1 16s Biochemical SocietyTransactions (1 992) 20
Mechanism of pyrimidine nucleoside uptake in intestinal brush border membrane vesicles. S. Tanna, C. Wood and M.J. Lawrence, Department of Pharmacy, Kings College London, University of London, London. SW3 6LX.
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.
2
1
0
00 Incubation time (sec)
Fig. 1. Time course of thvmidinc llptake into rabbit intestid BBMVs in the D R S ~nce of various ionic gradientS, Table 1 shows the effect of the various monovalent cations and anions on the rate of nucleoside uptake. Uptake at 10 secondswere used to calculate the rate.
Table 1. Uridine: Thymidine: Cytidine:
Rate of uDtake of fDmoVme Droteids). . ., aSCN NaCl KCI C h o w . s
Y .
0.20 0.19
0.11 0.12
0.16
0.11
0.04 0.03 0.03
0.04 0.03 0.03
These results suggest a high sodium specificity for nucleoside uptake by BBMV. In addition to the cation affecting uptake the extent of uptake was effected by the presence of different anions. In all cases uptake was higher in the presence of SCN ions compared to C1 ions. A similar electrogenic effect has been observed with nucleoside uptake in renal BBMV [4]. The effect of nucleoside concentration on uptake was studied. It was found that the sodium dependent uptake was saturable at concentrations above 500 p.M.In contrast uptake in the presence of other monovalent ions was linear as a function of concentration. It is interesting to note that the higher concentrations are comparable to those expected in vivo after oral administration of synthetic nucleoside drugs. From these results it is'concluded that the brush-border membrane of rabbit enteroctes contain a broad specificity sodium-dependent nucleoside transporter.
The authors wish to thank the Wellcome Foundation for their support. 1. Walker, R.T., De Clercq, E. & Eckstein, F. (1979) in Nucleoside Analogues: Biology and Medical Applications, Plenum Press, London. 2. Vijayalakshmi, D. & Belt J.A. (1978) J. Biol. Chem., 263, 19419-19423. 3. Kessler, M., Acuto, O., Storelli. C., Murer, H., Muller, M. & Semenza, G (1978) Biochim. Biophys. Acta.. 506, 136159. 4. Williams, T.C. & Jarvis, S.M. (1987) Biochem. SOC.Trans., 15. 1134-1135.
Mechanism of pyrimidine nucleoside uptake in intestinal brush border membrane vesicles. S. Tanna, C. Wood and M.J. Lawrence, Department of Pharmacy, Kings College London, University of London, London. SW3 6LX.
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.
2
1
0
00 Incubation time (sec)
Fig. 1. Time course of thvmidinc llptake into rabbit intestid BBMVs in the D R S ~nce of various ionic gradientS, Table 1 shows the effect of the various monovalent cations and anions on the rate of nucleoside uptake. Uptake at 10 secondswere used to calculate the rate.
Table 1. Uridine: Thymidine: Cytidine:
Rate of uDtake of fDmoVme Droteids). . ., aSCN NaCl KCI C h o w . s
Y .
0.20 0.19
0.11 0.12
0.16
0.11
0.04 0.03 0.03
0.04 0.03 0.03
These results suggest a high sodium specificity for nucleoside uptake by BBMV. In addition to the cation affecting uptake the extent of uptake was effected by the presence of different anions. In all cases uptake was higher in the presence of SCN ions compared to C1 ions. A similar electrogenic effect has been observed with nucleoside uptake in renal BBMV [4]. The effect of nucleoside concentration on uptake was studied. It was found that the sodium dependent uptake was saturable at concentrations above 500 p.M.In contrast uptake in the presence of other monovalent ions was linear as a function of concentration. It is interesting to note that the higher concentrations are comparable to those expected in vivo after oral administration of synthetic nucleoside drugs. From these results it is'concluded that the brush-border membrane of rabbit enteroctes contain a broad specificity sodium-dependent nucleoside transporter.
The authors wish to thank the Wellcome Foundation for their support. 1. Walker, R.T., De Clercq, E. & Eckstein, F. (1979) in Nucleoside Analogues: Biology and Medical Applications, Plenum Press, London. 2. Vijayalakshmi, D. & Belt J.A. (1978) J. Biol. Chem., 263, 19419-19423. 3. Kessler, M., Acuto, O., Storelli. C., Murer, H., Muller, M. & Semenza, G (1978) Biochim. Biophys. Acta.. 506, 136159. 4. Williams, T.C. & Jarvis, S.M. (1987) Biochem. SOC.Trans., 15. 1134-1135.
