Abstract
The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.
Highlights
The stimulation of D-glucose uptake into the membranes was specific for Na+
The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of n-glucose uptake
The present results show that the imposition of an extravesicular to intravesicular Na+ gradient brings about the transient accumulation of n-glucose in renal proximal tubule brush border membranes that reaches a level severalfold higher than that attained at equilibrium
Summary
Rabbit renal brush border membranes were isolated by a modification of the method described previously [10, 12]. The brush border membranes were washed off the pellets, resuspended in the sucrose, and centrifuged again for 5 min at. The supernatants were discarded and the loosely packed brush border membranes were washed off the diminishing densely packed contaminating portion of the pellets. The membranes were suspended in a medium of 300 mM mannitol, buffered with 1 mM Tris-Hepes’ (1 mM Hepes adjusted with Tris hydroxide) to pH 7.5, and centrifuged at 27,000 x g for 5 min. The supernatants were discarded and the pellets resuspended in the buffered mannitol medium. Uptake of o-glucose by the renal brush border membranes was measured by the Millipore filtration technique as detailed previously (6, lo), with two modifications. All water used for preparing media and reagents was triple distilled and deionized, and all solutions were filtered through 0.45.Frn Millipore filters [11] prior to use
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