Na+ gradient-dependent uptake of [3H]glycine was stimulated by an intravesicular-negative membrane potential. Efflux of [3H]glycine was enhanced by an intravesicular-positive membrane potential. Substrate velocity analysis of net Na+-dependent [3H]glycine uptake over the range of amino acid concentrations from 10 to 500 microM demonstrated a single saturable transport system with apparent Km = 84 microM and apparent Vmax = 143 pmol [3H]glycine X mg protein-1 X 15 s-1. Counterflow of [3H]glycine was demonstrated in the presence of Na+ when basolateral vesicles were preloaded with glycine but not with L-alanine or L-proline. These findings are consistent with carrier-mediated, electrogenic cotransport of Na+ and glycine in basolateral vesicles. Unlike the case for [3H]glycine, Na+ gradient-dependent uptake of neither L-[3H]alanine nor L-[3H]proline was observed in basolateral vesicles. Na+ gradient-dependent uptake of all three amino acids was demonstrated in brush border vesicles from the dog kidney. We conclude that variability exists between basolateral and brush border membranes in terms of the presence or absence of Na+-dependent transport systems for specific amino acids. This variability probably reflects differences between the functional significances of the Na+-dependent transport processes in the two membranes.

Inorganic anion transport in kidney and intestinal brush border and basolateral membranes

1980 ◽  
Vol 238 (6) ◽  
pp. F452-F460 ◽  
Author(s):  
S. Grinstein ◽  
R. J. Turner ◽  
M. Silverman ◽  
A. Rothstein
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Anion Transport ◽  
Significant Inhibition ◽  
Kidney Cortex ◽  
Basolateral Membrane Vesicles ◽  
Inorganic Anion ◽  
Transport Pathways ◽  
Basolateral Membranes

The efflux of inorganic anions from purified brush border and basolateral membrane vesicles from dog kidney cortex was measured under equilibrium exchange conditions. Marked differences in temperature sensitivity and effects of inhibitors were found between the Cl and SO4 transport pathways and between the two types of membranes. SO4 transport in both brush border and basolateral membranes was markedly reduced by cooling, but significant inhibition by 4,4'–diisothiocyano-2,2'–disulfonic stilbene (DIDS) was only observed in basolateral vesicles. In contrast, Cl efflux from both types of vesicles was neither substantially inhibited by DIDS nor by lowering the temperature to 0 degrees C. Phosphate efflux from basolateral membrane vesicles was found to be only partially sensitive to DIDS. Attempts to label the stilbene-sensitive SO4 pathway in basolateral vesicles using [3H2]DIDS as a marker were unsuccessful due to the nonspecific labeling of many membrane components. The asymmetry in inorganic anion transport behavior exhibited by brush border and basolateral membrane vesicles from dog renal proximal tubule was also observed in equivalent vesicles prepared from rat small intestine.

Na+ gradient-dependent Pi uptake in basolateral membrane vesicles from dog kidney

1984 ◽  
Vol 246 (5) ◽  
pp. F663-F669 ◽  
Author(s):  
S. J. Schwab ◽  
S. Klahr ◽  
M. R. Hammerman
Keyword(s):  
Brush Border ◽  
Renal Cortex ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Basolateral Membrane Vesicles ◽  
Basolateral Membranes ◽  
Solute Uptake ◽  
Dog Kidney ◽  
Proximal Tubular

To ascertain whether Na+ gradient-stimulated 32Pi uptake was demonstrable in renal basolateral membrane vesicles, we measured 32Pi uptake in basolateral membrane suspensions isolated from canine renal cortex and compared solute uptake in basolateral suspensions with that measured in brush border suspensions. Measurements revealed Na+ gradient-dependent 32Pi transport in basolateral preparations. D-[3H] Glucose uptakes in basolateral suspensions were not stimulated by the Na+ gradient in contrast to findings in brush border suspensions. Na+ gradient-dependent 32Pi transport in basolateral suspensions was electrogenic in contrast to that measured in brush border preparations. Unlike 32Pi uptake in brush border preparations, Na+ gradient-dependent 32Pi uptake in basolateral suspensions did not increase as extravesicular pH was increased from 6.5 to 7.5. Na+ gradient-dependent 32Pi uptake in basolateral membranes showed saturation over the range of [Pi] from 5 to 100 microM (apparent Km, 14 +/- 2 microM; apparent Vmax, 34 +/- 2 pmol Pi X mg protein-1 X 30s-1). Our findings are compatible with the presence of an electrogenic Na+-Pi cotransporter in the canine proximal tubular basolateral membrane.

scholarly journals Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex

1989 ◽  
Vol 264 (1) ◽  
pp. 223-231 ◽  
Author(s):  
T C Williams ◽  
A J Doherty ◽  
D A Griffith ◽  
S M Jarvis
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Facilitated Diffusion ◽  
Basolateral Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Outer Cortex ◽  
Overshoot Phenomenon

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

Urate transport in the proximal tubule: in vivo and vesicle studies

1985 ◽  
Vol 249 (6) ◽  
pp. F789-F798 ◽  
Author(s):  
A. M. Kahn ◽  
E. J. Weinman
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Anion Exchanger ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Transport Processes ◽  
Basolateral Membrane Vesicles ◽  
Urate Transport ◽  
Rat Proximal Tubule

The transport of urate in the mammalian nephron is largely confined to the proximal tubule. Depending on the species, net reabsorption or net secretion is observed. The rat, like the human and the mongrel dog, demonstrates net reabsorption of urate and has been the most extensively studied species. The unidirectional reabsorption and secretion of urate in the rat proximal tubule occur via a passive and presumably paracellular route and by a mediated transcellular route. The reabsorption of urate, and possibly its secretion, can occur against an electrochemical gradient. A variety of drugs and other compounds affect the reabsorption and secretion of urate. The effects of these agents depend on their site of application (luminal or blood), concentration, and occasionally their participation in transport processes that do not have affinity for urate. Recent studies with renal brush border and basolateral membrane vesicles from the rat and brush border vesicles from the dog have determined the mechanisms for urate transport across the luminal and antiluminal membranes of the proximal tubule cell. Brush border membrane vesicles contain an anion exchanger with affinity for urate, hydroxyl ion, bicarbonate, chloride, lactate, p-aminohippurate (PAH), and a variety of other organic anions. Basolateral membrane vesicles contain an anion exchanger with affinity for urate and chloride but not for PAH. Both membrane vesicle preparations also permit urate translocation by simple diffusion. A model for the transcellular reabsorption and secretion of urate in the rat proximal tubule is proposed. This model is based on the vesicle studies, and it can potentially explain the majority of urate transport data obtained with in vivo techniques.

Na+-independent D-glucose transport in rabbit renal basolateral membranes

1988 ◽  
Vol 254 (5) ◽  
pp. F711-F718 ◽  
Author(s):  
P. T. Cheung ◽  
M. R. Hammerman
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Brush Border ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Rabbit Kidney ◽  
Basolateral Membrane Vesicles ◽  
Proximal Tubular

To define the mechanism by which glucose is transported across the basolateral membrane of the renal proximal tubular cell, we measured D-[14C]glucose uptake in basolateral membrane vesicles from rabbit kidney. Na+-dependent D-glucose transport, demonstrable in brush-border vesicles, could not be demonstrated in basolateral membrane vesicles. In the absence of Na+, the uptake of D-[14C]glucose in basolateral vesicles was more rapid than that of L-[3H]glucose over a concentration range of 1-50 mM. Subtraction of the latter from the former uptakes revealed a saturable process with apparent Km of 9.9 mM and Vmax of 0.80 nmol.mg protein-1.s-1. To characterize the transport component of D-glucose uptake in basolateral vesicles, we measured trans stimulation of 2 mM D-[14C]glucose entry in the absence of Na+. Trans stimulation could be effected by preloading basolateral vesicles with D-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose, but not with L-glucose or alpha-methyl-D-glucoside. Trans-stimulated D-[14C]glucose uptake was inhibited by 0.1 mM phloretin or cytochalasin B but not phlorizin. In contrast, Na+-dependent D-[14C]glucose transport in brush-border vesicles was inhibited by phlorizin but not phloretin or cytochalasin B. Our findings are consistent with the presence of a Na+-independent D-glucose transporter in the proximal tubular basolateral membrane with characteristics similar to those of transporters present in nonepithelial cells.

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