Simon M. Jarvis

Nucleoside transport in animal cells

Nucleosides have a mult i tude of biochemical and physiological effects. For example, adenosine is believed to participate in processes such as modulation of immune response% regulation of blood flow, p late let aggregation, renal function, neuromodulation in the central nervous system, neurotransmission, muscle contraction, and numerous other biological processes ( i -3 ) . Inosine has been postulated as an in vivo energy source for adult pig erythrocytes, ceils unable to metabolize glucose (4 ,5) . In addit ion, nucleoside analogues are widely used as the rapeu t i c agents for neoplas t ic and viral diseases in man and to a lesser ex t en t as an t iparas i t ic agents (6) . However , be fore the above biological e f f e c t s of nucleosides and their analogues are in i t ia ted these c o m p o u n d s have general ly to be t ranspor ted out of or into cells. Thus , n u c l e o s i d e t rans loca t ion is an impor tan t de t e rminan t of the biological ac t iv i ty of nucleosides and nucleoside drugs. This review is no t a c o m p r e h e n s i v e survey of the large l i t e r a tu re on nucleoside uptake; for this readers should re fe r to two r ecen t review ar t ic les (7~g). Instead, we have a t t e m p t e d to highlight and discuss r ecen t o b s e r v a t i o n s r e l e v a n t to the g e n e r a l m e c h a n i s m s of nuc leos ide t r a n s p o r t . Most of t h e s e o b s e r v a t i o n s c o m e f ro m s t u d i e s on mammal ian red cells and as a consequence this review will c o n c e n t r a t e mainly on the proper t ies of the nucleoside t r anspor t e r in e r y t h r o c y t e s . In p a r t i c u l a r , we discuss the kinet ics and inhibition of nucleoside transport~ the proper t ies of the speci f ic potent nuc leos idet ranspor t i n h i b i t o r n i t r o b e n z y l t h i o i n o s i n e ( 6 [ L t n i t r o b e n z y l t h i o l ] 9 B D-r ibofuranosylpur ine; NBMPR)9 the regulat ion of nucleoside t ranspor t , and the molecular proper t ies of the t r anspor te r .

Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine and p-chloromercuriphenyl sulfonate.

SummaryThe sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial,p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 μM) by NBMPR was biphasic −35% of the transport activity was inhibited with an IC50 value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 μM. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparentKm for uridine 50±18 and 163±28 μM, respectively). The two transport systems also differed in their sensitivity topCMBS. NBMPR-insensitive uridine transport was inhibited bypCMBS with an IC50 of ∼25μM, while 1 mMpCMBS had little effect on NBMPR-sensitive transport by intact cells.pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by β-mercaptoethanol, suggesting that thepCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [3H]NBMPR binding to the cell membrane (apparentKd46±25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25±6 molecules/site per sec at 22°C.pCMBS had no effect on [3H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [3H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent Mr on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR andpCMBS.

2-Chloroadenosine, a permeant for the nucleoside transporter.

Human erythrocytes were shown to possess a saturable uptake mechanism for 2-chloroadenosine (apparent Km 23 microM, 22 degrees). Uptake by this route was inhibited by nitrobenzylthioinosine, uridine and adenosine, but adenine had no effect. In addition, uridine caused the countertransport of 2-chloroadenosine and vice versa. 2-Chloroadenosine was also shown to be an apparent competitive inhibitor of uridine influx (apparent Ki value of 33 microM) and high-affinity nitrobenzylthioinosine binding (apparent Ki 0.18 mM). The apparent Ki value for inhibition of uridine influx was close to the apparent Km value for 2-chloroadenosine uptake. Previous studies [Jarvis et al., Biochem. J. 208, 83 (1982)] have demonstrated that dog erythrocytes do not possess a saturable transport system for uridine and adenosine. Similarly, in the present study, the entry of 2-chloroadenosine into dog erythrocytes was slow and linear with concentration. Nitrobenzylthioinosine (NBMPR) had no effect on the uptake of 2-chloroadenosine into dog erythrocytes. These results demonstrate that 2-chloroadenosine enters human erythrocytes by the same nucleoside carrier as other nucleosides. It is suggested from these data that the previous explanation that the inability of nucleoside transport inhibitors to potentiate the pharmacological effects of 2-chloroadenosine was due to the failure of the nucleoside carrier to accept 2-chloroadenosine as a permeant may have to be reassessed.

Nucleoside transport in human erythrocytes: nitrobenzylthioinosine binding and uridine transport activities have similar radiation target sizes

Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.

Identification of the Adenosine Uptake Sites in Guinea Pig Brain

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high‐affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 ± 0.012 nM; Bmax values of 0.24 ± 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high‐affinity binding. Low concentrations of cyclohexoadenosine (10–300 nM) had no effect on NBMPR binding. These properties of the high‐affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free‐radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000–45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzyl‐thioguanosine.

Radiation inactivation of the human erythrocyte nucleoside and glucose transporters.

The human erythrocyte nucleoside and glucose transporters, identified previously as band 4.5 peptides (apparent Mr 66 000-45 000) on SDS-polyacrylamide gels, have been characterized in situ by radiation inactivation analysis. Target size analysis of lyophilized membranes indicates an apparent Mr of 110 000 +/- 12 000 and 124 000 +/- 11 000 for the nucleoside and glucose carriers, respectively. These data suggest that both transporters exist in the membrane as dimers.

Benzodiazepine inhibition of nucleoside transport in human erythrocytes

The interaction of several benzodiazepines (BDZs) with the nucleoside transport system of fresh erythrocytes from humans was investigated. The affinities of BDZs for the nucleoside transport system were estimated by measuring BDZ inhibition of (a) the site-specific binding of nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport, and (b) the uridine transport processes, zero-trans influx, zero-trans efflux, and equilibrium exchange influx. The BDZs inhibited both the inward and outward transport processes, and, for individual agents, inhibition constants (Ki) were similar for the inhibition of each transport process and for the inhibition of the site-specific binding of nitrobenzylthioinosine. The order of potencies of the BDZs in their interactions with the nucleoside transport mechanism (Ro 5-4864 greater than diazepam greater than clonazepam greater than lorazepam greater than flurazepam) is distinct from the potencies of these compounds at BDZ recognition sites. The affinities of the BDZs for the nucleoside transport system, which are about 1000-fold lower than for BDZ recognition sites, suggest that significant inhibition is unlikely to occur with the plasma concentrations (less than 1 microM) that result from usual anxiolytic doses of these agents.

Photoaffinity labelling of the nucleoside transporter of cultured mouse lymphoma cells

Nitrobenzylthioniosine (NBMPR), a potent and specific inhibitor of nucleoside transport, is bound reversibly by high affinity sites on nucleoside transporter proteins of erythrocyte membranes and, upon photoactivation, NBMPR molecules become covalently bonded to the sites. This study showed that [3H]NBMPR molecules reversibly bound to intact S49 and L5178Y mouse lymphoma cells became covalently bound upon exposure to UV light. Electrophoretic analysis of plasma membrane fractions from the labelled cells showed that 3H was present in polypeptides which migrated as a major band with an apparent M r of 45000–65000.

The Use of Ligands in the Study of the Nucleoside-Transport Complex

Permeation of physiological and cytotoxic nucleosides across the plasma membrane of animal cells is mediated largely by nucleoside-specific transporters sensitive to inhibition by nanomolar concentrations of nitrobenzylthioinosine (NBMPR) and related 6-thiopurine ribonucleosides. Transport inhibition by NBMPR results from reversible high-affinity binding of ligand to cell membranes, an association that represents a specific interaction with functional nucleoside transport proteins. The commercial availability of high-specific activity [3H]-NBMPR within the last few years has led to significant advances in our knowledge of the kinetic and molecular properties of this carrier system. In the present chapter we detail and discuss the methodologies associated with the use of this ligand.