Na+-dependent hexose transport in vesicles from cultured renal epithelial cell line

1982 ◽  
Vol 243 (5) ◽  
pp. C293-C298 ◽  
Author(s):  
A. Moran ◽  
J. S. Handler ◽  
R. J. Turner
Keyword(s):  
Apical Membrane ◽  
Membrane Vesicles ◽  
Sodium Concentration ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Vesicle Preparation ◽  
Dependent Component ◽  
Sodium Dependent ◽  
Renal Epithelial Cell Line ◽  
Apical Membrane Vesicles

Apical membrane vesicles were prepared from cultured epithelia formed by LLC-PK1 cells using a calcium precipitation technique. alpha-Methylglucoside uptake into this vesicle preparation was markedly stimulated by sodium and inhibited by phlorizin. In addition, a transient "overshoot" of intravesicular alpha-methylglucoside concentration above its equilibrium value was observed under initial sodium gradient conditions. The specificity of this sodium-dependent hexose transporter closely resembled that found in the mammalian kidney brush border membrane, e.g., alpha-methylglucoside, D-glucose, and D-galactose apparently share the transporter while 2-deoxy-D-glucose, mannose, and fructose do not. Kinetic analysis of the sodium-dependent component of alpha-methylglucoside flux into LLC-PK1 apical membrane vesicles indicates the existence of single transporter with Km congruent to 2 mM and Vmax congruent to 3 nmol.min-1.mg protein-1. Measurement of alpha-methylglucoside uptake as a function of sodium concentration is consistent with a sodium:sugar stoichiometry of approximately 2:1.l There is a good correlation over time between the development of the concentrating capacity of the intact epithelium for alpha-methylglucoside and the transport properties of the vesicle preparation.

Increased Na(+)-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells

1991 ◽  
Vol 260 (4) ◽  
pp. C738-C744 ◽  
Author(s):  
R. F. Reilly ◽  
J. G. Haggerty ◽  
P. S. Aronson ◽  
E. A. Adelberg ◽  
C. W. Slayman
Keyword(s):  
Apical Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Epithelial Cell Line ◽  
Marker Enzyme ◽  
Maximal Velocity ◽  
Apical Surface ◽  
Membrane Level ◽  
Apical Membrane Vesicles

In whole cell experiments, the PKE20 mutant of the renal epithelial cell line LLC-PK1 displays a severalfold elevation of Na(+)-H+ antiporter activity at the apical surface (J.G. Haggerty, N. Agarwal, R.F. Reilly, E. A. Adelberg, and C.W. Slayman. Proc. Natl. Acad. Sci. USA 85: 6797-6801, 1988). The present study was undertaken to explore the properties of the mutant at the membrane level. Apical membrane vesicles were prepared by the magnesium-aggregation technique, with a similar enrichment (ca. 10-fold) of the marker enzyme gamma-glutamyltranspeptidase in vesicles from parent and mutant cell lines. In both cases, 22Na influx was stimulated by an inside-acid pH gradient, inhibited by ethylisopropylamiloride (EIPA), and unaffected by valinomycin, indicating that it was mediated by Na(+)-H+ antiport. Quantitatively, PKE20 vesicles showed a 4.2-fold increase in the maximal velocity of Na(+)-H+ antiporter activity compared with the parent, with only minor increases in the activity of two other Na(+)-dependent transporters (14-56% for alpha-methylglucoside and L-glutamate). Dose-response curves for EIPA indicated that the increased Na(+)-H+ antiport activity in PKE20 vesicles was due to an increased activity of the relatively amiloride-resistant form of the Na(+)-H+ antiporter with little or no change in the amiloride-sensitive form. No differences in polypeptide composition of the two vesicle preparations could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, the results indicate that the mutation in PKE20 is expressed at the membrane level and is specific for the relatively amiloride-resistant Na(+)-H+ antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-dependent sulfate transport in renal outer cortical brush border membrane vesicles

1984 ◽  
Vol 247 (5) ◽  
pp. F793-F798 ◽  
Author(s):  
R. J. Turner
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Sodium Concentration ◽  
Brush Border Membrane Vesicles ◽  
Sulfate Transport ◽  
Dependent Component ◽  
Sulfate Flux ◽  
Sodium Dependent ◽  
Stimulation Of

The transport of sulfate into outer cortical brush border membrane vesicles (early proximal tubule) was studied. Sulfate uptake was markedly enhanced by sodium and slightly enhanced by lithium but showed no stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of sulfate flux indicated a single transport system obeying Michaelis-Menten kinetics. Studies of the dependence of sulfate flux on sodium concentration indicated that more than one sodium ion was involved in the sulfate transport event. Although sulfate influx was found to be stimulated by negative intravesicular potentials, evidence is presented that sodium/sulfate cotransport is nevertheless an electroneutral event presumably involving 2 Na+ per SO2-4. Under zero trans sodium conditions sodium-dependent sulfate influx was stimulated by the presence of intravesicular unlabeled sulfate (trans stimulation); however, a similar stimulation of sulfate efflux by the presence of unlabeled extravesicular sulfate was not observed. These latter results are consistent with an ordered binding scheme for the transporter, with sulfate binding before sodium on the extravesicular (urine) surface and dissociating before sodium on the intravesicular (cytosolic) surface.

scholarly journals H+-gradient-dependent active transport of tetraethylammonium cation in apical-membrane vesicles isolated from kidney epithelial cell line LLC-PK1

1985 ◽  
Vol 227 (1) ◽  
pp. 199-203 ◽  
Author(s):  
K Inui ◽  
H Saito ◽  
R Hori
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
Epithelial Cell Line ◽  
Overshoot Phenomenon ◽  
Kidney Epithelial Cell ◽  
Kidney Epithelial Cell Line ◽  
Apical Membranes ◽  
Apical Membrane Vesicles

Transport of [3H]tetraethylammonium (NEt4+), an organic cation, has been studied by using apical-membrane vesicles isolated from cultured kidney epithelial cell line LLC-PK1. The uptake of NEt4+ by apical-membrane vesicles was osmotically sensitive, time-dependent and saturable. The presence of an H+ gradient ([H+]i greater than [H+]o) induced a marked stimulation of NEt4+ uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was inhibited by HgCl2. These results suggest that apical membranes isolated from the LLC-PK1 cells retain the transport characteristics of NEt4+ similar to those observed in renal brush-border membranes.

Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy

1992 ◽  
Vol 20 (2) ◽  
pp. 218-221
Author(s):  
Henning F. Bjerregaard
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
South African ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Electrophysiological Measurements ◽  
Renal Epithelial Cell Line ◽  
Darby Canine Kidney

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

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