Chloride transport across rat ileal basolateral membrane vesicles.
The present study was designed to investigate Cl- 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.
Daher
M
M
Department of Pediatric Gastroenterology and Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
Acra
S
S
Dykes
W
W
Ghishan
F K
FK
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007082:Ileum / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009994:Osmolar Concentration; D011188:Potassium / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000502:physiology; D014634:Valinomycin / Q000494:pharmacology
D002712:Chlorides; D002034:Bumetanide; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D012964:Sodium; D011188:Potassium