Mechanism of pyrimidine nucleoside uptake in intestinal brush border membrane vesicles.

A large number of synthetic nucleoside analogues have been synthesized with potential for antiviral, antitumor and antiparasitic activity [ 11. The absorption of these compounds after oral administration is poorly understood. Indeed a lot of controversy exists as to whether these drugs are absorbed via predominantly active or passive means. In order to characterise the transport processes involved in the absorption of these drugs it is important to first understand the intestinal absorption mechanisms of endogenous nucleosides. The present study was undertaken in order to characterise the uptake mechanism in intestinal brush border membrane vesicles (BBMV). of three pyrimidine nucleosides; uridme, thymidme and cytidine. These nucleosides were chosen for the study as they are not rapidly mtabolised by BBMV [2]. BBMV were prepared from frozen small intestine of male white New Zealand rabbits [3]. Vesicles were stored frozen in aliquots, under liquid nitrogen until required. Uptake was measured at 25°C using a rapid filtration technique. The reaction was initiated by mixing 20pl of BBMV with 40 pl of incubation medium consisting of 5pM radiolabelled nucleoside, 10 mM Hepes-Tris (pH 7.4) and either 100 mM NaSCN, NaCI, KCI, choline chloride or 200mM mannitol. After the required incubation time the reaction was terminated with 1 ml of ice cold solution containing buffered 150 mM NaCl and 0.25 mM phloridizin. After washing with a total of 5ml of stop buffer the filters were dissolved in scintillant and counted. In order to determine non specific binding (nsb) of nucleeside to the filter, uptake was carried out in the absence of vesicles. Prewetting the filter with a 50pM nucleoside solution reduced nsb to the filter. Uptake data was corrected for nsb and expressed as pmol/mg of protein. All three nucleosides showed a marked stimulation in the presence of sodium ions with a transient overshoot of the intravesicular concentration above its equilibruim value. Accumulation of all nucleosides reached a maximum value between 10 and 20 sec, with equilibrium being reached within 5 minutes. The specificity of extravesiculax sodium in stimulating nucleoside uptake was studied by comparing uptake in the presence of chloride salts of various monovalent cations The uptake in the presence of the other monovalent cations showed no peak overshoot and was similar to that in the presence of no ions (ie mannitol). Fig. 1. shows the time course of thymidine uptake into rabbit intestinal BBMVs in the presence of the various ions.