Role of cysteine residues of rat A2a adenosine receptors in agonist binding

In the present study, we investigated the role of disulfide bridges and sulfhydryl groups in A2a adenosine receptor binding of the agonist 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadenosi ne (CGS 21680). To evaluate the presence of essential disulfide bridges, rat striatal membranes were incubated with [3H]CGS 21680 in the presence of dithiothreitol and binding of the agonist to membranes was measured. The amount of [3H]CGS 21680 which specifically bound, decreased progressively upon pretreatment of membranes with increasing concentrations of dithiothreitol. Pretreatment of rat striatal membranes with 12.5 mM dithiothreitol for 15 min at 25 degrees C resulted in a 2-fold decrease of A2a adenosine receptor affinity for [3H]CGS 21680, and a reduction in the maximal number of binding sites. The presence of agonist or antagonist ligands protected the A2a adenosine receptor sites from the effect of dithiothreitol. We also examined the susceptibility of A2a adenosine receptors to inactivation by the sulfhydryl alkylating reagent, N-ethylmaleimide. When rat striatal membranes were pretreated with N-ethylmaleimide for 30 minutes at 37 degrees C, a decrease in specific [3H]CGS 21680 binding was observed. Pretreatment of membranes with 1 mM N-ethylmaleimide also resulted in a 2-fold reduction of A2a adenosine receptor affinity for [3H]CGS 21680, as well as a slight decrease in the maximal number of binding sites. Neither agonist nor antagonist ligands were effective in protecting the receptor sites from inactivation by N-ethylmaleimide. In contrast, addition of 100 microM guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate were both effective in protecting the receptor sites from inactivation by N-ethylmaleimide. This protective effect was significant but not complete. Our data suggest that disulfide bridges play a role in the structural integrity of the A2a adenosine receptor, furthermore, reduced sulfhydryl groups appear to be important but we do not yet know if they are on the receptor or on the Gs alpha subunit.     

Modulation of [3H]quinpirole binding to dopaminergic receptors by adenosine A2A receptors

Alteration of ligand binding to dopamine D2 receptors through activation of adenosine A2A receptors in rat striatal membranes has been studied by means of kinetic analysis. The binding of dopaminergic agonist [3H]quinpirole to rat striatal membranes was characterized by the constants Kd = 1.50+/-0.09 nM and Bmax = 115+/-2 fmol/mg of protein. The kinetic analyses revealed that the binding had at least two consecutive and kinetically distinguishable steps, the fast equilibrium of complex formation between receptor and agonist (KA = 5.9+/-1.7 nM), followed by a slow isomerization equilibrium (Ki = 0.06). Activation of adenosine A2A receptors by CGS 21680 caused enhancement of the rate [3H]quinpirole binding, altering mainly the formation of the receptor-ligand complexes (KA) as well as the isomerization rate of this complexes (ki), while the deisomerization rate (k[-i]) and the apparent dissociation rate remained unchanged.     

3H]SCH 58261, a selective adenosine A2A receptor antagonist, is a useful ligand in autoradiographic studies

We have characterized the new potent and selective nonxanthine adenosine A2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A2A receptors ([3H]CGS 21680) or A1 receptors (N6-[3H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A2A versus A1 receptors (Ki values of 1.2 nM versus 0.8 microM). Moreover, receptor autoradiography showed that [3H]SCH 58261, in concentrations below 2 nM, binds only to the dopamine-rich regions of the rat brain, with a K(D) value of 1.4 (0.8-1.8) nM. The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 nM, the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [3H] SCH 58261 with the following estimated Ki values (nM): 2-hex-1-ynyl-5'-N-ethylcarboxamidoadenosine, 3.9 (1.8-8.4); CGS 21680, 130 (42-405); N6-cyclohexyladenosine, 9,985 (3,169-31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 microM) or Mg2+ (10 mM). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [3H]CGS 21680.     

The foot in hereditary motor and sensory neuropathies in children

We investigated the regulation of COX-2 expression and activity by adenosine receptors in rat microglial cells. The selective adenosine A2a-receptor agonist CGS21680 and the non-selective adenosine A1- and A2-receptor agonist 5'-N-ethylcarboxiamidoadenosine (NECA) induced an increase in COX-2 mRNA levels and the synthesis of prostaglandin E2 (PGE2). The adenosine A1-receptor agonist cyclopentyladenosine (CPA) was less potent, and the adenosine A1-receptor-specific agonist N6-2-(-aminophenylo)ethyladenosine (APNEA) showed only marginal effects. Microglia expressed adenosine A1-, A2a-, and A3-, but not A2b-receptor mRNAs, whereas astroglial cells expressed adenosine A2b- but not A2a-receptor mRNA. The adenosine A2a-receptor selective antagonist (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) inhibited both CGS21680-induced COX-2 expression and PGE2 release. CGS21680-increased PGE2 levels were inhibited by dexamethasone, by the nonsteroidal antiinflammatory drug meloxicam, and by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CGS21680 and NECA both increased intracellular cAMP levels in microglial cells. Dibutyryl cAMP as well as forskolin induced the release of PGE2. The results strongly suggest that adenosine A2a-receptor-induced intracellular signaling events cause an up-regulation of the COX-2 gene and the release of PGE2. Apparently, the cAMP second messenger system plays a crucial role in COX-2 gene regulation in rat microglial cells. The results are discussed with respect to neurodegenerative disorders of the CNS such as Alzheimer's disease, in which activated microglia are critically involved and COX inhibitors may be of therapeutic benefit.     

3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[l,5-c]pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg(-1) protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments. 3. Thermodynamic data indicated that [3H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors. 4. It was concluded that in human neutrophil membranes, [3H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg-1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments. 3. Thermodynamic data indicate that [3H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A-adenosine receptors. 4. It is concluded that in human lymphocyte membranes [3H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A-receptor. The presence of A2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.     

Effect of diffusion potential, osmosis and ion-exchange on transdermal drug delivery: theory and experiments

Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70-80%), NMDA-induced inward currents were inhibited by the adenosine A2A receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the A2A receptor selective antagonist 8-(3-chlorostyryl)caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP-beta-S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14-24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca2+ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, A2A receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca2+ pathway rather than the adenylate cyclase/PKA pathway.     

Inhibition of voltage-sensitive calcium channels by the A2A adenosine receptor in PC12 cells

The role of the A2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca2+ influx induced by membrane depolarization with 70 mM K+ could be inhibited with CGS21680, an A2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp-adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca2+ concentration induced by 70 mM K+, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca2+ influx without VSCC activity after cobalt or 70 mM K+ pretreatment. These data suggest that the A2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A2A receptors induce inhibition of VSCCs as well as ATP-induced Ca2+ influx, the two inhibitory effects are clearly distinct from each other.     

Adenosine A1 receptor-mediated modulation of dopamine D1 receptors in stably cotransfected fibroblast cells

The antagonistic interactions between adenosine A1 and dopamine D1 receptors were studied in a mouse Ltk- cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In membrane preparations, both the adenosine A1 receptor agonist N6-cyclopentyladenosine and the GTP analogue guanyl-5'-yl imidodiphospate induced a decrease in the proportion of dopamine D1 receptors in a high affinity state. In the cotransfected cells, the adenosine A1 agonist induced a concentration-dependent inhibition of dopamine-induced cAMP accumulation. Blockade of adenosine A1 receptor signal transduction with the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine or with pertussis toxin pretreatment increased both basal and dopamine-stimulated cAMP levels, indicating the existence of tonic adenosine A1 receptor activation. Pretreatment with pertussis toxin also counteracted the effects of low concentrations of the A1 agonist on D1 receptor-agonist binding. The results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the generation of second messengers.     

Effects of pH on responses to adenosine, CGS 21680, carbachol and nitroprusside in the isolated perfused superior mesenteric arterial bed of the rat

1. The receptors mediating the vasodilator responses to adenosine in the isolated mesenteric arterial bed of the rat were identified by use of selective agonists and antagonists and the involvement of the endothelium was examined. 2. Adenosine-mediated dilatation of the mesentery was potentiated by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), but in contrast, removal of the endothelium substantially reduced the responses to adenosine. 3. The order of potency of adenosine receptor agonists was: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-p-(-2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) > 2-chloro-N6-cyclopentyl-adenosine (CCPA) > or = adenosine, suggesting the presence of A2A receptors. 4. Adenosine-mediated dilatation was inhibited by the non-selective adenosine receptor antagonist, 8-phenyltheophylline (3 microM) and by the A2A receptor antagonist 8-(3-chlorostyryl)caffeine (500 nM), but was unaffected by the A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM). 5. Reducing the pH of the perfusate to 6.8 potentiated the actions of both CGS 21680 and adenosine, but the vasodilator effects of carbachol were the same at both pH values. The adenosine response at the lower pH as at pH 7.4, was unaffected by DPCPX. The actions of the nitrovasodilator, sodium nitroprusside, were also potentiated at pH 6.8 relative to those at the higher pH value but smaller responses were obtained at the lower pH value with forskolin, a stimulator of adenylyl cyclase, than at pH 7.4. 6. It is concluded that the adenosine receptor mediating dilatation of the rat mesenteric arterial bed is of the A2A subtype, that the response, under the conditions used, is apparently partly dependent on the endothelium (but not due to the release of nitric oxide), and that the response to activation of this receptor is potentiated by a reduction in pH which is similar to that seen in ischaemic conditions.     

A quantitative visualization study of flow in a scaled-up model of a centrifugal blood pump

There is increasing evidence that adenosine (ADO) and dopamine (DA) interact directly in the basal ganglia via actions at ADO A2a and DA D2 receptors, respectively. The purpose of this study was to determine 1) the extent to which these receptors modulate endogenous GABA release in discrete regions of the rat basal ganglia and 2) whether GABA release is modulated by a direct and opposing interaction between ADO A2a and DA D2 receptors. Tissue slices of striatum (STR) containing globus pallidus (GP; STR/GP) and micropunches of STR, GP, and substantia nigra pars reticulata (SNr) were studied. Radioligand binding demonstrated that ADO A1, ADO A2a, and DA D2 receptors were present in each of the tissue preparations with the exception of SNr, in which ADO A2a receptors were not detected. Stimulation of ADO A2a receptors with CGS 21680 (1-10 nM) increased electrically stimulated GABA release in STR/GP slices and GP micropunches. Consistent with the lack of A2a receptors in SNr, CGS 21680 had no effect on GABA release from this region. In contrast, stimulation of DA D2 receptors with N-0437 (1-100 nM) inhibited evoked GABA release from STR/GP slices and both GP and SNr micropunches. The D2-mediated inhibition of GABA release in GP was abolished in the presence of CGS 21680 (10 nM). These experiments demonstrate that stimulation of ADO A2a and DA D2 receptors has opposing effects on endogenous GABA release in STR and GP. These opposing actions may explain the antagonistic interactions between ADO and DA that have been observed in behavioral studies and support the hypothesis that the striatopallidal efferent system is an important anatomical substrate for the A2a/D2 receptor interaction.     

Blunt injury of the small intestine and mesentery--the trauma surgeon''s Achilles heel?

It has been previously demonstrated that activation of A1 adenosine receptors in frog melanotrophs causes inhibition of spontaneous action potential discharges and alpha-melanocyte-stimulating hormone secretion. In the present study, we have investigated the effect of adenosine on high-voltage-activated (HVA) calcium currents in cultured melanotrophs, using the whole-cell variant of the patch-clamp technique with barium as a charge carrier. Adenosine and the specific A1 adenosine receptor agonist R-PIA (50 microM each) produced a decrease of the amplitude of the barium current, while the selective A2 adenosine receptor agonist CGS 21680 did not affect the current. The inhibitory effect of R-PIA was observed throughout the activation range of the current, with stronger responses at more positive potentials. R-PIA inhibited both the L- and N-type components of the current, the effect on the N-component being two-fold higher than on the L-component. The inhibitory effect of R-PIA was rendered irreversible by addition of GTP gamma S (100 microM) to the intracellular solution. Pre-treatment of the cells with pertussis toxin (1 microgram/ml; 12 h) totally abolished the effect of R-PIA on the HVA calcium channels. Conversely, addition of a high concentration of cAMP (100 microM) together with the phosphodiesterase inhibitor IBMX (100 microM) to the intracellular solution did not modify the effect of R-PIA on the current. It is concluded that, in frog melanotrophs, adenosine induces inhibition of L- and N-calcium currents and that this effect is mediated by a pertussis toxin-sensitive G protein. Our data also indicate that the inhibitory effect of adenosine on the calcium currents is not mediated by inhibition of adenylyl cyclase.     

Further characterization of [3H]-CGS 21680 binding sites in the rat striatum and cortex

1. The putative high affinity binding site for the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N- ethylcarboxamidoadenosine (CGS 21680) in the rat cerebral cortex was characterized by use of a number of selective A1 and A2 adenosine receptor ligands, and compared to the characteristics of the more abundant striatal A2A receptor. 2. The binding of [3H]-CGS 21680 to cortical membranes was performed at pH 5.5, in order to increase the amount of specific binding. 3. Reduction of the pH from 7.4 to 5.5 increased the apparent affinity of the striatal binding side for both agonists and antagonists. The relative order of potencies of both groups of ligands were the same at both pH values, and were consistent with binding to the A2A receptor. There was no observable change in the Bmax, the values being 415 and 446 fmol mg-1 protein at pH 5.5 and 7.4 respectively. 4. The cortical binding site yielded a Bmax value of 117 fmol mg-1 protein. The relative order of potencies of the adenosine receptor ligands observed at this binding site were not the same as those observed in the striatum, exhibiting a profile with both A1 and A2 characteristics. 5. Further characterization of this cortical binding site in the presence of the A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed a more typical A2A profile. This indicated that under the conditions used there were two components of [3H]-CGS 21680 binding, approximately 20% of the A1 receptor and 80% to the A2A receptor. 6. It is concluded that in the cerebral cortex there is a CGS 21680 binding site showing the characteristic properties of the striatal A2A receptor, and no evidence was obtained for the existence of a novelA2A-like binding site.     

Adenosine A2A receptors facilitate 45Ca2+ uptake through class A calcium channels in rat hippocampal CA3 but not CA1 synaptosomes

In the hippocampus, the neuromodulatory role of adenosine depends on a balance between inhibitory A1 responses and facilitatory A2A responses. Since the presynaptic effects of hippocampal inhibitory A1 adenosine receptors are mostly mediated by inhibition of Ca2+ channels, we now investigated whether presynaptic facilitatory A2A adenosine receptors would modulate calcium influx in the hippocampus. The mixed A1/A2 agonist, 2-chloroadenosine (CADO; 1 microM) inhibited veratridine (20 microM)-evoked 45Ca2+ influx into hippocampal synaptosomes of the CA1 or CA3 areas by 24.2 +/- 4.5% and 17.2 +/- 5.8%, respectively. In the presence of the A, antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), the inhibitory effect of CADO (1 microM) on 45Ca2+ influx was prevented in CA1 synaptosomes, but was converted into a facilitatory effect (14.2 +/- 6.7%) in CA3 synaptosomes. The A2A agonist, CGS 21680 (3-30 nM) facilitated 45Ca2+ influx in CA3 synaptosomes, with a maximum increase of 22.9 +/- 3.9% at 10 nM, and was virtually devoid of effect in CA1 synaptosomes. This facilitatory effect of CGS 21680 (10 nM) in CA3 synaptosomes was prevented by the A2A antagonist 8-(3-chlorostyryl)caffeine (CSC; 200 nM), but not by the A1 antagonist, DPCPX (20 or 100 nM). The facilitatory effect of CGS 21680 on 45Ca2+ uptake by CA3 synaptosomes was prevented by the class A calcium channel blocker, omega-agatoxin-IVA (200 nM). These results indicate that presynaptic adenosine A2A receptors facilitate calcium influx in the CA3 but not the CA1 area of the rat hippocampus through activation of class A calcium channels.     

Leukocytes in neuronal ceroid-lipofuscinoses: function and apoptosis

The aim of present study was to assess the role of activation of adenosine A-1 and A-2a receptors in the modulation of dopamine (DA) and glutamate release in the rat striatum by microdialysis in freely moving animals. Adenosine A-1 receptor agonist N6-cyclopentyladenosine (CPA) and A-2a receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were administered locally into the striatum through microdialysis probe. CPA (10, 100 and 500 microM) did not affect basal levels of DA or glutamate. In contrast, CGS 21680 at the concentrations of 10, 50 and 100 microM increased in a dose-dependent manner the level of DA and at 100 microM also the level of glutamate. Both agonists at the concentration of 100 microM inhibited KCl-induced (100 mM) DA and glutamate release. The present results suggest, that in physiological conditions only excitatory effects of adenosine may be observed and adenosine A-2a receptors seem to be involved. During depolarization with KCl, adenosine, by inhibiting excessive outflow of neurotransmitters mediated via A-1 and A-2a receptors, manifests its protective role as homeostatic neuromodulator.     

A postsynaptic interaction between dopamine D1 and NMDA receptors promotes presynaptic inhibition in the rat nucleus accumbens via adenosine release

The mechanism underlying dopamine D1 receptor-mediated attenuation of glutamatergic synaptic input to nucleus accumbens (NAcc) neurons was investigated in slices of rat forebrain, using whole-cell patch-clamp recording. The depression by dopamine of EPSCs evoked by single-shock cortical stimulation was stimulus-dependent. Synaptic activation of NMDA-type glutamate receptors was critical for this effect, because dopamine-induced EPSC depressions were blocked by the competitive NMDA receptor antagonist D/L-2-amino-5-phosphonopentanoate (AP5). Application of NMDA also depressed the EPSC, and both this effect and the dopamine depressions were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), implicating adenosine release in the EPSC depression. A1 receptor agonists also depressed EPSCs by a presynaptic action, causing increased paired-pulse facilitation, but this was insensitive to AP5. Activation of D1 receptors enhanced both postsynaptic inward currents evoked by NMDA application and the isolated NMDA receptor-mediated component of synaptic transmission. The biochemical processes underlying the dopamine-induced EPSC depression did not involve either protein kinase A or the production of cAMP and its metabolites, because this effect was resistant to the protein kinase inhibitors H89 and H7 and the cAMP-specific phosphodiesterase inhibitor rolipram. We conclude that activation of postsynaptic D1 receptors enhances the synaptic activation of NMDA receptors in nucleus accumbens neurons, thereby promoting a transsynaptic feedback inhibition of glutamatergic synaptic transmission via release of adenosine. Unusually for D1 receptors, this phenomenon occurs independently of adenylyl cyclase stimulation. This process may contribute to the locomotor stimulant action of dopaminergic agents in the NAcc.     

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.     

Effect of adenosine receptor agonists on spontaneous and K(+)-evoked acetylcholine release from the in vivo rat cerebral cortex

Repeated applications of elevated K+ (100 mM) in artificial cerebrospinal fluid (CSF) were used to evoke an efflux of acetylcholine (ACh) from the in vivo rat cerebral cortex using a cortical cup technique. Elevated K+ reproducibly increased the levels of ACh in cup superfusates by a factor of 3-5-fold above basal levels (27.2 +/- 9.7 nM). The adenosine A1 receptor agonist N6-cyclopentyl adenosine (CPA), at a concentration of 10(-8) M, depressed basal, but not K(+)-evoked ACh efflux. 10(-6) M CPA increased basal, but did not alter K(+)-evoked, ACh efflux. The A2 selective agonist CGS 21680 did not alter either basal, or K(+)-evoked, ACh efflux. The inhibitory effects of 10(-8) M CPA on ACh efflux would be consistent with the presence of adenosine A1 receptors on cholinergic nerve terminals in the cerebral cortex. At a higher concentration (10(-6) M) CPA elevated basal release, possibly by activating low affinity A2 receptors. The failure of CGS 21680 (10(-8) M) to alter basal ACh release suggests an absence of high affinity A2 receptors in these terminals. Whereas elevated K+ in cup superfusates consistently enhanced ACh efflux from the cerebral cortex, this increase was not affected by either CPA or CGS 21680. High K(+)-evoked release of cerebral cortical ACh may be an inappropriate model for the study of adenosine's actions on neurotransmitter release.     

Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors

Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively activated mutants of receptors should provide a convenient means to detect inverse agonists. Incubation of cells either transiently or stably transfected with a constitutively activated mutant of the human beta 2-adrenoceptor with the beta 2-inverse agonists betaxolol or sotalol, which are both able to inhibit CAM beta 2-adrenoceptor-mediated basal adenylyl cyclase activity, resulted in a strong upregulation of levels of the receptor. In the stable cells lines this effect was prevented by co-incubation with neutral antagonists but could not be reproduced by an adenylyl cyclase P-site ligand which also inhibited basal adenylyl cyclase levels.     

Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion

Accumulation of adenosine and of deoxyadenosine in the absence of adenosine deaminase activity (ADA) activity results in lymphocyte depletion and in severe combined immunodeficiency (ADA SCID), which is currently explained by direct cell death-causing effects of intracellular products of adenosine metabolism. We explored the alternative mechanisms of peripheral T-cell depletion as due to inhibition of T-cell expansion by extracellular adenosine-mediated signaling through purinergic receptors. The strong inhibition of the T-cell receptor (TCR)-triggered proliferation and of upregulation of interleukin-2 receptor alpha chain (CD25) molecules, but not the direct lymphotoxicity, were observed at low concentrations of extracellular adenosine. These effects of extracellular adenosine (Ado) are likely to be mediated by A2a receptor-mediated signaling rather than by intracellular toxicity of adenosine catabolites, because (1) poorly metabolized adenosine analogs cause the accumulation of cAMP and strong inhibition of TCR-triggered CD25 upregulation; (2) the A2a, but not the A1 or A3, receptors are the major expressed and functionally coupled adenosine receptors in mouse peripheral T and B lymphocytes, and the adenosine-induced cAMP accumulation in lymphocytes correlates with the expression of A2a receptors; (3) the specific agonist of A2a receptor, CGS21680, induces increases in [cAMP]i in lymphocytes, whereas the specific antagonist of A2a receptor, CSC, inhibits the effects of Ado and CGS21680; and (4) the increases in [cAMP]i mimic the adenosine-induced inhibition of TCR-triggered CD25 upregulation and splenocyte proliferation. These studies suggest the possible role of adenosine receptors in the regulation of lymphocyte expansion and point to the downregulation of A2a purinergic receptors on T cells as a potentially attractive pharmacologic target.