Na+ uptake was studied in colonic enterocyte membrane vesicles prepared from normal and dexamethasone-treated rats. Vesicles from rats treated with dexamethasone demonstrated a fivefold greater 22Na+ uptake compared with vesicles from normal rats. Most of the tracer uptake in membranes derived from treated rats occurred through a conductive, amiloride-blockable pathway located in vesicles with low native K+ permeability and high Cl- permeability. Kinetic analysis of the amiloride inhibition curve revealed the presence of two amiloride-blockable pathways, one with a high affinity (Ki = 9 +/- 1.8 nM), accounting for 85% of the uptake, and one with a low affinity (Ki = 2.2 +/- 0.71 microM), accounting for only 12% of the uptake. Only the low-affinity pathway was detected with vesicles from normal rats. The high sensitivity to amiloride, the dependence on dexamethasone pretreatment, and the relative permeabilities to K+ and Cl- indicate that most of the 22Na+ uptake in membranes derived from treated rats is through a Na+-specific channel located in apical membrane vesicles. Preincubation of the isolated cells from dexamethasone-treated rats at 37 degrees C in Ca2+-free solutions before homogenization and membrane vesicle purification caused a 5- to 10-fold increase in amiloride-blockable 22Na+ uptake compared with vesicles derived from cells maintained at 0 degrees C. The addition of Ca2+, but not of Mg2+, to the incubation solution markedly reduced this temperature-dependent enhancement in 22Na+ uptake. The uptake of 22Na+ into vesicles from normal rats was unaffected by preincubation at 37 degrees C or the addition of Ca+ to the incubation solutions.(ABSTRACT TRUNCATED AT 250 WORDS)