A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO3- and SITS in rat intestinal epithelium.
A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.
Vaandrager
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Department of Biochemistry I, Medical Faculty, Erasmus University Rotterdam, The Netherlands.
De Jonge
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HR
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology*; D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions; D001639:Bicarbonates / Q000378:metabolism / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D007457:Iodine Radioisotopes / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials; D008871:Microvilli / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013267:Stilbenes / Q000494:pharmacology*; D013431:Sulfates / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology
D027321:Anion Transport Proteins; D000838:Anions; D001639:Bicarbonates; D002352:Carrier Proteins; D002712:Chlorides; D007457:Iodine Radioisotopes; D013267:Stilbenes; D013431:Sulfates; D014634:Valinomycin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D011189:Potassium Chloride