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.

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.

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.

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.

Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions.

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.

Effects of nitrobenzylthioinosine on neuronal injury, adenosine levels, and adenosine receptor activity in rat forebrain ischemia.

Adenosine levels increase in brain during cerebral ischemia, and adenosine has receptor-mediated neuroprotective effects. This study was performed to test the hypothesis that nitrobenzylthioinosine (NBMPR), a selective and potent inhibitor of one adenosine transporter subtype termed ENT1, or es, can protect against ischemic neuronal injury by enhancing adenosine levels and potentiating adenosine receptor-mediated effects, including attenuation of the cellular production and release of tumor necrosis factor-alpha (TNF-alpha). In rats, the phosphorylated prodrug form of NBMPR, NBMPR-phosphate, or saline was administered by intracerebroventricular injection 30 min before forebrain ischemia. Seven days following the ischemic episode, rats were killed, and neuronal damage in the CA1 region of the hippocampus was assessed. The number of pyramidal neurons was significantly (p < 0.001) greater in the NBMPR-P treatment group. A trend toward protection was still evident at 28 days postreperfusion. Adenosine increased significantly during ischemia to levels eight- to 85-fold above basal. NBMPR-P treatment did not cause statistically significant increases in ischemic adenosine levels; however, this treatment tended to increase adenosine levels in all brain regions at 7 min postreperfusion. Ischemia-induced expression of TNF-alpha was not altered by NBMPR-P treatment, and the nonselective adenosine receptor antagonist 8-(p-sulfophenyl) theophylline did not abolish the neuroprotective effects of NBMPR-P treatment. These data indicate that NBMPR can protect CA1 pyramidal neurons from ischemic death without statistically significant effects on adenosine levels or adenosine receptor-mediated inhibition of the proinflammatory cytokine TNF-alpha.

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 equilibrative, nitrobenzylthioinosine-sensitive nucleoside transporters (ENT1) in brain.

Nucleoside transport processes may play a role in regulating endogenous levels of the inhibitory neuromodulator adenosine in brain. The cDNAs encoding species homologues of one member of the equilibrative nucleoside transporter (ENT) gene family have recently been isolated from rat (rENT1) and human (hENT1) tissues. The current study used RT-PCR, northern blot, in situ hybridization, and [3H]nitrobenzylthioinosine autoradiography to determine the distribution of mRNA and protein for ENT1 in rat and human brain. Northern blot analysis indicated that hENT1 mRNA is widely distributed in adult human brain. 35S-labeled sense and antisense riboprobes, transcribed from a 153-bp segment of rENT1, were hybridized to fresh frozen coronal sections from adult rat brain and revealed widespread rENT1 mRNA in pyramidal neurons of the hippocampus, granule neurons of the dentate gyrus, Purkinje and granule neurons of the cerebellum, and cortical and striatal neurons. Regional localization in rat brain was confirmed by RT-PCR. Thus, ENT1 mRNA has a wide cellular and regional distribution in brain, indicating that this nucleoside transporter subtype may be important in regulating intra- and extracellular levels of adenosine in brain.

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.

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 receptor activity in Chinese hamster ovary cell lines transfected with human A1, A2A, or A2B receptors and a luciferase reporter gene.

Propentofylline is neuroprotective in vivo, but its mechanism of action is not completely understood. Previously, propentofylline was shown to block adenosine transport processes, to inhibit three adenosine receptor subtypes, and to inhibit cAMP phosphodiesterase. We tested the effect of propentofylline on adenosine receptor function in Chinese hamster ovary (CHO) cells transfected with human adenosine A1, A2A, or A2B receptors and a luciferase reporter gene under control of a promoter sequence containing several copies of the cAMP response element. We investigated the concentration-dependent inhibitory effects of propentofylline on cAMP phosphodiesterase, adenosine transport processes, and adenosine A1, A2A, and A2B receptors. At concentrations > or = 1 mM, propentofylline increased luciferase activity probably as a result of inhibition of cAMP phosphodiesterase. Inhibition of [3H]adenosine uptake by propentofylline was concentration dependent, with IC50 values of 37-39 microM for the three cell types. Agonist-activated adenosine A1 receptors were antagonized by 100 microM propentofylline, but inhibition of agonist-stimulated A2A or A2B receptors was not observed. In contrast, A1 and A2A receptor mediated effects of adenosine were enhanced by propentofylline at concentrations of 1 and 100 microM, respectively. These data indicate that the net effects of propentofylline in vivo will be dependent on the concentrations of propentofylline and adenosine available and on the subtypes of adenosine receptors, phosphodiesterases, and nucleoside transporters present.

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.

Uptake and release of [3H]formycin B via sodium-dependent nucleoside transporters in mouse leukemic L1210/MA27.1 cells.

At least seven functionally distinct nucleoside transport processes exist; however, mouse leukemic L1210/MA27.1 cells possess only one subtype, a Na+-dependent transporter termed N1/cif. The capacity of this transporter subtype to release nucleosides from L1210/MA27.1 cells was investigated with the poorly metabolized inosine analog [3H]formycin B. Uptake of [3H]formycin B into these cells was inhibited by replacement of Na+ in the buffer with choline, or by blocking Na+/K+ ATPase with 2 mM ouabain, inhibiting glycolysis with 5 mM iodoacetic acid or inhibiting nucleoside transport with 1 mM phloridzin. Sodium stimulated uptake with an EC50 value of 12 mM. To measure release of [3H]formycin B, cells were loaded with [3H]formycin B (10 microM) then washed and resuspended in buffer. Replacement of Na+ in the buffer with choline enhanced [3H]formycin B release by 20 to 47%, and significant stimulation of release was observed with Na+ concentrations of 30 mM or less. Resuspending loaded cells into Na+ buffer containing 2 mM ouabain or 10 microM monensin, a Na+ ionophore, significantly enhanced [3H]formycin B release during 20 min by 39% or 29%, respectively. Release of [3H]formycin B into choline buffer was inhibited 26.5% by 10 mM phloridzin and 39.6% by 10 mM propentofylline, compounds known to inhibit various transporters including Na+-dependent nucleoside transporters. Release was also inhibited significantly by 100 microM concentrations of dilazep, dipyridamole and nitrobenzylthioinosine, inhibitors with selectivity for Na+-independent nucleoside transporters. In the absence of Na+, the permeants adenosine and uridine enhanced [3H]formycin B release by up to 40.9% and 21.4%, respectively. These data indicate that in the absence of an inwardly directed Na+ gradient, Na+-dependent nucleoside transporters can function in the release of nucleosides.

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.

Nucleoside transporter-mediated uptake and release of [3H]L-adenosine in DDT1 MF-2 smooth muscle cells.

[3H]L-Adenosine, an enantiomer of the physiological D-adenosine, was shown previously to be taken up and released, at least in part, through nucleoside transporters in rat brain preparations. In the present study, we used clonal smooth muscle DDT1 MF-2 cells that contain almost exclusively equilibrative inhibitor-sensitive (es) nucleoside transporters to test the hypothesis that L-adenosine is a permeant for these bidirectional nucleoside transporters. DDT1 MF-2 cells accumulated approximately 3 times more [3H]D- than [3H]L-adenosine; 10 microM nitrobenzylthioinosine significantly (P < 0.01) inhibited the accumulation of [3H]D-adenosine by 86% and of [3H]L-adenosine by 63%. The IC50 values for the nitrobenzylthioinosine-sensitive portions of [3H]L- and [3H]D-adenosine accumulation were 1.6 and 2.0 nM, respectively. [3H]D-Adenosine accumulation was significantly (P < 0.05) inhibited by up to 72% with L-adenosine and [3H]L-adenosine accumulation was significantly (P < 0.01) inhibited by up to 52% with D-adenosine. Release of accumulated [3H]L-adenosine was temperature- and time-dependent, and was significantly (P < 0.05) reduced by 47% with dipyridamole, 39% with dilazep, and 45% with nitrobenzylthioinosine; the apparent IC50 value for nitrobenzylthioinosine was < 1 nM. These data indicate that a significant proportion of [3H]L-adenosine uptake and release in DDT1 MF-2 cells is mediated by es nucleoside transporters.

Ability of nitrobenzylthioinosine to cross the blood-brain barrier in rats.

Nucleoside transport inhibitors that cross the blood-brain barrier may be able to potentiate the neuroprotective effects of adenosine. We tested whether nitrobenzylthioinosine (NBMPR) crosses the blood-brain barrier in three types of experiments. First, intravenous injection of [3H]NBMPR and [14C]sucrose was performed. Brain volume of distribution and brain delivery were greater for [3H]NBMPR than for [14C]sucrose. Second, rats were injected intraperitoneally with NBMPR 5'-monophosphate (NBMPR-P), a prodrug form of NBMPR, or vehicle. Perchloric acid extracts of brains from rats treated with NBMPR-P inhibited [3H]NBMPR binding in competition binding assays nearly 3-fold more than extracts from brains of vehicle-treated animals. Third, cerebrospinal fluid (CSF) extracted from rats treated with NBMPR-P (10 mg/kg i.p.) contained 24.1 +/- 4.4 nM NBMPR while levels were undetectable in CSF from vehicle-treated rats. From these data, we conclude that NBMPR crosses the blood-brain barrier.

[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.

Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells.

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the 'equilibrative sensitive' or 'es' transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximately Km values of 21 microM, 12.5 microM and 15 microM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

Effects of iodotubercidin on adenosine kinase activity and nucleoside transport in DDT1 MF-2 smooth muscle cells.

Iodotubercidin is an adenosine kinase inhibitor that through its ability to increase levels of endogenous adenosine can enhance adenosine's receptor-mediated effects. We investigated whether iodotubercidin can inhibit [3H]adenosine accumulation by inhibiting transport processes in addition to inhibition of intracellular trapping of labeled adenine nucleotides. Under conditions in which extensive metabolism of intracellular adenosine was present, [3H]adenosine accumulation by DDT1 MF-2 cells was almost completely inhibited by iodotubercidin and the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine or by the nucleoside transport inhibitor nitrobenzylthioinosine. By using similar conditions, [3H]adenosine accumulation was significantly greater in Na+ buffer than in buffer containing N-methyl-D-glucamine in place of Na+; however, this effect may be explained by an observed 40% inhibition of adenosine kinase activity by N-methyl-D-glucamine. By using uptake intervals of 14 sec to represent the transport component of uptake, iodotubercidin decreased the affinity for adenosine, by about 3-fold, but had no effect on maximum velocity of transport. That these effects of iodotubercidin were due to direct interactions with nucleoside transporters was supported by findings that iodotubercidin inhibited [3H]nitrobenzylthioinosine binding to nucleoside transporters with a Ki value of 4 microM and inhibited [3H]uridine and [3H]formycin B uptake with IC50 values of 7 and 15 microM, respectively. These data suggest that iodotubercidin, at pharmacologically relevant concentrations, inhibits nucleoside transport independently of its well characterized inhibition of adenosine kinase and that N-methyl-D-glucamine must be used with caution in experiments to determine the possible presence of Na+ gradient-dependent concentrative nucleoside transporters.

Functional characterization of adenosine A2 receptors in Jurkat cells and PC12 cells using adenosine receptor agonists.

The effect of several adenosine analogues on cyclic AMP accumulation was examined in the rat phaeochromocytoma cell PC12 and in the human T-cell leukaemia cell Jurkat, selected as prototypes of cells predominantly expressing adenosine A2A or A2B receptors. Using the reverse transcription-polymerase chain reaction it was, however, demonstrated that the Jurkat cell and the PC12 cell express both A2A and A2B receptor mRNA, albeit in different relative proportions. In PC12 cells the concentration required for half-maximal response (EC50) for the full agonist 5'-N-ethyl-carboxamidoadenosine (NECA) was 30 times lower than in Jurkat cells. There was no significant difference in the pA2 for the antagonist 5-amino-9-chloro-2-(2-furanyl)- 1,2,4-triazolo(1,5-C)quinazolinemonomethanesulphonate (CGS 15943) between the two cell types. In the presence of forskolin (1 microM in PC12 cells; 10 microM in Jurkat cells) the EC50 value for NECA was reduced two-to sixfold. Forskolin also increased the maximal cAMP accumulation twofold in PC12 cells and sevenfold in Jurkat cells. A series of 2-substituted adenosine analogues CV 1808 (2-phenylamino adenosine), CV 1674 [2-(4-methoxyphenyl)adenosine], CGS 21680 ¿2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethyl- carboxamido adenosine¿, and four 2-substituted isoguanosines, SHA 40 [2-(2-phenylethoxy)adenosine; PEA], SHA 91 [2-(2-cyclohexylethoxy)adenosine; CEA], SHA 118 ¿2-[2-(p-methylphenyl)ethoxy]adenosine; MPEA¿, and SHA 125 (2-hexyloxyadenosine; HOA), all raised cAMP accumulation in PC12 cells, but had minimal or no effect in Jurkat cells. In the PC12 cells the addition of forskolin (1 microM) reduced the EC50 by a factor of 2(CV 1808) to 12 (SHA 125). In Jurkat cells all the analogues gave a significant, but submaximal, cAMP response in the presence of forskolin (10 microM), but they were essentially inactive in its absence. The results show that a series of 2-substituted adenosine analogues can be used to discriminate between A2A and A2B receptors. The two receptor subtypes appear to coexist, even in clonal cells selected for typical pharmacology. A2 receptor pharmacology can therefore be complex.