Urate Uptake in Brush Border Membrane Vesicles

Urate Uptake in Brush Border Membrane Vesicles PURPOSE AND RATIONALE 

The urate-anion exchanger system in brush border membrane vesicle, which mediates hydroxyl ion gradient-dependent urate uptake, is the most likely route for the mediation of urate transport in the first step of urate reabsorption in the proximal tubules. Luminal drugs which inhibit urate reabsorption are inhibiting the transport of urate by blocking the urate/anion exchanger. 

After preincubation of the brush border membrane vesicle preparation for 2 h, [2 14 C]urate uptake is initiated by adding 200 pi of incubation medium to 20 pi of the membrane suspension. The incubation medium has the following composition (mmol/1) 150 mannitol, 2 MgS04, 50 potassium phosphate buffer, pH 6.0 or 7.5, 0.02 [2-14 C]urate, and various concentrations of the inhibitor. At 10 s after the addition of the incubation medium, 200 pi portions of the suspension are pipetted onto the center of prewetted cellulose acetate filters kept under suction. The vesicles retaining on the filter are washed immediately with 5 ml of an ice-cold solution containing 150 mmol/1 mannitol and 50 mmol/1 potassium phosphate buffer, pH 6.0 or 7.5, which is used at the same pH as the incubation medium. Preincubations and incubations are performed at 23 1 °C. Each experiment is performed in triplicate. Corrections are made for the radioactivity bound to the filters in the absence of membrane vesicles. The term of the OH gradient-dependent urate uptake is defined as the difference between the uptakes in the incubation medium at pH 6.0 and that at pH 7.5. The OII gradient-dependent urate uptake at 10 s is assumed to present an initial velocity. 

From a concentration-response curve relating log concentration of drug to the logit activity of the OH gradient-dependent urate uptake for 10 s, 7C50 (concentration producing 50 % of inhibition) is determined by least-squares regression analysis. 

Koga H (1990) Uricosurics inhibit urate transporter in renal brush border membrane vesicles. Eur J Pharmacol 187 303-312 

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