A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO 3− and SITS in rat intestinal epithelium

Abstract

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 amount of 125I − driven by a large Cl − gradient (> 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. No 3 −, 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 SO 4 2−, SITS and HCO 3 − on Cl − I − exchange. Approximately 30% of the Cl − I − exchange was insensitive to SO 4 2− and showed a relatively low sensitivity to SITS (IC 50 = 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 SO 4 2− (IC 50 = 0.5 mM) and SITS (IC 50 = 2.5 μM) 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.