Potential signalling roles for UTP and UDP: sources, regulation and release of uracil nucleotides.

Increasing evidence for receptors for uracil nucleotides has focused interest on specific signalling mechanisms involving UTP and UDP. At least three metabotropic P2 receptors are stimulated by uracil nucleotides with equal or greater potency than by adenine nucleotides, and there might be ionotropic receptors as well. Regulation of uridine and uracil nucleotide levels is important when considering the receptor-mediated effects of these compounds. Cells can synthesize uracil nucleotides de novo or by salvage of uridine. UTP made from salvage might be preferentially used for RNA synthesis in the nucleus, while UTP synthesized de novo seems to be used for UDP-sugar and CDP-phospholipid production. UTP from both pathways can enter a free UTP pool, from which UTP can be released from cells. UTP and UDP can stimulate pyrimidinoceptors, but metabolism by ecto-nucleotidases limits their effects. Alternatively, UTP might be a substrate for ecto-protein kinases, and this could contribute to its extracellular regulation. Cells can reclaim uridine, using nucleoside transport processes, following dephosphorylation of UTP, UDP and UMP. In this article Christopher Anderson and Fiona Parkinson discuss how understanding the processes that regulate uridine and uracil nucleotide concentrations will enhance our ability to manipulate UTP/UDP signalling pathways for pharmacological effect.

Neuroprotective role of adenosine in cerebral ischaemia.

Several lines of evidence suggest that adenosine may be an endogenous protective agent in cerebral ischaemia. Adenosine is normally present in the extracellular fluid in most tissues of the body, including the brain, and its level increases dramatically following hypoxia or ischaemia. The rate of adenosine production is enhanced when the energy demand is larger than the rate of energy supply. Adenosine acts on specific receptors that are present in most cells in the body and that produce cellular effects that tend to antagonize a number of pathological events thought to be instrumental for ischaemic nerve cell death. Karl Rudolphi and colleagues review evidence for the neuroprotective potential of adenosine and indicate some targets for drug development.

Protein cysteine S-nitrosylation inhibits vesicular uptake of neurotransmitters.

Previous studies have shown that nitric oxide can induce cysteine S-nitrosylation of total protein in synaptosomes, suggesting that nitric oxide may contribute to the regulation of synaptic protein function. Vesicular neurotransmitter transporters pack neurotransmitters into synaptic vesicles and play an important role in neurotransmission. In the central nervous system, vesicular monoamine transporter 2 (VMAT2) is responsible for the uptake of monoamines, vesicular acetylcholine transporter (VAChT) is responsible for the uptake of acetylcholine, while vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) are responsible for the uptake of glutamate. The purpose of this study was to investigate the role of cysteine S-nitrosylation in the regulation of these vesicular neurotransmitter transporters. Using the biotin switch assay followed by avidin precipitation and immunoblotting we found that the nitric oxide donor nitrosoglutathione (GSNO) not only increased total cysteine S-nitrosylation, but also increased cysteine S-nitrosylation of VMAT2, VAChT, VGLUT1 and VGLUT2 in the mouse brain. Further, GSNO also decreased the vesicular uptake of [(3)H]dopamine, [(3)H]acetylcholine and [(3)H]glutamate. Our studies suggest that the cysteine S-nitrosylation may play an important role in the regulation of vesicular neurotransmitter transport.

Adenosine A1 and A2A receptors and nitrobenzylthioinosine-sensitive transporters in gerbil brain: no changes following long-term treatment with the adenosine transport inhibitor propentofylline.

There is evidence that adenosine is an endogenous neuroprotective substance in the gerbil and that propentofylline, a novel xanthine derivative that acts as a transport inhibitor, exerts part of its neuroprotective activity in this species by enhancing adenosine actions. Using autoradiography we have examined the distribution of adenosine A1 and A2A receptors and of equilibrative adenosine transporters in gerbil brain as well as the possible changes induced by repeated treatment with propentofylline. Nucleoside transporters, studied by [3H]NBMPR binding, were found to be widely distributed in the gerbil brain, with no clear relationship to the distribution of adenosine receptors. Adenosine A2A receptors, studied by [3H]CGS 21680 binding and by in situ hybridization, were found to be present in intrinsic neurons in the caudate putamen, nucleus accumbens and tuberculum olfactorium. Adenosine A1 receptors were studied by examining the binding of [3H]CHA, an agonist, and [3H]DPCPX, an antagonist. There was an overall similarity in the distribution of binding sites for these two ligands, and a similarity with the distribution in the rat. However, the antagonist was found to label certain structures, especially white matter structures, more than the agonist. It is argued that these binding sites for antagonists represent receptors that are in transit from the site of synthesis in the perikaryon to the destination in the nerve terminal, and are not coupled to G proteins. There were no differences in the binding of any of these ligands or in A2A mRNA following 2 weeks' treatment with propentofylline, indicating that the drug has minimal effects on adenosine mechanisms under basal physiological conditions. This also suggests that tolerance to adenosine-related effects of the drug is less likely to occur.

Differences between rat primary cortical neurons and astrocytes in purine release evoked by ischemic conditions.

In the brain, the levels of adenosine increase up to 100-fold during cerebral ischernia; however, the roles of specific cell types, enzymatic pathways and membrane transport processes in regulating intra- and extracellular concentrations of adenosine are poorly characterized. Rat primary cortical neurons and astrocytes were incubated with [(3)H]adenine for 30 min to radiolabel intracellular ATP. Cells were then treated with buffer, glucose deprivation (GD), oxygen-glucose deprivation (OGD), 100 micro M sodium cyanide (NaCN) or 500 micro M iodoacetate (IAA) for 1 h to stimulate the metabolism of ATP and cellular release of [(3)H]purines. The nucleoside transport inhibitor dipyridamole (DPR) (10 micro M), the adenosine kinase inhibitor iodotubercidin (ITU) (1 micro M), the adenosine deaminase inhibitor EHNA (1 micro M) and the purine nucleoside phosphorylase inhibitor BCX-34 (10 micro M) were tested to investigate the contribution of specific enzymes and transporters in the metabolism and release of purines from each cell type. Our results indicate that (a). under basal conditions astrocytes released significantly more [(3)H]adenine nucleotides and [(3)H]adenosine than neurons, (b). OGD, NaCN and IAA conditions produced significant increases in [(3)H]adenosine release from neurons but not astrocytes, and (c) DPR blocked [(3)H]inosine release from both astrocytes and neurons but only blocked [(3)H]adenosine release from neurons. These data suggest that, in these experimental conditions, adenosine was formed by an intracellular pathway in neurons and then released via a nucleoside transporter. In contrast, adenine nucleotide release and extracellular metabolism to adenosine appeared to predominate in astrocytes.

Adenosine receptors and nucleoside transport sites in cardiac cells.

1. Potential mechanisms responsible for the prominent depression of atrioventricular conduction by adenosine have been investigated in guinea-pig heart. 2. Adenosine A1 receptors and nucleoside transport (NT) sites were identified and enumerated in cardiac myocytes, atrioventricular conduction cells and coronary endothelial cells in 10 microns sections by autoradiographical analysis of the binding of the A1 selective antagonist 8-cyclopentyl-1,3-[3H]-dipropylxanthine ([3H]-DPCPX) and the NT ligand [3H]-nitrobenzylthioinosine ([3H]-NBMPR), respectively. 3. Atrioventricular conduction cells were identified by acetylcholinesterase histochemistry and endothelial cells by von Willebrand factor immunohistochemistry. 4. Site-specific binding of [3H]-DPCPX, when expressed as grains per cell nucleus was significantly higher (30 fold) in conduction cells than in surrounding myocytes. [3H]-DPCPX site density on endothelial cells in adjacent coronary vessels was not significantly different from myocytes. 5. In contrast, autoradiography of [3H]-NBMPR sites in these areas indicated that, relative to myocytes, conduction cells and endothelial cells were significantly enriched (2 fold and 4.5 fold, respectively) in NT sites. 6. The pronounced dromotropic effect of adenosine in guinea-pig heart is correlated with a higher density of adenosine A1 receptors in atrioventricular conduction cells than in myocytes. The NT capacity of these cells, as estimated by [3H]-NBMPR binding site density, is not increased in proportion to A1 receptors.

Heterogeneity of nucleoside transport inhibitory sites in heart: a quantitative autoradiographical analysis.

1. The distribution of nucleoside transport inhibitory sites in rat and guinea-pig cardiac sections was investigated by use of [3H]-nitrobenzylthioinosine ([3H]-NBMPR) autoradiography. The distribution of these sites was heterogeneous in guinea-pig sections and homogeneous in rat sections. 2. The areas of high density of nucleoside transport inhibitory sites found in guinea-pig cardiac sections were compared to the distribution of an endothelial cell marker, von Willebrand Factor, determined by radioimmunocytochemistry. These two markers were co-localized suggesting that coronary endothelial cells from guinea-pig have a high density of NBMPR binding sites and thus may have a high nucleoside transport capacity. 3. Nucleoside transporter subtypes with differing affinity for NBMPR or dipyridamole were investigated by quantitative autoradiography. Sites in rat tissues had high affinity for NBMPR (KD = 0.6 nM) but were of low sensitivity to dipyridamole (Ki = 3.1 microM). In guinea-pig sections, areas of high and low [3H]-NBMPR binding site density were analyzed separately. In both areas, sites had high affinity for NBMPR (KD = 1.4 nM, 4.5 nM, respectively) and dipyridamole (Ki = 108 nM, 245 nM, respectively). 4. While differences in density of nucleoside transport inhibitory sites are detectable between distinct regions of the heart, subtypes differing in affinity for NBMPR or dipyridamole were not evident. Therefore, more detailed structure activity studies are required to determine if subtypes of these sites exist within a single heart.

Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors.

1. Adenosine kinase (AK) inhibitors can enhance adenosine levels and potentiate adenosine receptor activation. As the AK inhibitors 5' iodotubercidin (ITU) and 5-amino-5'-deoxyadenosine (NH(2)dAdo) are nucleoside analogues, we hypothesized that nucleoside transporter subtype expression can affect the potency of these inhibitors in intact cells. 3. Three nucleoside transporter subtypes that mediate adenosine permeation of rat cells have been characterized and cloned: equilibrative transporters rENT1 and rENT2 and concentrative transporter rCNT2. We stably transfected rat C6 glioma cells, which express rENT2 nucleoside transporters, with rENT1 (rENT1-C6 cells) or rCNT2 (rCNT2-C6 cells) nucleoside transporters. 3. We tested the effects of ITU and NH(2)dAdo on [(3)H]-adenosine uptake and conversion to [(3)H]-adenine nucleotides in the three cell types. NH(2)dAdo did not show any cell type selectivity. In contrast, ITU showed significant inhibition of [(3)H]-adenosine uptake and [(3)H]-adenine nucleotide formation at concentrations < or =100 nM in rENT1-C6 cells, while concentrations > or =3 microM were required for C6 or rCNT2-C6 cells. 4. Nitrobenzylthioinosine (NBMPR; 100 nM), a selective inhibitor of rENT1, abolished the effects of nanomolar concentrations of ITU in rENT1-C6 cells. 5. This study demonstrates that the effects of ITU, but not NH(2)dAdo, in whole cell assays are dependent upon nucleoside transporter subtype expression. Thus, cellular and tissue differences in expression of nucleoside transporter subtypes may affect the pharmacological actions of some AK inhibitors.

Effects of mono- and divalent ions on the binding of the adenosine analogue CGS 21680 to adenosine A2 receptors in rat striatum.

The effect of monovalent and divalent cations on equilibrium binding of the adenosine A2-selective agonist ligand CGS 21680 (2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxami doadenosine) to membranes prepared from rat striatum was examined. Competition experiments with cyclohexyladenosine, 2-chloroadenosine, N-ethylcarboxamidoadenosine and CGS 21680 suggest that at 2 nM [3H]CGS 21680 binds to a single site with the pharmacology of an A2a receptor. Magnesium and calcium ions caused a concentration-dependent increase in binding that reached about 10-fold at 100 mM. Manganese ions had a biphasic effect on binding with a maximal increase at 5 mM. Lithium, sodium and potassium ions all caused a concentration-dependent decrease of binding. Sodium was most potent, potassium least. At 200 mM ion concentration, the inhibition of binding was 88% by sodium, 47% by lithium and 29% by potassium ions. The effect of sodium chloride was the same as that of sodium acetate. The effect of sodium ions was essentially similar to that of Gpp(NH)p. However, sodium ions produced a larger effect than even maximally effective concentrations of Gpp(NH)p. The maximal inhibition by Gpp(NH)p was about 55% at 2 nM radioligand concentration irrespective of the magnesium concentration. The maximal effect of sodium ions was reduced by increasing concentrations of magnesium ions. Increasing magnesium ion concentration from 1 to 100 mM increased the half-maximally effective concentration of Gpp(NH)p almost 10-fold and that of sodium ions less than 2-fold. Furthermore, sodium ions and Gpp(NH)p had additive effects. The binding of an agonist to striatal A2a receptors shows an unusually large dependence on both divalent and monovalent cations that can only partly be explained by a change in the coupling to Gs proteins.

Inhibitory effects of propentofylline on [3H]adenosine influx. A study of three nucleoside transport systems.

The neuroprotective effects of adenosine are well-recognized. Recently, propentofylline, a xanthine derivative, has been shown to increase extracellular concentrations of adenosine in ischemic brain and to limit the extent of neuronal damage in experimental models of cerebral ischemia. Since the concentration of adenosine in brain is controlled, in part, by nucleoside transporter proteins, the action of propentofylline was proposed to be due to inhibition of mediated transfer of adenosine across cell membranes. To determine the likelihood of this mechanism, we examined the inhibitory effects of propentofylline on [3H]adenosine transport by the three best-characterized nucleoside transport processes, es, ei, and cif in cultured cell lines under conditions where only a single transporter type was operative. Propentofylline inhibited [3H]adenosine uptake by each of the three transport processes in a concentration-dependent manner. The greatest inhibitory potency was for es transporters (L1210/B23.1 cells), with an IC50 value of 9 microM, followed by ei transporters, with IC50 values of 170 microM (L1210/C2 cells) and 166 microM (Walker 256 cells). Propentofylline was a weak inhibitor of cif transporter, with an IC50 value of 6 mM. These results demonstrate that propentofylline is an inhibitor of adenosine transport processes and suggest that its neuroprotective effects may be due to an increase in extracellular concentrations of adenosine by virtue of inhibition of es transporter function.

Stimulation of nucleoside efflux and inhibition of adenosine kinase by A1 adenosine receptor activation.

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors. Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT1 MF-2 smooth muscle cells were incubated with 10 microM [3H]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (1AA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 microM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [3H]adenosine. The adenosine A1 receptor selective agonist N6-cyclohexyladenosine (CHA, 300 nM) increased the release of [3H]purine nucleoside induced by IAA treatment by 39% (P < 0.05). This increase was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 microM). Treatment of cells with UTP (100 microM), histamine (100 microM), or phorbol-12-myristate-13-acetate (PMA, 10 microM) also increased [3H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [3H]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated cells. These data indicated that adenosine A1 receptor activation increased nucleoside efflux from metabolically stressed DDT1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.

Distribution of mRNA encoding a nitrobenzylthioinosine-insensitive nucleoside transporter (ENT2) in rat brain.

Nucleoside transporters may play a role in regulating levels of extracellular adenosine and adenosine receptor activity. Two members of the equilibrative nucleoside transporter family have recently been cloned. ENT1 is potently inhibited by nitrobenzylthioinosine (NBMPR) (K(i) approximately 1 nM) and was previously found to have a wide distribution in rat and human brain. ENT2 is insensitive to inhibition by NBMPR at low nanomolar concentrations and there is limited information describing its distribution in rat brain. The present study used RT-PCR, northern blot and in situ hybridization and detected rENT2 transcript in several brain regions including hippocampus, cortex, striatum and cerebellum. Our results indicate a wide cellular and regional distribution for ENT2 in rat brain, similar to ENT1, indicating that control of adenosine levels in brain is achieved by multiple transport processes.

Demonstration of the existence of mRNAs encoding N1/cif and N2/cit sodium/nucleoside cotransporters in rat brain.

Nucleoside transport may be involved in the regulation of extracellular levels of adenosine, an inhibitory neuromodulator in the central nervous system. Previous reports have provided functional evidence for Na+-dependent nucleoside transport in rat brain. We isolated total RNA from various regions of rat brain and tested for the presence of mRNA for two recently cloned Na+/nucleoside cotransporters using reverse transcriptase PCR (RT-PCR). Messenger RNA for a pyrimidine-selective Na+/nucleoside cotransporter mRNA (rCNT1) was detected in samples from each brain region tested by RT-PCR amplification of a 309-bp DNA product. Southern blot and sequence analysis confirmed that this product was derived from rCNT1 mRNA. A purine-selective Na+/nucleoside cotransporter mRNA (rCNT2, also termed SPNT) was detected throughout brain by amplifying a 235-bp DNA product, the sequence of which was identical to that published. These experiments demonstrate the presence of both rCNT1 and rCNT2 mRNA in rat brain.

Adenosine permeation of a dynamic in vitro blood-brain barrier inhibited by dipyridamole.

Adenosine is an inhibitory neuromodulator in the central nervous system and has been reported to have neuroprotective properties. Using a dynamic in vitro blood-brain barrier, we investigated the hypothesis that inhibition of adenosine transporters on the lumenal side of the blood-brain barrier may decrease the loss of adenosine from the brain. Our results indicate that lumenal administration of dipyridamole, a nucleoside transport inhibitor, can inhibit adenosine permeation from the extracapillary space into the lumen.

Effect of the neurotoxin AF64A on intrinsic and extrinsic neuronal systems of rat neostriatum measured by in vivo microdialysis.

In the present in vivo microdialysis study the aziridinium ion of ethylcholine mustard, AF64A and the excitotoxin ibotenic acid were compared for their effects on extracellular striatal acetylcholine, choline, gamma-aminobutyric acid (GABA), dopamine and its metabolites, glutamate and aspartate, measured in the same perfusate sample, under basal and high KCL conditions. Ten days following unilateral striatal injections of AF64A (2 x 0.08 to 2 x 8 mM) there was a dose-dependent decrease in the extracellular striatal levels of acetylcholine and GABA, the two major intrinsic striatal neurotransmitter systems. No significant effects were observed on any of the monitored neurotransmitter systems following the lowest (2 x 0.08 mM) dose of AF64A, while at the intermediate (2 x 0.8 mM) dose, AF64A produced a unilateral > 50% and > 70% decrease in basal extracellular striatal acetylcholine and GABA levels respectively. The effects of K(+)-depolarization on extracellular acetylcholine and GABA levels were diminished by approximately 50%. At the highest dose (2 x 8 mM), extracellular striatal acetylcholine levels were non-detectable under basal conditions, while the GABA levels were decreased by > 50%, when compared with the contralateral intact side. However, at this dose, GABA levels were bilaterally decreased compared to levels observed in control animals. Basal extracellular striatal dopamine and glutamate levels, representing the two major extrinsic neurotransmitter systems innervating the neostriatum were only affected by the highest dose of AF64A. The excitotoxin ibotenic acid (2 x 28.4 mM) produced a strong unilateral decrease in extracellular striatal acetylcholine (> 80%) and GABA (> 90%) levels, without significantly affecting basal dopamine and glutamate levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Subtypes of nucleoside transport inhibitory sites in heart: a quantitative autoradiographical analysis.

We evaluated the interaction of several nucleoside transport inhibitors and substrates with the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) to nucleoside transport sites in guinea pig cardiac sections. Using quantitative autoradiography, we determined inhibition constants for inhibition of [3H]NBMPR binding to both coronary endothelial cells and cardiac myocytes. We studied the interactions of NBMPR, nitrobenzylthioguanosine, dipyridamole, dilazep, hexobendine, lidoflazine, mioflazine, soluflazine, adenosine, inosine and uridine for these two cell types. Of the compounds tested in this study, lidoflazine (8.2X) and hexobendine (6.3X) have the greatest selectivity for coronary endothelial cell nucleoside transporters. All other compounds had 3-fold or less selectivity. Therefore, there is evidence of nucleoside transporter subtypes between endothelial cells and myocytes. This heterogeneity of transport inhibitory sites on nucleoside transporters may allow the development of agents to modulate selectively some of the cardiovascular effects of adenosine.

Effect of adenosine receptor agonists on release of the nucleoside analogue [3H]formycin B from cultured smooth muscle DDT1 MF-2 cells.

Adenosine has receptor-mediated effects in a variety of cell types and is predominantly formed from ATP by a series of nucleotidase reactions. Adenosine formed intracellularly can be released by bidirectional nucleoside transport processes to activate cell surface receptors. We examined whether stimulation of adenosine receptors has a regulatory effect on transporter-mediated nucleoside release. DDT1 MF-2 smooth muscle cells, which possess nitrobenzylthioinosine-sensitive (ES) transporters as well as both adenosine A1 and A2 receptors, were loaded with the metabolically stable nucleoside analogue [3H]formycin B. N6-cyclohexyladenosine (CHA), a selective adenosine A1 receptor agonist, produced a concentration-dependent inhibition of [3H]formycin B release with an IC50 value of 2.7 microM. Further investigation revealed CHA interacts directly with nucleoside transporters with a Ki value of 3.3 microM. Neither 5'-N-ethylcarboxamidoadenosine (NECA), a mixed adenosine A1 and A2 receptor agonist, nor CGS 21680, a selective adenosine A2A receptor agonist, affected nucleoside release. We conclude that release of the nucleoside formycin B from DDT1 MF-2 cells is not regulated by adenosine A1 or A2 receptor activation.

Effects of propentofylline on adenosine A1 and A2 receptors and nitrobenzylthioinosine-sensitive nucleoside transporters: quantitative autoradiographic analysis.

Previous studies have demonstrated that the xanthine compound, propentofylline, has beneficial effects in models of cerebral ischemia and can enhance some and exhibit other effects of adenosine. We investigated the in vitro effects of propentofylline and its hydroxy metabolite, A72,0287, on the binding of [3H]cyclohexyladenosine ([3H]CHA), [3H]2-[p-(2-carbonyl-ethyl)-phenylethyl-amino]-5'-N- ethylcarboxamido adenosine ([3H]CGS 21680) and [3H]nitrobenzylthioinosine ([3H]NBMPR) to adenosine A1 and A2 receptors and NBMPR-sensitive nucleoside transporters, respectively, in 10-microns coronal rat brain sections. Both xanthines had micromolar affinity for each of these sites with approximately 10-fold lower affinity for A2 receptors than for A1 receptors and [3H]NBMPR binding sites. Saturation analysis of [3H]CHA or [3H]CGS 21680 binding in the presence of increasing concentrations of propentofylline produced significant increases in KD values without affecting Bmax values; thus propentofylline is a competitive inhibitor at A1 and A2 receptors. The effects on A2 receptors apparently require higher concentrations (Ki approximately 200 microM) than the effects on A1 receptors (Ki approximately 20 microM). Propentofylline was also found to be a competitive inhibitor of [3H]NBMPR binding. Therefore we conclude that propentofylline interacts with adenosine-responsive systems to increase interstitial adenosine concentrations and to selectively inhibit A1 receptors.

[3H]adenosine transport in DDT1 MF-2 smooth muscle cells: inhibition by metabolites of propentofylline.

Adenosine receptor signal transduction mechanisms have previously been characterized in Syrian hamster smooth muscle DDT1 MF-2 cells but adenosine transport in these cells has not. DDT1 MF-2 cells possess a high density (370,000 sites/cell) of high affinity (Kd value of 0.26 nM) binding sites for [3H]nitrobenzylthioinosine, a marker for the equilibrative and inhibitor-sensitive subtype of nucleoside transporters. Transport of [3H]adenosine was insensitive to Na+ and was inhibited by the nucleoside transport inhibitors nitrobenzylthioinosine, dilazep and dipyridamole with IC50 values of 1, 13 and 270 nM, respectively. Propentofylline, a neuroprotective compound that can inhibit nucleoside transporters, is rapidly metabolized in vivo to the racemate (+/-)-A720287. Based on recent findings that some transport inhibitors exhibit marked stereoselectivity, we tested the degree to which individual stereoisomers of (+/-)-A720287 affect adenosine transport. Propentofylline inhibited [3H]adenosine transport in DDT1 MF-2 cells with an IC50 value of 24 microM. (+/-)-A720287 and the individual stereoisomers (+)-833791 and (-)-844261 had similar potency to propentofylline for inhibition of [3H]adenosine transport in DDT1 MF-2 cells as well as in clonal mouse leukemia L1210/B23.1 cells, cells which possess only the equilibrative and inhibitor-sensitive subtype of nucleoside transporters. Thus, the neuroprotective effects of propentofylline may be due, in part, to the primary metabolites of propentofylline.

The binding of the adenosine A2 receptor selective agonist [3H]CGS 21680 to rat cortex differs from its binding to rat striatum.

The binding of the reportedly A2A selective agonist CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'N-ethylcarboxamidoadenos ine) to cortex and striatum was examined in parallel using quantitative receptor autoradiography. [3H]CGS 21680 bound to a single site in rat striatum with KD 2.3 nM and Bmax 320 fmol/mg grey matter. In addition [3H]CGS 21680 bound to a single site in the cerebral cortex with KD 47 nM and Bmax 100 fmol/mg grey matter. In cat cortex [3H]CGS 21680 (2 nM) binding was strong and particularly evident in the most superficial layers. The potency order for inhibition of 2 nM [3H]CGS 21680 binding to rat striatum was NECA (5'-N-ethylcarboxamidoadenosine; IC50 9.0 nM) > 2-CADO (2-chloroadenosine; 87 nM) > R-PIA (N6-(R)-phenylisopropyladenosine; 110 nM). The potency order for inhibition of 2 nM [3H]CGS 21680 binding to rat cortex was NECA (3.0 nM) > 2-CADO (14 nM) > or = R-PIA (16 nM). Gpp(NH)p (5'-guanylyl imidodiphosphate) inhibited [3H]CGS 21680 binding to both cortex and striatum, but more potently in cortex (IC50 100 nM vs. 470 nM). The present results show that there is a cortical binding site for [3H]CGS 21680 which appears to be different from the the striatal A2A receptor, the A2B receptor and the A1 receptor.