THIAMINE OUTFLOW FROM THE ENTEROCYTE - A STUDY USING BASOLATERAL MEMBRANE-VESICLES FROM RAT SMALL-INTESTINE

1. Rat small intestinal basolateral membrane vesicles (BLMVs) were pre pared and found to be 31 % non-vesiculated and 69 % vesiculated, 4.9 % right side out and 63.8% inside out. 2. Thiamine uptake by BLMVs foll owed a hyperbolic time course reaching equilibrium after 60-90 min inc ubation. Uptake was not affected by the transmembrane potential or by the presence or absence of Na+ or K+ in the incubation medium.3. At co ncentrations below 1.25 muM, [H-3]thiamine was taken up mainly by a sa turable mechanism with an apparent Michaelis-Menten constant (K(m)) = 1.32 muM and maximal flux (J(max)) = 1.93 pmol (mg protein)-1 (4 s)-1. At higher concentrations, a non-saturable mechanism prevailed. 4. Onl y 29 % of [H-3]thiamine taken up by the vesicles was membrane bound, t he remaining being translocated into the vesicular space. No thiamine phosphoesters could be detected inside the vesicles. 5. In the absence of ATP, the Na+-K+-ATPase inhibitors ouabain, frusemide and vanadate reduced thiamine uptake by 35, 30 and 15 % respectively. 6. In experim ents conducted with K+ inside the vesicles and Na+, Mg2+ and ATP outsi de, the time course of thiamine uptake by BLMVs displayed an overshoot (80-90% increment) at 30 s incubation as compared to controls. When A TP was replaced with phosphocreatine, or when NaCl was replaced with i sosmotic amounts of KCl, the overshoot disappeared. 7. The thiamine an alogues pyrithiamine, amprolium and 4'-oxythiamine decreased the ATPas e-dependent transport of [H-3]thiamine by 100, 86 and 31 % respectivel y. 8. These results provide evidence that the transport of thiamine by BLMVs is coupled directly to the hydrolysis of ATP (primary active tr ansport).