Trasporto della tiamina in vescicole di membrane microvillari di rene di ratto.

Microvillous membrane vesicles prepare from the cortex of rats, were suspended in different media, controlling the composition of the intravesicular fluid by prolonged equilibration at 4°C in preincubation buffers of desired composition. Until used they were held on ice. For the uptake experiments the vesicles were incubated at 25°C with tritiated thiamin for suitable times. Then, they were separated with a rapid filtration procedure for the radiometric measusement of the radioactivity taken up. Thiamin uptake was Na+-independent and 53% membrane bound. The concentration curve had a biphasic course which was hyperbolic (saturable) at physiological concentrations (<1.25 uM) and linear at higher ones. Thiamin uptake was three times increased by an outwardly directed H+ gradient (pHin 6 : pHout 7.5), which enhanced only thiamin translocation and maintained biphasic the concentration curve. However, the saturable component displayed kinetic costants 12-fold higher than those in the absence of gradient. Moreover, the saturable component remained unmodified by typical organic cations transported by renal microvillous vesicles, but inhibited by inhibitors of intestinal thiamin/H+, renal guanidine/H+ and Na+/H+ antiporters.