Uptake by central nervous tissues as a mechanism for the regulation of extracellular adenosine concentrations

Neurochem. Int. Vol. 6, No. 5, pp. 613~32, 1984 Printed in Great Britain. All fights reserved

0197-0186/84 $3.00+ 0.00 Copyright © 1984 Pergamon Press Ltd

COMMENTARY UPTAKE BY CENTRAL NERVOUS TISSUES AS A MECHANISM FOR THE REGULATION OF EXTRACELLULAR ADENOSINE CONCENTRATIONS P. H. Wu and J. W. PHILLIS Department of Physiology, Wayne State University, School of Medicine, Detroit, MI 48201, U.S.A. (Received 29 January 1984; accepted 27 March 1984)

Abstract--The various facets of the uptake of adenosine by central nervous tissues are described. The uptake process includes the transport of nucleoside across neuronal and glial plasma membranes and its metabolism within the cell. Much of the transported adenosine is phosphorylated into adenosine nucleotides. Inhibitors of adenosine uptake increase extraceUular levels of adenosine and can thus potentiate its pharmacological actions. This may be an important component in the actions of various groups of psychoactive drugs.

Nucleosides, namely purine nucleosides (adenosine the same type of mechanism. Phillis et al. (1979) and guanosine) and pyrimidine nucleosides (cytidine have demonstrated that iontophoretic application of and uridine), are important constitutents of the cen- adenosine onto rat brain cortical neurons will depress tral nervous system (CNS). It is known that the brain the spontaneous discharges of these neurons. Simullacks the complete apparatus for pyrimidine synthesis taneous applications of known adenosine uptake (Mcllwain and Bachelard, 1971), therefore the pyri- inhibitors such as dipyridamole, papaverine and midines present in the CNS have to be derived either hexobendine enhance the depressant action of adenofrom the re-utilization of pyrimidine or transported sine. The adenosine uptake inhibitors can inhibit from the periphery. An understanding of the mech- neuronal firing even in the absence of iontophoretianisms that control the supply of these nucleic acid caUy applied adenosine, suggesting that in normal precursors in the brain is of great importance. Re- conditions, neurons are subject to a continuous tonic cently, the status of nucleosides in the CNS has been regulation by endogenously released adenosine extended, so that now they are not only considered (Phillis et al., 1979). These experiments have clearly to serve as precursors for nucleic acid synthesis, but demonstrated the importance of the uptake system in one of the nucleosides, adenosine, has gained recog- regulating extracellular adenosine levels. Nimit et al. nition as a neuromodulator for central nervous func- (1981) have also shown a potentiating effect of adenotions (Phillis and Wu, 1981). In the light of the sine uptake inhibitors on adenosine stimulation of growing acceptance of adenosine as a regulator of cyclic A M P formation in brain slices. Wu et al. (1982) neuronal activity in the CNS, the mechanisms in- have demonstrated that adenosine inhibits K ÷volved in the regulation of extracellular concen- evoked Ca 2+ uptake by rat brain synaptosomes. This trations of adenosine have received more attention. It inhibition of Ca 2+ uptake was potentiated by diis known that the extracellular concentration of many pyridamole, a potent adenosine uptake inhibitor. The putative neurotransmitters including monoamines evidence therefore suggests that adenosine is a major and amino acids are regulated by re-uptake systems regulator of neuronal activity and that the uptake (Iversen, 1967) in the nerve endings. The re-uptake process is an important factor in terminating adenosystem will terminate the action of a putative neuro- sine's action. Despite the great importance of the transmitter by removing it from the synaptic cleft, adenosine uptake processes in the CNS, our underthus preventing its continuing action. Many neuro- standing of the mechanisms that control the uptake chemical and neurophysiological studies have shown system is limited. that adenosine's action can also be terminated by The voluminous literature on the nucleoside up613

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P.H. Wu and J. W. PHILLIS

take (transport) is derived mainly from studies on peripheral tisues (see reviews, by Plagemann and Wohlhueter, 1980; Paterson, 1981). These findings will be used to form a basis for comparison with and discussion of the results observed in the CNS. Uptake versus transport: a definition Numerous articles deal with either uptake or transport of nucleosides. It is generally accepted that the uptake of nucleosides involves their transport though the plasma membrane by specific carriers and their subsequent intracellular metabolism (i.e. phosphorylation, deamination, etc.). Therefore, it is useful to define the terms uptake and transport at the outset of their discussion. Plagemann and Wohlhueter (1980) and Berlin and Oliver (1975) have defined uptake as the accumulation of radioactivity derived from exogenous labelled substrate within the cell, regardless of metabolic conversion. Thus uptake represents a composite process which involves a series of steps including a nonconcentrative transport system and various cytoplasmic enzymes. Transport is defined as the transfer of a substrate (or the translocation of a chemical substance) across the cell membrane in either direction. It is mediated by a saturable and selective carrier. The importance of defining these terms can be seen from previous reports of discrepancies in a given system where one paper reports the uptake rate as opposed to transport of nucleosides, because the initial uptake velocities do not always reflect those of transport of substrates into the cell. Berlin and Oliver (1975) and Plagemann and Richey (1974) have suggested that the initial uptake velocities can be utilized to represent the velocity of the transport process. However, recent studies have shown that transport may be so rapid that intraeellular steady-state concentrations of free substrate fall below the estimated Michaelis-Menten constant of transport (Plagemann and Wohlhueter, 1980). Therefore, in assessing any results involving the accumulation of extracellular nucleoside within cells, it is necessary to consider the method that was employed to carry out the initial investigation. Metabolism o f adenosine in CNS Metabolism of adenosine and other nucleosides takes place very readily in many cell types including those in central nervous tissues. In peripheral tissues, adenosine, deoxyadenosine and cytidine are phosphorylated or deaminated, and guanosine, inosine, uridine and thymidine phosphorylated and then subsequently further metabolized. Enzymes involved in

the metabolism of adenosine and adenosine related nucleotides are AMP deaminase (3.5.4.6), adenosine deaminase (3.1.4.4), 5'-nucleotidase (3.1.3.5), adenosine kinase (2.7.1.20), and possibly nucleosidase (3.2.2.1). The predominant role that each of these enzymes plays in the metabolism of adenosine is dependent on the type of tissue preparation and experimental conditions. The brain o f intact animals. When the blood-brain barrier was by-passed by perfusing [U-~4C]adenosine through the cerebrospinal fluid (CSF) convexitycisternal system, Winn et al. (1980) were able to show by comparisons of the specific activities of adenosine and adenine nucleotides in the cerebrospinal perfusion fluid, that the incorporation of adenosine was 7-fold greater than that of other nucleosides or hypoxanthine, and that adenosine was directly phosphorylated to AMP. Berne et al. (1974) have also shown that the intrathecal administration of [U-~4C]adenosine resulted in heavy labeling of the brain, with 87~ of the radioactivity incorporated in the form of nucleotides. These studies seem to suggest that the principal metabolites for adenosine are in the form of phosphorylated products. lsolated brain slices. Santo et al. (1968) showed that [~4C]adenosine can be readily taken up by brain slices. Most of the labelled 14C found in the tissues had been incorporated into adenine nucleotides (some 79-85~). Similarly, using guinea-pig brain slices, Shimizu et al. (1972) demonstrated that the majority of radiolabelled [~4C]adenosine added to the incubation mixture was recovered from brain slices as [~4C]nucleotides. Therefore, it was concluded that adenosine was actively incorporated into slices and phosphorylated almost quantitatively to adenine nucleotides (Shimizu et al., 1969). Radioactivity found in mouse brain slices after incubation with [14C]adenosine was also associated with adenosine nucleotides. Banay-Schwartz et al. (1980) showed that [~4C]adenosine was rapidly converted to adenine nucleotides. When 0.01 mM of [~4C]adenosine was incubated at 37°C for 5 min with mouse brain slices, a 77~ conversion took place; and if [~4C]adenosine was incubated for 10 min at 37°C, only 9 ~ of radioactivity present in the brain slices was in the form of unmetabotized [~4C]adenosine. These findings are consistent with the conclusion that, for in vitro experiments using brain slice preparations from various animal species, phosphorylation of adenosine to nucleotides is the principal metabolic pathway. Synaptosomalpreparations. Various brain synaptosomal preparations have been used to investigate the uptake and metabolism of adenosine. These prepara-

Uptake of adenosine by central nervous tissues tions include synaptosomes and synaptosomal beds. Kuroda and Mcllwain (1974) showed that after a 20 min incubation with [14C]adenosine at 37°C, the [t4C]adenine derivatives extracted from synaptosome beds consist of labelled adenine nucleotides (77.7%), inosine (2.8%), cyclic AMP (0.2%), hypoxanthine (0.4%), adenosine (16.8%) and adenine (2.1%). The composition of the radioactive metabolites did not change even when the concentration of [14C]adenosine used in the incubation was increased 10-fold. Bender et al. (1980) also showed that adenine nucleotides were the major metabolites of adenosine incubated with a rat brain synaptosome suspension. In this experiment approx 51% of radioactivity recovered from the synaptosomes after their incubation with [3H]adenosine was in the form of adenosine. Hypoxanthine, cyclic AMP, inosine and nucleotides contributed 3.1, 1.2, 16 and 28.7% of the radioactivity respectively. The incubation was carried out in a medium saturated with 95% 02 and 5% CO2 for 15 min at 37°C. In other experiments, when a shorter (30s) incubation time was used, adenosine contributed up to 70% of total radioactivity recovered from the labelled synaptosomes. Hypoxanthine, cyclic AMP, inosine and nucleotides contributed 1.5, 2.5, 14.0 and 12.0% respectively (Bender et al., 1981a). These observations seem to suggest that the extent of conversion of adenosine to adenine nucleotides is dependent on the incubation time used. Consistent with this observation, Barberis et al. (1981) demonstrated that adenine nucleotide levels increased in guinea pig synaptosome beds with an increase in the length of incubation. Adenine nucleotide concentration increased from approx 46% of total radioactivity at 1 min to approx 92% at 60 min incubation at 37°C, whereas adenosine levels decreased from 47% at 1 min to 4.6% at 60min of incubation. Isolated brain capillaries. Adenosine metabolism is known to occur in the peripheral vascular system (see review by Olsson and Patterson, 1976). The metabolism of adenosine in brain capillaries had not been studied until very recently. Wu and Phillis (1982) showed that adenosine can be taken up by isolated rat brain capillaries. The adenosine was very rapidly metabolized. After incubation of [3H]adenosine with isolated brain capillaries for 10 min at 37°C, nucleotides were the major metabolites consisting of 55.6% of the total radioactivity extracted from the tissue. Adenosine, hypoxanthine, cyclic AMP and inosine contributed 35.8, 4.7, 0.9 and 3.0% respectively. This observation is consistent with previous findings that prolongation of incubation time will result in the

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formation of nucleotides from nucleoside substrates and that phosphorylation is a very active process in the cellular metabolism of nucleosides. Factors that effect adenosine metabolism. The metabolism of adenosine incorporated into brain tissues varies with the type of brain tissue preparation, the length of incubation, the temperature and the adenosine concentration used for the incubation. Generally, a greater percentage of adenosine is converted to nucleotides in intact animal brain, brain slices and brain vascular tissue. In the case of synaptosomal suspensions, Bender et al. (1981) have shown that with a 30 s incubation time at least 70% of the adenosine remains in the tissue without being metabolized. Even when the incubation time is increased to 15 min, adenosine still contributes up to 50% of the total adenosine metabolites extracted from the synaptosomes. Barberis et al. (1981) showed that in guinea pig brain synaptosome beds, up to 90% of adenosine initially taken up by the tissue preparation was converted to adenine nucleotides. These results may be due to species differences. Olsson and Patterson (1976) have indicated a need for a more detailed examination of the oxygen-adenosine interaction with special emphasis on the importance of controlling pO2 in studies of the metabolic effects of adenosine in vascular smooth muscle. It is possible that the same type of emphasis should also be stressed in studies of the metabolism of adenosine in various tissue preparations. Model systems for studying the transport of nucleoside (adenosine) As indicated in the previous section, transport is defined as the transfer of substrate across the membrane. It does not involve the metabolism of transferred substrate. Based on this definition, several models have been proposed for the study of nucleoside transport (Lieb and Stein, 1974; Eilam and Stein, 1974). The experimental designs are strictly applicable only to ceils which fail to metabolize the substrate or in which the metabolism is inhibited, for instance by depletion of ATP with KCN and iodoacetate treatment. One model that has been developed for the study of nucleoside transport is the human erythrocyte. In this section, we intend to introduce only those procedures which have been developed to test the model. A detailed description of the various parameters which regulate the transport process of the model can be found in a review article by Plagemann and Wohlhueter (1980) and the original articles by Lieb and Stein (1974) and Eilam and Stein (1974).

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Zero-trans influx and efflux. The zero-trans (Zt) procedure is usually applied to measure the transport of a substrate from one side of the membrane where substrate concentration is varied (the cis side) to the other side where substrate concentration is initially zero (the trans side). The assumption has been made in this case that the intracellular nucleoside concentration (the trans side) is low and is considered to be zero. Uridine has been used as the substrate for the estimation of various kinetic parameters in human erythocytes, employing the Zero-trans (Zt) procedures, because it is known that uridine cannot be metabolized in these cells. The estimated kinetic constants vary from Michaelis-Menten constants of 710 (Oliver and Paterson, 1971) to 400/~M (Cabantchik and Ginsburg, 1977). Apparently, the difficulty in getting an accurate measurement of the kinetic constants is due to the rapidity of nucleoside transport in most animal cells. It has been found that a modification of Lieb and Stein's equation was necessary (Eilam and Stein, 1974; Wohlhueter et al., 1978). Despite various modifications made to improve the accuracy of the procedure for the estimation of the kinetic constants, Plagemann and Wohlhueter (1980) concluded that although the velocities estimated from the procedure yielded reasonable Michaelis-Menten hyperbolae, the estimated maximum velocities grossly underestimated the velocity of zero-trans (outside~inside). Equilibrium exchange inward and outward. This procedure differs from Zero-trans by assuming that the substrate concentration at the two sides of the membrane is held equal. The movement of substrate back and forth is a function of time and substrate concentration. Eilam and Stein (1974) have solved the equation using a simple carrier model. Later, Cabantchik and Ginsburg (1977) applied this approach to measure the equilibrium exchange of uridine by human erythrocytes. Wohlhueter et al. (1979) used the equation to study the thymidine equilibrium exchange in thymidine kinase-deficient Novikoff cells. The Michaelis-Menten constant and maximum velocity showed considerable differences in various studies. For example, the Michaelis-Menten constant was 5300#M in one study (Pickard and Paterson, 1972) whereas it was 1310#M in another (Cabantchik and Ginsburg, 1977). Inflnite-trans procedure. Infinite-trans procedure allows one to measure the movement of substrate at a given concentration from one side of the membrane (outside, cis side) to the other side (inside, trans side) where the internal concentration of the substrate is greater than the Michaelis-Menten constant of the

transport system (i.e. the internal concentration of substrate---,~). The important concept of the infinite-trans procedure is that transporting the substrate against an internal concentration gradient (countertransport) is generally considered one of criteria for defining a carrier-mediated transport system. In the infinite-trans procedure, the substrate is transported against such a gradient inside (trans side) of the cell membrane. This procedure allows a more accurate assessment of the kinetic parameters than those calculated by the Zero-trans procedure which often underestimates the velocity of transport system and thus presents a misleading conclusion of carrier asymmetry (Plagemann and Wohlhueter, 1980). Infinite-cis procedure. Two infinite-cis protocols have been used to study nucleoside transport in animal cells. Lieu et al. (1971) measured the movement of uridine in the presence of various nucleotides. This procedure measures the net movement of substrate from the cis side of the membrane where it is present at a concentration much higher than the Michaelis-Menten constant of the transport system. Cabantchik and Ginsburg (1977) also applied this procedure to measure the velocities of infinite-cis and infinite-trans of net uridine transport in human erythrocytes. The velocity was estimated by measuring the initial rate of transport of radioactive substrate from one side (cis-side) of the membrane, where radioactively labelled substrate was present at a very high concentration, to the trans-side where radioactively labelled substrate concentration was varied. The other protocol which is referred to as "accelerated exchange diffusion" measures the unidirectional flux of substrate. In this case, the movement of radioactivity from the cis-side of the membrane, where labelled substrate is present at a concentration greater than that of the Michaelis-Menten constant, to the trans-side, where the concentration of non-labelled substrate concentration is varied, is measured (Cass and Paterson, 1972). An unique feature of the infinite-cis procedure is that it allows one to determine the symmetry of the carrier with respect to the mobility of "loaded" and "unloaded" carrier. All of the above mentioned procedures have been applied to measure the transport of uridine into human erythrocytes (Cabantchik and Ginsburg, 1977). The conclusions derived from these experiments have been (1) the transport of nucleosides is consistent with the idea of a simple carrier mediated mechanism, (2) the carrier of the human red blood cells exhibits asymmetry, in that the loaded carrier moves faster than the unloaded carrier and the

Uptake of adenosine by central nervous tissues unloaded carrier moves approx four times more rapidly in the direction of outside to inside than that of inside to outside, (3) the loaded carder moves with equal rapidity in either direction.

Phosphorylation of nucleosides The model systems described above operate under the assumption that nucleosides were "transported" across the membranes, and were not further "metabolized" to other metabolites. Events other than the simple permeation of substrates were not considered. However, it is known that many nucleosides can be metabolized by either membrane bound or cytosolic cellular enzymes to form other metabolites thus "trapping" the modified form of substrate inside the cell. Phosphorylation of nucleosides is an important ongoing process in the cell. Therefore, a model system proposing coupled transport and phosphorylation must be considered. The advantage of proposing such a model is that, although it only represents an oversimplified version of cellular process, it does represent a system that addresses itself to the physiological realities. Wohlhueter and Plageman (1980) proposed this model to investigate (l) the relationship between the rate of intracellular phosphorylation and extracellular substrate concentration, (2) the relative kinetic properties of transport and phosphorylation, (3) selective inhibitors that affect different targets, i.e. inhibitors influencing transport velocity only, or transport Km only, (4) the contribution of each step toward determining the over-all rate of substrate uptake into the cell. By integrating the influx, effiux and phosphorylation components of the reaction, Wohlhueter and Plagemann (1980) were able to conclude that (1) when the velocity of phosphorylation increased relative to velocity of transport system, the concentration of intracellular free substrate concentration will approach zero, while the velocity of uptake reflects that of permeation; (2) if the velocity of transport system is much greater than that of enzyme phosphorylation, then intracellular substrate concentration will approach that in the extracellular compartment and the velocity of uptake reflects that of phosphorylation. While the model system has been proven useful in Novikoff cells (Wohlhueter and Plagemann, 1980), the condition in normal cells usually falls between the above mentioned extremes.

Uptake of adenosine by brain tissues It is difficult to measure the transport of adenosine in mammalian brain tissue because, according to

617

one of the criteria which define transport, it requires that the substrate must not be metabolized inside the cell. The problem is that in brain tissues, adenosine is very rapidly transformed into other metabolites. Although shortening the incubation time may be able to alleviate part of the problem, the transport of nucleoside may be so rapid that a 1-5 s period of incubation it is already over the allowable time limit. It has been reported that in the absence of metabolism, and at concentrations of exogenous substrate less than the Michaelis-Menten constant for "Zerotrans", the transport systems operate so rapidly that the equilibrium for substrate across the membrane can be achieved within 3-20 s at 37°C (Wohlhueter and Plagemann, 1980). The uptake of adenosine and other nucleosides into intact animal brain has been demonstrated by Cornford and Oldendorf (1975) who showed by injecting radioactively labelled nucleosides into the common carotid artery along with second reference isotopes, that the uptake of adenosine and nucleotides into brain was mediated by a measurable, saturable uptake system. They also concluded, by using other nucleosides for their uptake studies, that these nucleosides may utilize a common carrier system. In an attempt to examine the transport of nucleosides by the brain-blood-barder of the mammalian CNS, Wu and Phillis (1982) have shown that the uptake of adenosine by isolated brain capillaries does indeed suggest the presence of a measurable, saturable and carrier-mediated uptake system. The nucleosides seem to utilize common carrier sites. The Km and Vm,xvalues of the uptake process are shown in Table 1. Mammalian brain slices have often been used to study the uptake of adenosine into nervous tissues. Shimizu et al. (1972) demonstrated a high affinity uptake of adenosine into brain slices with a K~ value of 19/zM. By comparing the K~ value of the uptake to that of the enzyme adenosine kinase (Kin, 20/~M), they concluded that phosphorylation of adenosine to ATP could be the main mechanism for regulating the adenosine uptake into brain tissue (rate limiting step). In mouse brain slices adenosine was taken up by the tissue via a saturable uptake system with a K~ value of 1.4 x 10-4 M and V ~ value of 7.5 x 10 -5 mol/min/ml tissue water (Banay-Schwartz et al., 1980). Adenosine was found to have the most rapid uptake and reached the highest levels in mouse brain slice preparations among the several nucleosides which were used in the study. Brain synaptosomal preparations have often been used to examine the uptake of adenosine. Bender et al. (1980) observed

618

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Fig. 1. Some biochemical properties of the rapid adenosine uptake system in rat brain cortical synaptosomes. A. Time course of [3H]adenosineuptake from incubation medium into rat cerebral cortical synaptosomes. Fifty microliters (0.4--0.5mg protein) of rat cerebral cortical synaptosomes was preincubated for 2 min at 37°C. Ten microliters of [3H]adenosine (sp. act. 1/~Ci/nmol) was added to the incubation mixture to produce a final concentration of 1/~M of adenosine. The reaction was allowed to continue for different periods of time. (--O--), Total uptake of [3H]adenosine into synaptosomes; (--A--), total uptake of [3H]adenosine into synaptosomes in the presence of 2 raM-cyanide; (--II--), binding of [3I-I]adenosineto the ruptured synaptosomes (blank) values). The results are the mean + SEM of eight separate experiments. B. Initial rate of uptake of adenosine by rat cerebral cortical synaptosomes. Fifty microliters (0.4-0.5 mg protein) of rat brain cortical synaptosomes were incubated for 30 s in the presence of various adenosine concentration. Results are expressed as mean + SEM of eight separate experiments. (--I1--), Total uptake of [3H]adenosine;(--Q--), binding of [3H]adenosineto the ruptured synaptosomes (blank values); (--&--), net uptake of adenosine. C. Lineweaver-Burk plot for adenosine uptake into rat brain cortical synaptosomes. Values were obtained from the net uptake data presented in Fig. ! B. The graph reveals that uptake of adenosine into rat brain cortical synaptosomes had a K= of 0.9/~M and a V~x of 5.26pmol/mg protein/30 s. (Bender et al., 1981a)

that rat brain cortical synaptosomes take up adeno- about I min after the start of incubation (See Fig. 1). sine at two high affinity uptake sites when the brain This uptake system is designated as a rapid adenosine uptake system in rat brain synaptosomal preparapreparation was incubated at 37°C for 15 min. The tions. A detailed study of this adenosine uptake adenosine uptake appears to follow the system has revealed that adenosine is taken up by a Michaelis-Menten kinetics showing Km values for high affinity uptake process showing a Km value of one site of 1.0 #M (high affinity A site) and for the other site of 5.3 # M (high affinity B site). The VmaX 0.9/~M and V~x value of 5.26 pmol/mg protein/30 s. values for these two sites are 1.7 pmol/mg protein/ The uptake is apparently not affected by the inclusion min and 6.8pmol/mg protein/min, respectively. of 2raM cyanide (Bender et al., 1981a), suggesting that the uptake of adenosine may be mediated by a Bender et al. (1981a) have also observed that the uptake of adenosine into rat brain cortical syn- facilitated diffusion process, which is saturable and aptosomes occurs very rapidly. It reaches saturation temperature dependent. A more in depth look into

Uptake of adenosine by central nervous tissues the mechanism of the rapid adenosine uptake will be presented in the next section. Barberis et al. (1981) showed that there is a high affinity adenosine uptake system in guinea-pig brain neocortical synaptosomal preparations. The system "transports" adenosine with a Km value of 21 #M. They suggested that because of the close agreement between the Km values of adenosine uptake system and that of partially purified adenosine kinase, the transport system for adenosine may be tightly coupled to the adenosine kinase system. It is interesting to note that the uptake and "transport" of adenosine in guinea-pig brain slice preparations (Shimizu et al., 1972) or in neocortical synaptosome preparations (Barberis et al., 1981) do show close agreement in their Km values. However, the Km value for adenosine uptake in guinea-pig brain seems to be higher than that of rat brain cortical synaptosomes (approx 20-fold difference in Km value). It is not known at this point whether the uptake system in guinea-pig brain tissue is different from that in rat brain tissue. Species differences in the uptake of adenosine by heart tissues have been reported (Hopkins and Goldie, 1971). Adenosine uptake by a glial cell preparation has also been investigated (Hertz, 1978). He showed that adenosine was taken up by cultured astrocytes using a high affinity uptake process with a Km value of 3.4 # M and Vm~ value of 0.36 nmol/mg protein/min. Lewin and Bleck (1979) reported that adenosine was taken up by cultured astrocytoma cells via a high affinity uptake system also (See Table 1). Nonmammalian nervous tissue is able to take up adenosine. It has been shown by Zimmermann et al. (1979) that the purine salvage pathway plays an important metabolic role in the nerve endings of the T o r p e d o electric organ. Adenosine can be taken up by this

619

tissue preparation via a high affinity system exhibiting a saturable uptake process with a Km value of 2 # M and Vm,xvalue of 30 pmol/mg protein/min. These reports indicate that an uptake system for adenosine is present in various nerve tissue preparations. Although there is a probability of animal species differences in the affinity and coupling mechanisms of the uptake system, it is clear that adenosine can be taken up by brain cells (nerve endings, astrocytes, capillaries, etc.) via a high affinity uptake system. Such an uptake system has the properties of a substrate saturable, carrier mediated, facilitated diffusion process.

B i o c h e m i c a l p r o p e r t i e s o f adenosine u p t a k e s y s t e m in CNS

The biochemical characterization of the adenosine (nucleoside) uptake (transport) system had been carried out mainly in peripheral tissue preparations. The conclusion arrived at from previous investigations has been that the uptake of adenosine into cells is mediated by a specific nucleoside carrier in the plasma membrane which is responsible for the transport (uptake) of adenosine and related nucleosides (Berlin and Oliver, 1975). Kessel and Shurin (1968) showed that the accumulation of cytosine arabinoside and deoxycytidine in a subline of L1210 murine leukemia cells was mediated by substrate-saturable and temperature sensitive mechanisms. The metabolic inhibitors 2,4-dinitrophenol, potassium cyanide and iodoacetate did not affect the uptake process. Lieu et al. (1971), by studying the efflux of uridine from erythrocytes, showed that counterflow occurred when a second nucleoside was added to cells which had been loaded with radioactively labelled uridine.

Table 1. Kineticparameters for adenosine uptake in various nervoustissue preparations Vmax Tissue preparation Km(,aM) mol/mgprotein/s mol/ml tissue H20/s Ref. Rat cortical synaptosomcs 0.9 0.175 x 10-12 Bender et al. 1981 Rat cortical synaptosom¢s High affinityA 1.0 0.028 x 10-~2 Bender et al. 1980 High affinityB 5.3 0.113 x 10-.2 Mouse brain slices 140 1.25 x 1 0 - 6 Banay-Schwartz et al. 1980 Cultured astrocytomacells 0.95 1.83 x 10-j2 Lewin and Bleck, 1979 Astrocyt¢s (primarycultures) 3.4 6.0 x 10-12 Hertz, 1978 Guinea-pigbrain synaptosomes 21 Barberis et al. 1981 Guinea-pigbrain slices 19 N.A. Shimizu et al. 1972 Rat brain crude mitochondrial fraction 600 N.A. Davies and Taylor, 1979 Rat blood-brainbarrier 18 N.A. Comford and Oldendrof, 1975 Rat brain capillaries 4.74 0.036 x 10-Iz Wu and Phillis, 1982 Cultured mouse cerebral endothelium 5.0 19 x 10-jz Beck et al. 1983 Torpedo electric organ 2 5.0 x 10-12 Zimmermannet al. 1979 Torpedo electric organ 2.4 0.29 x 10-t2 Meunier and Morel, 1978

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P.H. Wu and J. W. PHILLIS

They concluded that nucleosides share a common membrane carrier. Experiments on the accumulation of adenosine and thymidine into rabbit PMN cells have shown that the uptake is rather specific to nucleosides because free purine and pyrimidine bases have essentially no inhibitory effect on the nucleoside accumulation (Taube and Berlin, 1972). In a study using rat brain cortical synaptosomes, Bender et al. (1981a,b) have shown that the adenosine uptake system in brain tissue shares many of the features that are present in peripheral tissues. The adenosine uptake system in the CNS is a carrier mediated, temperature dependent uptake process which specifically accepts nucleosides as substrates. Nucleotides, purine and pyrimidine bases do not affect the uptake of adenosine. Uptake of adenosine by CNS synaptosomes was reduced by 80% when the incubation was carried out at 0 ~ 10°C. Adenosine uptake increased very rapidly to a maximum when the temperature rose to 50°C. A further increase in incubation temperature to 65°C inhibited the uptake of adenosine by approx 80%. This effect of temperature on the uptake system suggests that adenosine uptake is mediated by a facilitated diffusion. The average Q~0 value for the uptake system was 1.77 which is consistent with uptake being a carrier mediated, facilitated diffusion process (Zimmermann et al., 1979). The necessary activation energy (Ea), as calculated by the integrated Arrhenius equation, was estimated to be 8870 Cal/mol, which is in close agreement with the Ea value obtained from Novikoff rat hepatoma cells (Plagemann, 1970) (Fig. 2). The Q~0 value for the adenosine uptake process in rat brain synaptosomes is also in agreement with that of Torpedo cholinergic nerve endings which showed a Q10 value of 1.8 (Zimmermann et al., 1979). An analysis of the Arrhenius plot of adenosine uptake in rat brain synaptosomes has suggested that no transitional temperature was present in the uptake process. Similar results were obtained with human erythrocyte ghosts (Schrader et al., 1972) and rabbit polymorphonuclear leukocytes (Berlin, 1973). The sudden drop in the uptake rate after 50°C indicates a possible denaturation of the carrier protein. Plagemann and Erbe (1972, 1973) have reported an involvement of sulfhydryl groups with the nucleoside transport carrier. Bender et al. (1981b) also showed that by treating synaptosomes with sulfhydryl reagents, such as p-chloromercuribenzoate and Nethylmaleimide, the uptake of adenosine was competitively inhibited suggesting that substrate binding site on the carrier protein contains - SH group. Such a concept has also been endorsed by Jarvis and

Young (1982) when they presented a molecular model for the nucleoside transport in erythrocytes. One of the important questions has been the substrate specificity of the carrier protein (transporter) for the adenosine uptake system. Results from many studies have indicated that adenosine was transported (taken up) faster and in larger quantities than the other nucleosides (Banay-Schwartz el al., 1980). The debate has been centered on the question of a specific adenosine uptake site which is distinct from that of other nucleosides. The general consensus has been that nucleosides share a common carrier for their transport system, and it appears that the carrier shows a preference for adenosine as its substrate (Banay-Schwartz et al., 1980; Oliver and Paterson, 1971; Lieu et al., 1971). On the contrary, Plagemann and Erbe (1973) addressed the same question by using a kinetic experimental approach in which members of a group of nucleosides were compared, each with respect to inhibition of the transport of the others. They then compared the K~ and K~ values for competing permeants and were able to conclude that guanosine and inosine were transported by a system distinct from other systems for the transport of adenosine and of pyrimidine nucleosides in the cultured Novikoff hepatoma cells. Taube and Berlin (1972) used the same approach as did Plagemann and Erbe (1973); however, Taube and Berlin arrived the conclusion that in rabbit polymorphonuclear leukocytes, nucleosides were transported by a single type of Temp(°C) 60 i

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Fig. 2. Temperature dependence of [3H]adenosine uptake in rat brain cortical synaptosomes. Suspensions of rat brain cortical synaptosomes were incubated with [3H]adenosine 1/aM for 30 s at 0, 10, 22, 37, 50 and 65°C. The sample was equilibrated at the indicated temperature for 5 min before the addition of [3H]adenosine. The initial velocities were plotted against inverse absolute temperature. The curve is the regression line on the uptake data and corresponds to an Arrhenius activation energy of 8.87 K cal/mol (from Bender et al., 1981b).

Uptake of adenosine by central nervous tissues t r a n s p o r t m e c h a n i s m with a b r o a d specificity. B a s e d o n a n accelerative e x c h a n g e diffusion study, Oliver a n d P a t e r s o n (1971) a n d Cass a n d P a t e r s o n (1972, 1973) r e p o r t e d t h a t t h y m i d i n e a n d u r i d i n e were e q u i v a l e n t s u b s t r a t e s for t h e e r y t h r o c y t e t r a n s p o r t system, which accepted both purine and pyrimidine n u c l e o s i d e s as s u b s t r a t e . H o w e v e r , o n e s h o u l d n o t a c c e p t the c o n c l u s i o n w i t h o u t a n y e x c e p t i o n s . A s a m a t t e r o f fact, P a t e r s o n et al. (1975) h a v e i n d i c a t e d that the uridine and thymidine transport systems of H e L a cells are different b e c a u s e these p e r m e a n t s are n o t m u t u a l l y c o m p e t i t i v e . T h u s , it a p p e a r s t h a t s o m e cells m a y h a v e s y s t e m s with b r o a d specificity. I n the

Analogues

621

n e r v o u s tissue p r e p a r a t i o n s , t h e issue is even less conclusive t h a n for p e r i p h e r a l cell p r e p a r a t i o n s . B e n d e r et al. (1981) h a v e e x a m i n e d a series o f n u c l e o s i d e s for their abilities to c o m p e t e w i t h t h e a d e n s o s i n e u p t a k e s y s t e m in rat b r a i n s y n a p t o s o m e s . T a b l e 2 s h o w s t h a t cytidine, inosine, g u a n o s i n e , uridine, t h y m i d i n e , f o r m y c i n a n d t u b e r c i d i n are c o m petitive i n h i b i t o r s o f the a d e n o s i n e u p t a k e process. A d e n o s i n e i s the m o s t suitable s u b s t r a t e for t h e u p t a k e p r o c e s s as it s h o w s a n affinity for the s y s t e m o f 1/~M. T h e o t h e r nucleosides are less active s h o w ing affinities to t h e s y s t e m of: c y t i d i n e ( 3 0 0 # M ) , inosine (340/zM), guanosine (325/zM), uridine

Table 2. Inhibition of adenosine uptake by various analogues IC~0 Value (uM)* Ki Value 0~M)* Type inhibition* > 100 2 1 C 340 170 C 33 17 C 91 16 > 250

% Inhibition~" Adenine 21 Adenosine 66 cAMP AMP ADP ATP Adenosine-Ytetraphosphate > 250 Flavin moninucleotide > 250 fl,y-Methylene-5"-ATP > 250 ~t,fl-Methylene-5'-ADP 4,900 2,500 2-Azidoadenosine 60 30 2-Azido-AMP 900 450 2-Azido-ATP 2,800 1,400 Formycin 4,400 400 C Tubercidin 34 30 C Purine riboside 1,300 60 2-Fluoroadenosine 85 43 2-p-Methoxy-phenyladenosine 45 23 Nitrobenzylthioguanosine 50 25 Nitrobenzylthioinosine 59 30 Guanine 26 Guanosine 650 330 C 23 GMP > 250 GDP > 250 GTP > 250 cGMP > 250 Uracil 6 Uridine 560 280 C 42 UMP > 250 UDP > 250 UTP > 250 Inosine 680 340 C IMP > 250 IDP > 250 ITP > 250 Cytidine 600 300 C 19 Thymidine 850 430 C 48 *Rat brain cortical synaptosomes were preincubated in the presence or absence of various agents at 37°C for 2 rain. The uptake was initiated by addition of [3H]adenosine to the medium, to a final concentration of I x 10-6 M. The incubation was allowed to 30 s. ICs0 values were obtained from the semilogarithmic plots of dose-response curve determined from the inhibition of uptake. (Bender et al., 1981). C: competitive inhibition. tSlices of mouse brain were incubated in a medium containing the inhibitor for 30 rain; then the 10-SM [~adenosine was added, and incubation was continued for an additional 5 rain (5 min uptake). The concentration of inhibitors was 10-2 M, except that guanosine and inosine were 5 x 10 -~ M. (BanaySchwartz et aL, 1980).

622

P.H. Wu and J. W. PHILLIS be transported. Nucleotides would have to be broken down to nucleosides before being transported. This concept is consistent with that concluded from work with peripheral tissue preparations. However, despite the general agreement of results found in CNS tissues with those in peripheral tissues, it is not yet possible to conclude that the molecular structure of CNS nucleoside carrier (transporter) is the same as the one found in peripheral tissues. Further investigation of this subject should yield some interesting and useful findings, providing some insight into the possible functional differences in nucleoside transport systems in CNS and peripheral tissues.

(287/zM) and thymidine (425/tM). Purine and pyrimidine bases show very little affinity for this uptake system (Bender et al., 1980, 1981a). This finding is consistent with the view that adenosine shares a common uptake system with other nucleosides. The possibility that nucleotides can also be taken up by the nucleoside uptake system must be addressed. Adenosine-5'-monophosphate (AMP) has been shown to be competitive inhibitor of adenosine uptake showing a K~ value of 48 # M. The K~ value for adenosine-5'-monophosphate to inhibit adenosine uptake is much lower than that of other nucleosides suggesting that nucleotides may also be used as substrate for the uptake. However, a subsequent study (Bender et al., 1981b) has shown that although nucleotides can inhibit adenosine uptake, they must be degraded to nucleosides before any effect on the uptake system can be observed. A stable analog of ATP, fl, 7-methylene 5'-ATP, showed very low affinity for the uptake system. Furthermore, ct,flmethylene 5'-ADP, an inhibitor of 5'-nucleotidase, can abolish the inhibitory action of A M P on adenosine uptake by rat brain synaptosomes (Fig. 3). These results imply that the carrier system for adenosine uptake is rather selective in its choice of substrates. Nucleosides can be used as substrates for the system. Purine and pyrimidine bases and nucleotides cannot

Inhibitors of adenosine uptake Articles dealing with this subject have recently been published by Phillis and Wu (1982, 1983). In order not to be repetitive, only the highlights of results obtained from the research on adenosine uptake inhibition will be described. Inhibition of uptake by coronary vasodilators. Dipyridamole, hexobendine, lidoflazine and papaverine have been used to study the inhibition of nucleoside transport and adenosine uptake into many tissues (Scholtissek, 1968; Plagemann and Roth, 1969; Plagemann and Erbe, 1974; Zylka and Plagemann,

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Fig. 3. Effect of nucleotide and nucleotide analogues on the rapid uptake of [3H]adenosine in rat brain cortical synaptosomes. A. Inhibition of the rapid adenosine uptake into rat brain synaptosomes by adenine nucleotides. Synaptosomes were preincubated and incubated in the presence of varying concentrations, 5 × 10-6 M to 4 x 10-4 M of (--/X--) AMP, (--C)--) ADP, and ( - - 0 - - ) ATP. Results are expressed as the semilogarithmic plots of concentration-response curves of four separate experiments in triplicate. B. Effect of ~,,~-methylene-ADP on AMP inhibition of the rapid adenosine uptake in brain synaptosomes. The rapid uptake of [3H]adenosine into brain synaptosomes was determined in the presence of varying concentrations of AMP (--O--); varying concentrations of AMP and 10-4M ~¢d3-methylene-ADP (--O--); and varying concentrations of ~,/~-methylene-ADP (--ID--). Results are the serailogarithmic plots of data derived from four separate experiments in triplicate. (Bender et al., 1981b)

Uptake of adenosine by central nervous tissues 1975; Peters and Hausen, 1971; Schrader et al., 1972; Roos and Pfleger, 1972; Rau and Scholtissek, 1970; Crifo et al,, 1973; Kessel and Hall, 1970; Kessel and Dodd, 1972; Turnheim et al., 1978; Plagemann and Sheppard, 1974; Sixma et al., 1976; Woo et al., 1974; Plagemann and Wohlhueter, 1980). In central nervous tissues, Huang and Duly (1974) showed that dipyridamole is an effective inhibitor of adenosine uptake by brain slices. Bender et al. (1980; 1981a) have shown that dipyridamole is a potent inhibitor of adenosine uptake into rat brain synaptosomes. Kinetic studies of the mechanism of inhibition have yielded variable results. Bender et al. (1980) demonstrated that dipyridamole inhibits the uptake of adenosine by synaptosomes in long incubation (15 min) experiments non-competitively, whereas, in a short incubation (30 s) experiment, Wu et al. (1981) showed that dipyridamole inhibited adenosine uptake into rat brain synaptosomes competitively. There is no adequate explanation for such differences, however, it is known that dipyridamole may have more than one site of action. It inhibits adenosine deaminase in dog erythrocytes (Kubler and Bretschneider, 1964) as well as brain phosphodiesterase (Fredholm et al., 1976). Plagemann and Richey (1974) concluded that dipyridamole caused a decrease in the maximum velocity of nucleoside transport by affecting the movement of carrier (transporter). They further qualified the statement by saying that dipyridamole either affected the structure of the membrane directly or interacted rather nonspecifically with integral membrane proteins including transport carriers (Plagemann and Wohlhueter, 1980). There is evidence (Paterson, 1979) suggesting that dipyridamole interacts with a specific site on the carrier because it can inhibit competitively the binding of a nucleoside carrier probe---p-nitrobenzylthioinosine (NBMPR). Papaverine was found to be a competitive inhibitor of adenosine uptake by synaptosome preparations (Bender et al,, 1980; Wu et al., 1981), and by cultured astrocytes (Hertz, 1978). Hexobendine and lidoflazine were also found to be competitive inhibitors of adenosine uptake by brain synaptosomes during short (30s) incubation periods, however, hexobendine was a non-competitive inhibitor of adenosine uptake by brain synaptosomes at long (15min) incubation periods. Other inhibitors of adenosine (nucleoside) uptake are cytochalasin B, streptovaracin, phloretin, phloridzin, aflatoxins, podophyllotoxin and acronycin (Plagemann and Wohlhueter, 1980). Some dipyridamole analogs such as RE 244-BS, RE 642-BS and RE 86-BS are also very potent inhibitors of adenosine uptake having

623

IC50 values in the 0.27-2.7#M range (Phillis and Wu, 1983). Antipsychotic and sedative agents. In this category of drugs, spiroperidol and sulpiride show potent inhibition of adenosine uptake with ICs0 values of 0.7 #M and 0.85 #M respectively. Trifluoperazine is one of the most potent inhibitors amongst the phenothiazines tested. It has an ICs0 value of 1.1 #M. Penfluridol, a diphenylbutylpiperidine, also shows strong inhibition of adenosine uptake with an ICs0 value of 5.6 #M whereas pimozide was less active showing an ICs0 value greater than 0.1 raM. Of particular interest have been the thioxanthenes, et-flupenthixol, a pharmacologically active isomer with an ICs0 value of 11 # M and fl-flupenthixol, an inactive isomer, with an ICs0 value of 60 #M. The difference in the potency of these two drugs was smaller than expected. However, the IC20values show that ~t-flupenthixol has an IC20 value of 0.37/zM and fl-flupenthixol an IC20 value of l0 #M. There is an approximate 30-fold difference in their activities and such a difference correlates well with their sedative potencies. Anxiolytic, hypnotic and sedative agents. The most important compounds in this group of agents are the benzodiazepines. Mah and Daly (1976) showed that diazepam inhibits adenosine uptake by brain slices. In subsequent studies, Phillis et al. (1980, 1981) have compared the abilities of a series of benzodiazepines to inhibit adenosine uptake by rat brain cortical synaptosomes. The order of potency as indicated by IC20 values is clonazepam, nitrazepam, lorazepam, chlordiazepoxide, RO-5-3636, oxazepam and RO 11-6893. The difference in the inhibitory potencies of the two stereoisomers, RO 11-6896 and RO-I 1-6893 is of special significance as RO 11-6896 is known to be much more effective therapeutically than RO 11-6893. The potencies of the benzodiazepines as adenosine uptake inhibitors show a good correlation with their clinical, anticonflict, pharmacological and receptor binding potencies (Sepinwall and Cook, 1980; Mohler and Okada, 1978; Braestrup and Squires, 1978; Duka et al., 1979). This correlation between the various pharmacological properties of the benzodiazepines and their ability to inhibit adenosine uptake implies that inhibition of adenosine uptake may be an important factor contributing to the central actions of the benzodiazepines. Further demonstration of the interactions between benzodiazcpin¢ binding sites and adenosine uptake sites was provided by Wu et al. (1981) and Davies et al. (1980). A survey of the actions of several competitive adenosine uptake inhibitors (dipyridamole, hexo-

624

P.H. Wu and J. W. PHILLIS

bendine, papaverine and 6-(2-hydroxy)-5-nitrobenzylthioguanosine) has shown that these substances are competitive inhibitors of [3H]diazepam binding to brain membranes. There is a large group of non-benzodiazepine anxiolytics and sedatives which include SQ 20009, SQ 66007, RO-20-1924, ZK 62711, ICI 63197, rolipram, zopiclone, CL 218,872, meprobamate and thalidomide. All these compounds have been shown to be potent inhibitors of adenosine uptake by rat brain synaptosomes (Phillis and Wu, 1983). Steroids. Some of the steroids have anesthetic actions (Selye, 1942) and it is posible that these exert their sedative actions via inhibition of adenosine uptake in CNS tissues. Dexamethasone acetate, progesterone, 17-/~-estradiol, 17-~-ethinylestradiol and diethylstilbestrol dipropionate inhibit adenosine uptake at IC20 values ranging from 4.8 to 6pM. A1phaxalone and tetrahydrocortisone are weaker inhibitors with IC20 values of 4 0 # M and 10mM respectively. A kinetic analysis of the uptake inhibition indicates that steroids inhibit adenosine uptake competitively (Bender, Phillis and Wu, unpublished observations). The mechanisms which mediate steroid inhibition of adenosine uptake are unknown. In view of similar potencies for the inhibition of uptake among the steroids, it is possible that inhibition could be of a non-specific type. Plagemann and Wohlhueter (1980) suggested that membrane lipid transitions might affect the movement of transport carrier. A decrease in membrane fluidity would hinder the movement of the carrier. If steroids act at the sites adjacent to the carrier to slow down the movement of the transporter, this could then account for the competitive inhibition of adenosine uptake by the steroids. It is also possible that steroids may inhibit adenosine kinase and thus reduce the uptake rate. These possibilities need further exploration. Antidepressants. Both tricyclic and non-tricyclic antidepressants were tested for their ability to inhibit adenosine uptake by brain synaptosomes. Among the tricyclic antidepressants, nortriptyline was the most potent inhibitor or adenosine uptake with an IC20 value of 0.8 #M. The other tricyclic antidepressants were less potent with IC20 values of 26#M for clomipramine; 10#M for iprindole, 17#M for amoxapine and greater than 0.1 mM for imipramine. The non-tricyclic antidepressant, viloxazine was very weak in inhibiting adenosine uptake with an IC20 value of greater than 0.1 mM. The mechanism and the sites at which the antidepressants exert their actions have not been elucidated.

Antibiotics and nucleoside analogs. There is a long list of compounds which have been found to inhibit the uptake of adenosine and other nucleosides (for review see Plagemann and Wohlhueter, 1980). Some of the common inhibitory analogs which are effective inhibitors of adenosine uptake systems in peripheral tissue preparations are also active in the central nervous tissue preparations. These agents include cordycepin (3'-deoxyadenosine), 6-mercaptopurine riboside, purine riboside, puromycin, showdomycin, tubercidin, 2-chloroadenosine, 2-azidoadenosine, 2'deoxyadenosine, formycin, 2-fluoradenosine, nitrobenzylthioguanosine and nitrobenzylthioinosine. Toyocamycin also shows potent inhibition of adenosine uptake in rat brain synaptosomes (Phillis and Wu, 1983). Kinetic analysis of mechanisms involved in the inhibition of adenosine uptake has shown that most of these compounds are competitive inhibitors of CNS tissue uptake of adenosine. They are also simple competitive inhibitors of peripheral nucleoside transport or uptake systems (Plagemann and Wohlhueter, 1980). Does calmodulin regulate adenosine uptake? Calmodulin is an ubiquitous protein which mediates a variety of cellular processes in biological systems (Cheung, 1982). Since there are difficulties in studying the calmodulin mediated reactions via using isolated and purified preparations, calmodulin antagonists have been widely used to provide the necessary evidence which supports the involvement of calmodulin. The various proposed calmodulin antagonists include trifluoperazine, W-7 [N-(6-aminohexyl)-5chloro-l-naphthalene-sulfonamide] and R24571 (1[biz (P-chtorophenyl)methyl])- 3- (2,4-dichloro -/~-(2,4 dichlorobenzoxyl)phenethyl)imidazolinium chloride. As was cited in the previous section, adenosine uptake by rat brain cortical synaptosomes is inhibited by phenothiazines and butyrophenones including trifluoperazine, penfluridol and chlorpromazine (Phillis and Wu, 1981). Since the potency order for phenothiazine inhibition of adenosine uptake bears some similarity to those reported for inhibition of calmodulin activated systems (Weiss et al., 1982), the possibility exists that adenosine uptake in rat brain may also be regulated by calmodulin. In order to explore this possibility, Wu et al. (1983) studied the inhibition of adenosine uptake using trifluoperazine, W-7 and R24571. The results indicate that these calmodulin inhibitors are able to inhibit the uptake of adenosine at concentrations known to inhibit other calmodulin regulated processes. The potency order for these compounds to inhibit adenosine uptake

Uptake of adenosine by central nervous tissues follows the descending order of trifluoperazine > R24571 >W-7. The kinetic parameters for the inhibition of adenosine uptake as revealed by a Lineweaver-Burk plot (Wu et aL, 1983), indicate that these compounds were capable of inhibiting uptake competitively. Competitive inhibition of the uptake process suggests that these agents are acting on the cartier protein. Alternatively, Levin and Weiss (1976, 1977) have indicated that although the kinetics of a reaction may suggest competition between drugs and calmodulin-dependent enzymes, the agents may actually act directly on the calmodulin. Based on this proposal, it is possible that agents interact with a specific region of the calmodulin molecule which is involved in the association with receptor protein (e.g. phosphodiesterase) and thus inhibit the adenosine uptake. Definitive proof will have to await the isolation and purification of the nucleoside transporter. The association and dissociation of calmodulin or calmodulin like protein(s) from the cartier system can then be used as conclusive evidence indicating that adenosine uptake sites are regulated by calmodulin. It is important to note that the adenosine uptake system may to some extent couple to other enzyme systems (i.e. adenosine kinase). The inhibition of adenosine uptake could be a result of calmodulin regulation of kinase activity. These fundamental questions are in need of further clarification. Study of adenosine uptake by a high affinity probe p-Nitrobenzylthioinosine (NBMPR) has been shown to reversibly inhibit the transport of nucleosides in human erythrocytes (Paterson and Oliver, 1971; Pickard et al., 1973; Cass and Paterson, 1972, 1973; Turnheim et aL 1978). It is one of the most potent nucleoside uptake inhibitors for these cells. The dissociation constant of the binding on erthrocyte membranes is approx. 1 nM. Nucleoside uptake is inhibited by NBMPR in an apparently competitive manner. However, Wohlhueter et al. (1978) have shown that the inhibition of thymidine transport in ATP-depleted Chinese hamster ovary cells (CHO) by NBMPR conforms to a pattern of simple, non-competitive inhibition in that NBMPR diminishes the maximum velocity of transport without affecting the substrate/carrier affinity constant. Thymidine partially protects the transport system. Because of the formation of a tight bond between NBMPR and the carrier, it has been suggested that NBMPR can be used as a high affinity probe to study the transport and uptake of nucleosides (Paterson, 1980; Eilam and Cabantchik, 1977). Several lines of evidence also support the concept

625

that NBMPR is a useful high affinity probe for studying nucleoside transport. In human erythrocytes, there is strict proportionality between the amount of NBMPR bound to high affinity sites and the degree of transport inhibition, suggesting that high affinity binding may represent a specific interaction with the nucleoside transport system. Jarvis and Young (1980) observed that in the nucleoside impermeable type sheep erythrocyte, there was an absence of NBMPR binding sites whereas the binding site is present in the nucleoside permeable erythrocytes. The observation provides strong evidence that NBMPR interacts specifically with a functional nucleoside transport system. Similar observations were made by Cass et al. (1981), who indicated the absence of binding sites for NBMPR on nucleoside transport-deficient mouse lymphoma cells. Jarvis and Young (1982) were able to estimate the number of nucleoside transport carriers in human erythrocytes by using NBMPR binding. They calculated that the translocation capacity for each transport system in human erythrocytes was approx 180 molecules/site/s at 25°C. Over the years, NBMPR has been proven to be a useful tool for studying nucleoside transport systems in various cell types. Despite the evidence indicating the usefulness of NBMPR as a high affinity probe for nucleoside transport systems there are several questions that remain unanswered. Cass and Paterson (1976) showed that deoxycytidine, a permeable nucleoside has no effect on NBMPR binding to human erythrocytes. Further, in HeLa cells, the relationship between NBMPR binding and transport inhibition is rather complex, in that, there was only partial loss of transport activity, despite saturation of high affinity NBMPR binding sites (Lauzon and Paterson, 1977; Paterson, 1980). Therefore, it remains to be established whether NBMPR binds to the actual nucleoside permeation site of the nucleoside transport mechanism, or to a modifier site as suggested by the inability of deoxycytidine to inhibit binding. There are very few reports using NBMPR as high affinity probe to study nucleoside translocation in nervous tissue. Wu and Phillis (1982) have demonstrated that [3H]p-nitrobenzylthioinosine ([3H]NBMPR) binds to rat brain cortical synaptosomal membranes at two binding sites, one of which is a high affinity site (Ka = 0.05 nM), the other is a low affinity site (K~ = 190/zM) (Fig. 4). Nucleosides are capable of preventing the binding of NBMPR to rat brain synaptosomal membranes. The concentrations of nucleosides which achieved a 50~o inhibition of [3H]NBMPR in the brain membranes

626

P.H. Wu and J.

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Fig. 4. Kinetics of [3H]NBMPR binding to rat brain cortical synaptosomal membranes. Isolated rat cerebral cortical synaptosomal membranes were incubated with [3H]pnitrobenzylthioinosine ([3H]NBMPR) at concentrations ranging from 2.5 to 50 nM. Following the incubation and the termination of the reaction, the [3H]NBMPR labelled m e m b r a n e s were collected on a GF/C giass-fibre filter. Results are the mean + SE of five experiments in triplicate. (Wu and Phillis, 1982) are: adenosine (1.8 raM), inosine (10mM), uridine (35 mM) and thymidine ( > 50 mM). Hypoxanthine and adenine are devoid of inhibitory activity on [3H]NBMPR binding (See Table 3). These observations are consistent with the concept that the nucleoside carrier (transporter) is rather specific in choosing its substrate. However, when one compares the ability of nucleosides to inhibit [3H]NBMPR binding with their inhibition of adenosine uptake

Nucleoside Adenosine Inosine Uridine Thymidine Hypoxanthine Adenine

W.

PHILLIS

by rat brain synaptosomes, it becomes apparent that nucleosides are much weaker inhibitors of [3H]NBMPR binding than adenosine uptake (Table 3). The IC50 value for uridine to inhibit adenosine uptake was 0.57 mM, whereas it inhibited [3H]NBMPR binding with an IC50 value of 35 mM. Likewise, adenosine, inosine and thymidine all showed 50- to 1800-fold differences in their abilities to inhibit adenosine uptake and [3H]NBMPR binding. This observation suggests that [3H]NBMPR may not be a suitable ligand for studies of the adenosine uptake site. Indeed, Wohlhueter e t al. (1978) have indicated that in some cell preparations, N B M P R binds virtually irreversibly, whereas in other cells, i.e. Novikoff cells, such binding did not take place and N B M P R may simply have acted as an alternate substrate for the carrier. Therefore, results from studying adenosine uptake' system by [3H]NBMPR should be interpreted with great caution. Although N B M P R bound to rat synaptosomes at a high affinity site, it is a rather weak inhibitor of adenosine uptake in the same system (IC50= 100#M). If N B M P R indeed binds to adenosine uptake sites and if the transport carrier is the primary site for N B M P R interaction, one would expect that the saturation of high affinity N B M P R binding sites should result in an inhibition of adenosine uptake, i.e. in n M range of N B M P R concentration. However, this is not the case in rat brain synaptosomes. It is interesting to note that N B M P R binding in rat brain synaptosomes bears some similarity to that of HeLa cells, in that both systems are only partially and weakly inhibited by N B M P R binding. Further experimentation needed to clarify this matter. Dipyridamole has been shown to competitively inhibit the high affinity binding of [3H]NBMPR to

Table 3 IC~ value for inhibition of NBMPR binding (/~M) 1,800 12.000 35,000 > 50,000 -t -t

IC~ value for inhibition of adenosineuptake into synaptosomes* (/zM) 1 680 570 850 N.A. N.A.

Nucleosides (l-50mM) were added to the incubation medium containing 10 nM [3H]nitrobcnzylthioinosine([3I-I]NBMPR) in the presence of rat brain cerebral cortical synaptosomalmembranes. The reaction mixture was incubated for a

period of 30rain at 37°C. For determination of nonspecificbinding, a final concentration of NBMPR 50#M was included in the incubation mixture. Followingthe reaction, membranes were isolated by filtrationon a GF/C filter. Results are the average of 2-3 experimentsin triplicate. OVu and Phillis, 1982). *Data were taken from Bender et aL (1981). tNo significantinhibition at concentrationof 0.1 M.

Uptake of adenosine by central nervous tissues transport sites in HeLa cell cultures (Paterson, 1980). However, in the rat brain synaptosomal membrane preparation, dipyridamole is a weak inhibitor of [3H]NBMPR high affinity binding, even though it is a potent inhibitor of adenosine uptake by brain synaptosomes. These findings also indicate that the high affinity NBMPR binding site may not be associated with the permeation sites in rat synaptosomes. Recently, Clanachan and Hammond (1983) have observed thermodynamic differences between the binding interaction of NBMPR and dipyridamole with nucleoside transport system in human erythrocytes. Results indicated that the binding of NBMPR to human erythrocytes is driven by a large loss in enthalpy whereas the binding of dipyridamole is quite different. The evidence suggested that NBMPR bound to carrier and induced a conformational change in the transporter system. Dipyridamole binding did not produce a conformational change in the drug-transporter complex. The thermodynamic differences in the actions of NBMPR and dipyridamole could explain our observation that dipyridamole is a weak NBMPR displacer in rat synaptosome preparation incubated at 37°C. Alternatively, it is possible that dipyridamole acts only on the externally facing (c/s-side) aspect of the carrier molecule. Since [3H]NBMPR can bind to both c/s-side and trans-side of carrier protein in a membrane preparation, dipyridamole would only inhibit the binding of [3H]NBMPR to those carrier sites at the cis-side of the membrane (which accounts for approx 30% of total [3H]NBMPR binding, Wu and Phillis, 1982b). On the other hand, hexobendine was found to be able to inhibit [aH]NBMPR binding at the synaptosomal membrane preparations with an IC50 value similar to that of inhibition of adenosine uptake into intact synaptosomes (Wu and Phillis, 1982b). The sidedness of a transport system has also been addressed by Deve and Krupka (1978). They indicated that competitive inhibitors commonly have four different affinities for the substrate sites on the two membrane faces. DISCUSSION Rate limiting step of nucleoside uptake Transport is the first step of the multistep mechanism of nucleoside accumulation into cells. It has been proposed that kinetic studies of the uptake of radiolabelled nucleosides into cells can be approximated by using rapid sampling procedures, so that the kinetics of initial rate of uptake may be approaching that of transport rate (Paterson et al., N,C.I. 6/$~B

627

1981). It has become apparent that nucleoside transport rates often exceed those of metabolic trapping of the nucleoside (Rozengurt et al., 1977; Koren et al., 1978). Thus, it is evident that while transport might in some instances be rate-limiting in the nucleoside uptake process, it is frequently not the case in cells which trap nucleosides by metabolism of the permeant. Various types of peripheral cells are available for studying nucleoside transport. These cell types are not capable of "trapping" (phosphorylation) of nucleosides, i.e. uridine and thymidine permeation in human erythrocytes (Oliver and Paterson, 1971), thymidine permeation in rat hepatocytes (Ungemach and Hegner, 1978) or some mutant cell lines (Wohlheuter et al., 1979). In these special cell types or cell lines, the nucleoside accumulated by the cell represents the consequence of the "transport" mechanism. However, neuronal and glia cells are capable of phosphorylation of nucleosides taken up by the cells. Thus, it is difficult, if not impossible, to study the "transport" mechanism of nucleoside accumulation. Although our initial studies have shown that the rapid adenosine uptake process may be representative of adenosine transport system in nerve tissues, the studies also showed that phosphorylation of adenosine did take place even with a very short time course of incubation. Furthermore, nerve tissue differs from peripheral tissues in many respects due to its neurotransmission function. Nucleosides may play a role other than substrates for DNA or RNA synthesis; they may play roles as neuromodulators or neurotransmitters. In the case of adenosine, it has been demonstrated and proposed as a neuromodulator in the central nervous system (Phillis and Wu, 1981). Since adenosine plays roles other than that of a substrate for cellular synthesis, the possibility of a specialized compartmentation must be considered. Bender et al. (1981a) showed that there was more than one source of adenosine which could be released by depolarization. Brain synaptosomes incubated for 30 s accumulated [3H]adenosine in a pool which was released upon K÷-depolarization or veratridine depolarization. The release of [3H]adenosine from this "pool" was not Ca 2÷ dependent, however, when the brain synaptosomes were incubated with [3H]adenosine for 15 min, the release of [3H]adenosine by K ÷ and veratridine was Ca2÷-dependent. This study proposed that there might be more than one compartment for adenosine accumulation or storage. Therefore, the kinetic properties of the storage compartment(s) must be incorporated into the model initially proposed for nucleoside permeation studies. Figure 5 illustrates such a new

628

P.H. Wu and J. W. PHILLIS

C15

I/ll#l /l l l l l #1,

iron5

i

!Membrane I c ,-~--c c~--c

lk

ADO ADO I i

PHOSPHORYLATION l / DEAMINATION ADO l ~ STORAGE STORAGE ? ?

I/ll/llll#/#/lllh Fig. 5. Schematic presentation of the sample carrier model for the uptake of adenosine. Subsequent storages and metabolism of adenosine were included to illustrate the complexity of adenosine (nucleoside) uptake system in CNS.

version of the adenosine uptake system in the nervous tissue. It is evident from the scheme shown here that the rate limiting step of adenosine permeation may not necessarily be the adenosine "transport" step, nor may it be a phosphorylation reaction. It is possible that the inhibition of adenosine uptake in nervous tissue by some agents may involve more than one site of action, and that studies on such inhibition should not be restricted to the examination of a single step in the reaction such as inhibition of transport, phosphorylation or deamination, although the transport step does represent the initial reaction of nucleoside translocation cascade.

Roles of glial cells in the regulation of adenosine uptake It has been estimated that in nervous tissue the ratio between glia cells and neurons is approx 10:1. The recent finding of a high affinity glial adenosine uptake mechanism is therefore of great significance for the regulation of extracellular adenosine concentrations and thus of adenosine effects. The demonstration of high affinity glial adenosine uptake was described by Schultz et al. (1972) using C-6 rat ghoma cells and subsequently by Clark et al. (1974) using astrocytoma cell line. Lewin and Bleck (1979) reported the uptake of adenosine by cultured astrocytoma cells. The kinetic parameters for the adenosine uptake indicated that adenosine was taken up by the cells via a high affinity uptake system showing a K m value of 1.04/~M and Bm~x value of 0.14/~mol/g protein/min. They also demonstrated that cultured astrocytoma cells were able to metabolize adenosine to inosine and hypoxanthine which were then released. They concluded that astrocytoma

cells may play a role in inactivating adenosine. However, astrocytoma cells are not normal cells and their function may not be representive of the normal physiological condition. Hertz (1978) used astrocytes in primary cultures grown from the cerebral hemispheres of newborn mice. Cultured astrocytes have been shown to take up glutamate, GABA and taurine. Hertz et al. (1978) demonstrated that the culture consists mainly of glial cells resembling mature astrocytes. Uptake studies using cultured astrocytes have shown that astrocytes possess a minor, unsaturable component and an intense, high affinity uptake of adenosine. The uptake followed Michaelis-Menten kinetics showing a K m value of 3.4 p M and Vmax value of 0.36 nmol/min/mg protein. The uptake in glial cells was inhibited by papaverine. It was concluded that this glial adenosine uptake process may be of physiological importance because of its high affinity and large capacity. Since the adenosine uptake blockers inhibited both uptake of adenosine by neurons as well as by glial cells, and since the glial cells were present in larger numbers than the neurons, the glial adenosine uptake system may be of great significance in contributing to the removal of extracellular adenosine in the brain/n

vivo. Adenosine uptake inhibition has been proposed to be an important factor in the mechanism of action of several centrally active agents. The inhibition of uptake has been explained through an action via synaptosomes (implying the neuronal component of nervous tissue). It is possible that in an intact nervous system, the inhibition of adenosine uptake by glial cells might be a more important factor in controlling the extracellular adenosine concentration.

Physiological and pharmacological implications of adenosine uptake system in CNS Experimental observations on in vivo neurons have shown that adenosine's primary action is to reduce the release of other neurotransmitters through an action on presynaptic nerve terminals. Drugs which block the uptake and metabolism of adenosine depress the firing of central neurons and potentiate adenosine's depressant action. Adenosine receptor antagonists excite central neurons. These observations suggest that the excitability of central neurons is subject to regulation by endogenously released adenosine. A survey of several groups of centrally acting drugs has demonstrated that many sedative, anxiolytic and antidepressant compounds are potent inhibitors of adenosine uptake by rat brain synaptasomes. Poten-

Uptake of adenosine by central nervous tissues tiation of the effects of endogenously released adenosine may be an important factor in the central actions of these compounds.

629

reported experimental work and E. M. Carethers for the competent typing of the manuscript. We are grateful to Dr Robin Barraco for reading the manuscript. The experimental work was supported by Medical Research Council of Canada and Wayne State University School of Medicine.

CONCLUSION Upon examining various aspects of the adenosine uptake system in CNS, one recognizes that the mechanism involved in uptake of adenosine is very similar to the system which mediates peripheral nucleoside transport. The uptake system is a carrier mediated, facilitated diffusion process. The carrier in the CNS tissue seems to be sensitive to temperature, pH, and it is vulnerable to the sulfhydryl reagents like Nethylmaleimide and p-chloromercuribenzoate. The Ea (activation energy) for the adenosine uptake is 8870 cal/mol for CNS preparations and 9200 cal/mol (Plagemann, 1970) for a peripheral uptake system. Studies on the specificity of substrate requirements for the uptake show good agreement with those reported for the peripheral system, namely, nucleosides share a common carrier but purine and pyrimidine bases cannot interact with the carrier. Compounds which inhibit adenosine uptake in the peripheral tissue preparation are also inhibitors of adenosine uptake in the central nervous tissue. However, there is evidence that suggests a difference between the adenosine uptake system in peripheral and synaptosomal preparations. Isolated brain capillaries utilize an uptake system which resembles the peripheral uptake system, in that the bulk adenosine taken up by the preparation was converted to nucleotides (56%) in the capillaries, in comparison to 28% of nucleotides which were present in synaptosomes after 15 rain of incubation at 37°C. This difference may indicate that, in the peripheral tissue, the purine salvage pathway plays a more important role in regulating adenosine uptake. Further, dipyridamole, papaverine and hexobendine are more effective in inhibiting adenosine uptake in capillaries than in synaptosomes. Our observations suggest the uptake of adenosine may be "coupled" to different metabolic functions. Further investigation of these uptake systems may lead to the development of drugs potentiating central or peripheral effect of adenosine. A survey of several groups of centrally active drugs has demonstrated that many are potent adenosine uptake inhibitors. The pharmacological and physiological consequences of the inhibition of adenosine uptake warrants further investigation. Acknowledgements--We wish to thank Mr A. S. Bender and

Ms E. Davis for their excellent technical assistance in the

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