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 Vmax of 5.6 × 0.7 nmol/mg protein/5 sec and a Km 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.
|Original language||English (US)|
|Number of pages||7|
|Journal||Proceedings of the Society for Experimental Biology and Medicine|
|State||Published - Dec 1992|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)