Chloride Transport Across Rat Ileal Basolateral Membrane Vesicles

The present study was designed to investigate CI”⁻ transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl^- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO₃⁻ gradient, or their combination could elicit any stimulation of Cl⁻ transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl⁻ uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na⁺ and HCO₃⁻ gradients did not stimulate Cl⁻ uptake compared with the combination of K⁺ and HCO₃⁻ gradients or no HCO₃⁻ gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl⁻ uptake. Cl⁻ uptake was stimulated in the presence of combined Na⁺ and K⁺ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl⁻ uptake in the presence of combined Na⁺ and K⁺ gradients. Kinetic studies of bumetanide-sensitive Cl⁻ uptake showed a V_max of 5.6 × 0.7 nmol/mg protein/5 sec and a K_m of 30 × 8.7 mM. Cl⁻ uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K⁺ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl⁻ transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na⁺/K⁺/2 Cl⁻ process. Cl⁻ uptake is not enhanced by pH gradient, HCO₃⁻ gradient, their combination, or outwardly directed HCO₃⁻ and Na⁺ gradients.