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Abstract: Caffeine, which belongs to the methylxantine family of compounds, is commonly ingested in a range of beverages such as coffee, tea, and cola drinks. It is also used therapeutically and is frequently employed in the treatment of respiratory disturbances in human neonates. The aim of the present work has been to examine the ontogeny of the adenosine A1 receptor system in the brainstem of the newborn rat following postnatal treatment with caffeine to mimic the therapeutic administration of caffeine to premature human infants. The effect of this postnatal exposure to caffeine on the gradual appearance of adenosine A1 receptors was analysed by determining immunohistochemically the distribution of the receptors. The main difference between control animals and animals exposed to caffeine was the transient increase (only at postnatal day 6) in the number of immunopositive neurons in two brainstem areas, the ventrolateral medulla and the rostral dorsolateral pons, in caffeine-treated rat pups, or more specifically, the parabrachial and Kölliker-Fuse nuclei, both of which are classically associated with respiratory control. With previous research highlighting the important role played by the rostral pons in respiratory modulation by the adenosine A1 receptor system, it is thus possible that postnatal exposure to caffeine modulates the ontogeny of the adenosine A1 receptor network. This could imply that the role of caffeine to decrease the incidence of neonatal respiratory disturbances may be due to the earlier than normal development of the adenosinergic system in the brain. [Copyright &y& Elsevier]

1. This study examined whether Paeoniflorin (PF), the major active components of Chinese herb Paeoniae alba Radix, has neuroprotective effect in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). 2. Subcutaneous administration of PF (2.5 and 5 mg kg(-1)) for 11 days could protect tyrosine hydroxylase (TH)-positive substantia nigra neurons and striatal nerve fibers from death and bradykinesia induced by four-dose injection of MPTP (20 mg kg(-1)) on day 8. 3. When given at 1 h after the last dose of MPTP, and then administered once a day for the following 3 days, PF (2.5 and 5 mg kg(-1)) also significantly attenuated the dopaminergic neurodegeneration in a dose-dependent manner. Post-treatment with PF (5 mg kg(-1)) significantly attenuated MPTP-induced proinflammatory gene upregulation and microglial and astrocytic activation. 4. Pretreatment with 0.3 mg kg(-1) 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor (A1AR) antagonist, 15 min before each dose of PF, reversed the neuroprotective and antineuroinflammatory effects of PF. 5. In conclusion, this study demonstrated that PF could reduce the MPTP-induced toxicity by inhibition of neuroinflammation by activation of the A1AR, and suggested that PF might be a valuable neuroprotective agent for the treatment of PD. [ABSTRACT FROM AUTHOR]

Abstract: Three series of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene and 2-amino-5,6,7,8-tetrahydrocyclohepta[b]thiophenes with 3-carboxylates and carboxamides have been prepared using the Gewald synthesis and evaluated as A1AR allosteric enhancers. The structure–activity relationships of these classes of compound are described. A number of compounds, notably 7b, are more potent and efficacious than PD81,723 (1). [Copyright &y& Elsevier]

Abstract: Initial mutagenesis studies exploring the ligand recognition model of A1 adenosine receptor (A1R) mainly focused on the residues in the 5th–7th transmembrane domains (TMs5–7). Little is known about the role of residues in TM2. To explore the importance of reserved hydrophobic region in TM2 of A1R, we mutated the hydrophobic residues at positions 65 and 69 to hydrophilic residues (L65T, Leu-65 to Thr-65; I69T, Ile-69 to Thr-69; I69S, Ile-69 to Ser-69) to change the hydrophobicity at the outer end of TM2. Binding assays showed that the affinities of mutant receptors were significantly decreased for ribose group-containing agonists (2-chloro-N 6-cyclopentyladenosine (CCPA) and 5′-N-ethyl-carboxamidoadenosine (NECA)) but not for antagonists, N 6-cyclopentyl-9-methyladenine (N-0840), an adenine derivative lacking ribose group, and 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), a xanthine derivative. This observation suggests that the hydrophobic region at the outer end of TM2 may mediate the recognition of the ribose group of CCPA and NECA. [Copyright &y& Elsevier]

The interaction of a new nonribose ligand (LUF5831) with the human adenosine A1 receptor was investigated in the present study. Radioligand binding experiments were performed in the absence and presence of diverse allosteric modulators on both wild-type (wt) and mutant (T277A) adenosine A1 receptors. Thermodynamic data were obtained by performing these assays at different temperatures. In addition, cyclic adenosine monophosphate (cAMP) assays were performed. The presence of allosteric modulators had diverse effects on the affinity of LUF5831, N6-cyclopentyladenosine (CPA), a full agonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an inverse agonist/antagonist, for the adenosine A1 receptor. PD81,723, for example, increased the affinity of CPA, while the affinity of LUF5831 was decreased. However, the affinity of DPCPX was decreased even more. In addition, LUF5831 was shown to have an affinity for the mutant (T277A) adenosine A1 receptor (Ki=122+/-22 nM), whereas CPA's affinity was negligible. The results of temperature-dependent binding assays showed that the binding of LUF5831 was entropy driven, in between the behaviour of CPA binding to the high- and low-affinity states of the receptor, respectively. The inhibition of the forskolin-induced production of cAMP through activation of the wt adenosine A1 receptor showed that LUF5831 had a submaximal effect (37+/-1%) in comparison to CPA (66+/-5%). On the mutant receptor, however, neither CPA nor LUF5831 inhibited cAMP production. This study indicates that the nonribose ligand, LUF5831, is a partial agonist for the adenosine A1 receptor. [ABSTRACT FROM AUTHOR]

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R-A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R-A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R-A2ARheteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine. [ABSTRACT FROM AUTHOR]

Rationale: There is no consensus on the contribution of adenosine A1 and A2A receptor blockade to motor-activating effects of caffeine. Objective: Our aim was to use a detailed and continuous observational method to compare the motor effects induced by caffeine with those induced by selective A1 and A2A receptor antagonists. Methods: The behavioral repertoire induced by systemic administration of caffeine (3, 10, and 30 mg/ kg), A1receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.2, 4.8 and 7.2 mg/kg), and A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl- 1-propargylxanthine phosphate disodium salt (MSX-3; 1, 3, and 10 mg/kg) was analyzed. The effects of pretreatment with the selective A1 receptor agonist N6-cyclopentyladenosine (CPA; 0.1 mg/g) and the selective A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxyamidoadenosine (CGS 21680; 0.2 mg/kg) on the pattern of motor activation induced by caffeine, CPT, or MSX-3 were also examined. Results: The pattern of behavioral activation induced by caffeine was better mimicked by CPT than by MSX-3. Coadministration of CPT and MSX-3 gave different results depending on the dose and the type of behavioral response. CPA was more effective at decreasing the activating effects of caffeine and CPT than those of CGS 21680. On the other hand, CGS 21680 was more effective at decreasing the activating effects of MSX-3 than those of caffeine or CPT. Factor analysis revealed a complex three-dimensional behavioral profile for caffeine that was similar to the profile for CPT and was different from the profile for MSX-3. Conclusions: The results indicate a predominant role for A1 receptors in the motor-activating effects of acutely administered caffeine. [ABSTRACT FROM AUTHOR]

Long-lasting, activity-dependent changes in synaptic efficacy at excitatory synapses are critical for experience-dependent synaptic plasticity. Synaptic plasticity at excitatory synapses is determined both presynaptically by changes in the probability of neurotransmitter release, and postsynaptically by changes in the availability of functional postsynaptic glutamate receptors. Two kinds of synaptic plasticity have been described. In homosynaptic or Hebbian plasticity, the events responsible for synaptic strengthening occur at the same synapse as is being strengthened. Homosynaptic plasticity is activity-dependent and associative, because it associates the firing of a postsynaptic neuron with that of the presynaptic neuron. Heterosynaptic plasticity, on the other hand, is activity-independent and the synaptic strength is modified as a result of the firing of a third, modulatory neuron. It has been suggested that long-term changes in synaptic strength, which are associated with gene transcription, can only be induced with the involvement of heterosynaptic plasticity. The neuromodulator adenosine plays an elaborated pre- and postsynaptic control of glutamatergic neurotransmission. This paper reviews the evidence suggesting that in some striatal excitatory synapses, adenosine can provide the heterosynaptic-like modulation essential for stabilizing homosynaptic plasticity without the need of a "third, modulatory neuron". [ABSTRACT FROM AUTHOR]

The effects of paeoniflorin (PF), a compound isolated from Paeony radix, on neurological impairment and histologically measured infarction volume following transient and permanent focal ischemia were examined in Sprague–Dawley rats.In transient ischemia model, rats were subjected to a 1.5-h occlusion of the middle cerebral artery (MCA). The administration of PF (2.5 and 5 mg kg−1, s.c.) produced a dose-dependent decrease in both neurological impairment and the histologically measured infarction volume. Similar results were also obtained when PF (2.5, 5, and 10 mg kg−1, s.c.) was given in permanent ischemia model.The neuroprotective effect of PF (10 mg kg−1, s.c.) was abolished by pretreatment of DPCPX (0.25 mg kg−1, s.c.), a selective adenosine A1 receptor (A1R) antagonist.PF (10, 40, and 160 mg kg−1, i.v.) had no effect on mean arterial pressure (MAP) and heart rates (HR) in the conscious rat. Additionally, PF (10−3 mol l−1) had no effect on noradrenaline- (NA-) or high K+ concentration-induced contractions of isolated rabbit primary artery.In competitive binding experiments, PF did not compete with the binding of [3H]DPCPX, but displaced the binding of [3H]NECA to the membrane preparation of rat cerebral cortex. This binding manner was distinguished from the classical A1R agonists.The results demonstrated that activation of A1R might be involved in PF-induced neuroprotection in cerebral ischemia in rat. However, PF had no ‘well-known’ cardiovascular side effects of classical A1R agonists. The results suggest that PF might have the potential therapeutic value as an anti-stroke drug.British Journal of Pharmacology (2005) 146, 604–611. doi:10.1038/sj.bjp.0706335; published online 8 August 2005 [ABSTRACT FROM AUTHOR]

Abstract: Three subtypes of adenosine receptors (A1, A2A and A3 ARs) are functionally expressed in cardiomyocytes. Adenosine released during ischemia and ischemia/reperfusion plays a major role in cardioprotection. Phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB) and MEK/ERK1/2 pathways are involved in cell survival. Since the role of these pathways in AR-mediated preconditioning is poorly understood, we have investigated whether PI-3K/PKB and/or MEK1/ERK1/2 pathways are involved in AR-induced cardioprotection in neonatal rat cardiomyocytes. Cells were pre-treated (15 min) with adenosine (non-selective), CPA (A1), CGS 21680 (A2A) or Cl-IB-MECA (A3) before 4 h hypoxia (0.5% O2) and 18 h reoxygenation (HX4/R). HX4/R-induced increase in LDH release was significantly reduced by adenosine (70%), CPA (59%) and Cl-IB-MECA (46%). The MEK1 inhibitor PD 98059 suppressed the effects of adenosine, CPA, and Cl-IB-MECA on LDH release, whereas the PI-3K inhibitor wortmannin did not reverse this cardioprotection. Western blotting of phosphorylated ERK1/2 and PKB during HX4/R supported the involvement of ERK1/2 and not PKB in A1 and A3 agonist-mediated cardioprotection. In addition, adenosine, CPA and Cl-IB-MECA inhibited HX4/R-induced caspase 3 activity by 75%, 70% and 59%, respectively, and this inhibition was abolished by PD 98059. Interestingly, wortmannin inhibited by 66% the anti-apoptotic response triggered by Cl-IB-MECA but had no effect on adenosine or CPA-induced inhibition of caspase 3. CGS 21680 did not modify cell survival or caspase 3 activity. In conclusion, these data show that the preconditioning effect of adenosine requires A1 and A3 but not A2A ARs and involves an anti-apoptotic effect via MEK1/ERK1/2 pathway in neonatal rat cardiomyocytes. In addition, A3AR-induced preconditioning also involves a PI-3K dependent pathway. [Copyright &y& Elsevier]

To investigate the effects of renal ischemic preconditioning (IPC) on both renal hemodynamics and the renal interstitial concentrations of adenosine and adenine nucleotides induced by ischemia-reperfusion injury. Renal hemodynamics responses to ischemia-reperfusion injury in mongrel dog models were determined with or without multiple brief renal ischemic preconditioning treatments, as well as the adenosine A1 receptor antagonist (KW-3902), respectively. The renal interstitial concentrations of adenosine and adenine nucleotides in response to ischemia-reperfusion injury, either following 1–3 cycles of IPC or not, were measured simultaneously using microdialysis sampling technology. One 10-min IPC, adenosine A1 receptor antagonist (KW-3902) also shortened the recovery time of renal blood flow (RBF) and urine flow (UF), as well as mean blood pressure (BP). Advanced renal IPC attenuated the increment of adenosine and adenine nucleotides, as well as recovery time during the 60-min reperfusion which followed the 60-min renal ischemia. All of these recovery times were dependent on the cycles of 10-min IPC. The renal interstitial concentrations of adenosine and adenine nucleotides increased and decreased during renal ischemia and reperfusion, respectively. A significant relativity in dog models exists between the cycles of 10-min renal IPC and the recovery time of BP, UF, and RBF during the 60-min renal reperfusion following 60-min renal ischemia, respectively. Renal IPC can protect against ischemia-reperfusion injury and the predominant effect of endogenous adenosine induced by prolonged renal ischemia; renal adenosine A1 receptor activation during the renal ischemia-reperfusion injury is detrimental to renal function. [ABSTRACT FROM AUTHOR]

Abstract: Adenosine A1 and A2A receptor agonists and antagonists have been reported to alter learning and memory. The aim of our study was to investigate the involvement of adenosinergic system in memory retrieval into posterior cingulate cortex (PCC) of Wistar rats. To clarify this question, we tested specifics agonist and antagonists of adenosine A1 and A2A receptors in rats submitted to a one-trial inhibitory avoidance task. The stimulation of adenosine A1 and A2A receptors by CPA and CGS21680, respectively, impaired memory retrieval for inhibitory avoidance task, into PCC. These findings provide behavioral evidence for the role of adenosinergic system in the memory retrieval into PCC. [Copyright &y& Elsevier]

2-Amino-4,5,6,7-tetrahydrobenzo(β)thiophen-3-yl 4-chlorophenylmethanone (T62) is a member of a group of allosteric modulators of adenosine A1 receptors tested in animal models of neuropathic pain to increase the efficacy of adenosine. To determine its mechanisms at the level of receptor-G-protein activation, the present studies examined the effect of T62 on A1-stimulated[35S]guanosine-5′-O-(γ-thio)-triphosphate ([35S]GTPγS)binding in brain membranes, and by[35S]GTPγS autoradiography using the A1 agonist, phenylisopropyladenosine (PIA), to activate G-proteins. In hippocampal membranes, T62 increased both basal and PIA-stimulated[35S]GTPγS binding. The effect of T62 was non-competitive in nature, since it increased the maximal effect of PIA, with no effect on agonist potency. GTPγS saturation analysis showed that T62 increased the number of G-proteins activated by agonist but had no effect on the affinity of activated G-proteins for GTPγS.[35S]GTPγS autoradiography showed that the neuroanatomical localization of T62-stimulated[35S]GTPγS binding was identical to that of PIA-stimulated activity. The increase in PIA-stimulated activity by T62 varied between brain regions, with areas of lower A1 activation producing the largest percent modulation by T62. These results suggest a mechanism of allosteric modulators to increase the number of activated G-proteins per receptor, and provide a neuroanatomical basis for understanding potential therapeutic effects of such drugs. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work was to study whether caffeine or theophylline chronically consumed during pregnancy affect inhibitory adenylyl cyclase pathway mediated by adenosine, in rat brain of both mothers and full-term fetuses. Immunoblotting analysis revealed a significant decrease in αGi1,2 subunit level (27–29% in mothers, 15–18% in fetuses), associated with a significant increase in the mRNA level coding αGi1 in both maternal and fetal rat brain (12–22%) after methylxanthine intake. No significant differences in αGi3 level were detected in any case. On the other hand, forskolin- and forskolin plus guanosine-5′-O(3-thiotriphosphate) tetralithium salt-stimulated adenylyl cyclase activity was significantly decreased (30–36%) in maternal brain. Moreover, adenylyl cyclase inhibition elicited by N6-cyclohexyladenosine, specific adenosine A1 receptor agonist, was also significantly decreased in caffeine- (40.5%) and theophylline- (55.0%) treated mothers, suggesting a desensitization of adenosine A1 receptor/adenylyl cyclase pathway in maternal brain. However, no significant differences were detected in fetal brain between control and treated animals. Therefore, caffeine or theophylline chronic intake during pregnancy differently modulates inhibitory adenylyl cyclase pathway mediated by adenosine in maternal and fetal brain causing a loss of the system responsiveness only in maternal brain but down-regulating Gi1 protein in both mother and fetus brain. [Copyright &y& Elsevier]

Abstract: ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin–Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive–Ca2+ pathway through the stimulation of A1 receptors. [Copyright &y& Elsevier]

Abstract: We studied the wild-type human adenosine A1 receptor and three mutant receptors, in which the glycine at position 14 had been changed into an alanine, a leucine, or a threonine residue. All receptors were characterized in radioligand binding experiments, the wild-type and the Gly14Thr mutant receptor in greater detail. Both receptors were allosterically modulated by sodium ions and PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), although in a different way. All mutant receptors appeared to be spontaneously or “constitutively” active in a [35S]GTPγS binding assay, the first demonstration of the existence of such CAM (constitutively active mutant) receptors for the adenosine A1 receptor. The Gly14Thr mutant receptor was also constitutively active in another functional assay, i.e., the inhibition of forskolin-induced cAMP production in intact cells. Importantly, this mutant displayed a peculiar “locked-on” phenotype, i.e., neither agonist nor inverse agonist was capable of modulating the basal activity in both the GTPγS and the cAMP assay, unlike the wild-type and the two other mutant receptors. [Copyright &y& Elsevier]

To study the modulating effect of adenosine deaminase (ADA) on yhe adenosine A1 receptor (A1R) in HEK293 cells stably expressing the human A1R.cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned A1R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of adenosine deaminase on A1R was studied by using [3H]DPCPX binding assay and an intracellular calcium assay.HEK293 cells stably expressing human A1R were obtained. Saturation studies showed that theKD value andBmax value of [3H]DPCPX were 1.6±0.2 nmol/L and 1.819±0.215 nmol/g of protein respectively, in the absence of ecto-ADA respectively, and 1.3±0.2 nmol/L and 1.992±0.130 nmol/g of protein in the presence of ecto-ADA respectively, suggesting that theKD value andBmax value of [3H]DPCPX were unaffected by ecto-ADA. In the case of [3H]DPCPX competition curves obtained from intact cells or membranes, A1R agonist CCPA/[3H]DPCPX competition curve could be fitted well to a one-site model in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with aKH value of 0.74 (0.11–4.8) nmol/L (intact cells) or 1.8 (0.25–10) nmol/L (membrane) and aKL value of 0.94 (0.62–1.41)μmol/L (intact cells) or 0.77 (0.29–0.99)μmol/L (membrane). TheKL value is not significantly different from the EC50 value of 0.84(0.57–1.23)μmol/L (intact cells) or 0.84 (0.63–1.12)μmol/L (membrane) obtained in the absence of ecto-ADA. Similar results were obtained from the CPA/[3H]DPCPX competition curve in the absence or presence of ecto-ADA on intact cells or membranes. Intracellular calcium assay demonstrated that the EC50 value of CPA were 10 (5–29) nmol/L and 94 (38–229) nmol/L in the presence or absence of ecto-ADA, respectively.A1R stably expressed in the HEK293 cells display a low affinity for agonists in the absence of ADA and high and low affinities for agonists in the presence of ADA. The presence of ADA may promote the signaling through the adenosine A1 receptor in HEK293 cells. [ABSTRACT FROM AUTHOR]

Abstract: The effects of the hexapeptide angiotensin II (3–8) ANG IV, the selective A1 receptor agonist cyclohexyladenosine (CHA) and the combination of ANG IV + CHA on pentylenetetrazol (PTZ)-generalized seizures; kindling development and maintenance were studied. By using in vitro quantitative receptor autoradiography, the regulation of adenosine A1 receptor density at different time points during the kindling procedure and postkindling period was determined. ANG IV and CHA effectively reduced clonic seizures in PTZ-generalized seizure model, in PTZ-kindled mice as well as during kindling development and a week later by rechallenge with PTZ. Furthermore, coadministration of ANG IV and CHA had a strong anticonvulsant effect, both compounds acting synergistically. A significant increase of adenosine A1 receptor density was detected in somatosensory cortex, hippocampus, amygdala and geniculate nuclei early in the kindling procedure (after the 3rd injection), which persisted at least 1 month after the end of kindling procedure. In addition, a delayed up-regulation of adenosine A1 receptor binding was observed a week after kindling in the mamillary bodies and a month later in the motor cortex. The pretreatment with ANG IV caused a down-regulation of adenosine A1 receptor density to the control level in most time points and brain areas. In conclusion, PTZ kindling-induced increase of adenosine A1 receptor binding at different time points and in specific brain structures might represent an adaptive mechanism for coping with the hyperexcitability typical for this phenomenon. The antiepileptogenic effect of ANG IV could be realized partly through an adenosine-dependent mechanism. [Copyright &y& Elsevier]

1. The aim of the present study was to investigate the potential energy preserving effect of permanent bilateral common carotid artery occlusion (BCCAO) towards additional systemic hypotension of severe duration (30 min). In addition, the role of adenosine A1 receptors in cerebral ischaemic preconditioning was investigated in male Wistar rats. Thus, oligaemic rats were assigned randomly to continuous treatment with the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) or the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT), receiving daily intraperitoneal infusions of 0.1 mg/kg bodyweight CCPA or CPT or placebo (200 µL aqueous 2-hydropropyl-β-cyclodextrin) at a delivery rate of 0.5 µL/h over 14 days.2. Haemodynamic parameters and arterial blood gases were monitored. Rat cortical energy metabolites ATP, ADP, AMP, phosphocreatine and adenosine were measured using HPLC techniques. Adenosine A1 receptor expression was determined by immunhistochemistry and quantified by western blotting.3. Two weeks of permanent BCCAO induced an‘energy saving’ effect in rat cortical ATP concentrations. Under subchronic conditions, significant increases were detected in ADP and AMP concentrations after CCPA compared with placebo. Because similar changes were also seen after CPT, this adenosine A1 receptor-mediated effect does not seems to be specific. Furthermore, no differences in adenosine A1 receptor expression could be detected.4. Adenosine was not specifically involved in the‘preconditioning-like’ effect via the modulation of the adenosine A1 receptor in the present oligaemia model. Obviously, adenosine A1 receptor-specific effects after delayed cerebral ischaemic preconditioning do not seem to play an essential role if BCCAO is followed by a prolonged additional severe ischaemic event. [ABSTRACT FROM AUTHOR]

1 The present study investigates the effect of varying ligand structure on the ability of agonists to activate guanine nucleotide-binding proteins of the Gi, Gs and Gq families via the A1 adenosine receptor. 2 In CHO cells expressing this receptor, inhibition or potentiation of forskolin-stimulated cAMP accumulation was used as an end point to measure the activation of Gi and, in Pertussis toxin (PTX)- treated cells, Gs, respectively, Stimulation of inositol phosphate accumulation in PTX-treated cells was used as an index of Gq activation, 3 CPA (N6-cyclopentyladenosinc). NECA (5'-N-ethyl-carboxyiimidoadenosine) and eight analogues of these ligands presented a range of guanine nucleotide-binding protein (G-protein)-iictiv;Uing profiles. Some ligands could only activate Gi (e.g. 2'deoxyCPA). some primarily Gi and Gs (and only weakly Gq) (e.g. 3'deoxyCPA), highlighting the importance of the ribose hydroxyls in agonist activation of multiple G proteins, CHA (N6-cyclohexyladenosine) activated Gi. Gs and Gq, but was more efficacious than CPA in activating Gs. 4 The NECA analogues 5'-.N-cyclopropyl-carboxaniidoadenosine, 5'-N-cyclobutyl-carboxamidoadenosine and 5'-N-cyclopentyl-carboxamidoudenosine (CPeCA) also activated all three G proteins, although their ability to activate Gs and Gq (relative to CPA) was reduced with increasing substituent size, such that CPeCA produced only a small stimulation (at 100 μM) at Gq, but was a full agonist, relative to CPA, at Gi and Gs. 5 This study suggests that the A, adenosine receptor can adopt agonist-specific conformations, arising from small changes in ligand structure, which lead to the differential activation of Gi, Gs and Gq. [ABSTRACT FROM AUTHOR]

The objective of this investigation was to characterise the pharmacokinetic–pharmacodynamic correlation of 5′-deoxy-N6-cyclopentyl-adenosine (5′dCPA) in the chronic constriction injury model of neuropathic pain. Following intravenous administration of 5′dCPA (0.30 or 0.75 mg kg−1), the time course of the drug concentration in plasma was determined in conjunction with the effect on (1) the mechanical paw pressure and (2) the Von Frey Hair monofilament withdrawal threshold. Population pharmacokinetic–pharmacodynamic analysis was applied to derive individual concentration–effect relationships. For mechanical paw pressure a composite model consisting of an Emax model for the anti-hyperalgesic effect in combination with a linear model for the anti-nociceptive effect accurately described the data. The EC50 for the anti-hyperalgesic effect was 178±51 ng ml−1 and the slope of the anti-nociceptive effect 0.055±0.008 g ml ng−1. For the Von Frey Hair monofilament withdrawal threshold responders and non-responders were observed. Typically, in responders, full pain relief was observed at concentrations exceeding 100 ng ml−1. The high plasma concentrations required for the anti-hyperalgesic effect relative to the receptor affinity are consistent with restricted transport of 5′dCPA to the site of action in the spinal cord and/or the brain. [Copyright &y& Elsevier]

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A1, A2A and A3) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A1 selective agonist) and Cl-IB-MECA (A3 selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A2A selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks Gi/Go-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A1 and A3 receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O2) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A1 and A3 receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a Gi/Go-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning. [Copyright &y& Elsevier]

Abstract: Fully quantitative analysis of the adenosine A1 receptor (A1R) in the brain with 11C-MPDX and positron emission tomography is reported. The kinetics is described using a two-tissue three-compartment model, and estimated binding potentials correspond well with the estimates made by Logan plot. The image of the binding potential of the MPDX is physiologically reasonable. We conclude that MPDX is applicable to the visualization of the A1Rs in the brain with Logan plot. [Copyright &y& Elsevier]

2-Amino-3-benzoylthiophenes are allosteric enhancers of agonist binding to the adenosine A1 receptor. New compounds bearing an heteroaroyl instead of the benzoyl moiety at the 3-position of the thiophene were synthesized. The phenyl ring was replaced with heterocycles that possess heteroatoms able to form hydrogen bonds (2-furanyl, 2-benzofuranyl, 2-pyridinyl in compounds 2–13) or with a thienyl moiety as isoster of the phenyl ring (2-thienyl, 3-thienyl and 5-halo-2-thienyl in compounds 14–29). The effect of several alkyl substituents at positions 4 and 5 of the thiophene ring to increase enhancer activity was determined. The ability of the new molecules to reduce the cAMP content in CHO cells expressing the human adenosine A1 receptor was evaluated. Compounds 2–13 with hydrogen bond-forming heteroatoms did not show significant activity as allosteric enhancers. On the other hand, compounds 15–16 and 19–20 with an unsubstituted thienyl moiety as replacement for the phenyl ring were nearly as efficacious as PD 81,723, the prototypical A1 allosteric enhancer. Alkyl substituents at positions 4 and 5 of the thiophene ring were tolerated while a substituted piperidine ring was not tolerated. We conclude that hydrogen bonds could not be formed in the domain of the receptor that accommodates the phenyl ring of 2-amino-3-benzoylthiophene derivatives, indicating that this domain is hydrophobic. [Copyright &y& Elsevier]

We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated Gi1 α-subunits (A1–Giα) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first analysed whether such fusion products are still subject to allosteric modulation, and, secondly, we investigated the potential utility of the fusion proteins to study constitutive receptor activity. We determined the pharmacological profile of nine different A1–Giα fusion proteins in radioligand binding studies. In addition, we performed [35S]GTPγS binding experiments to study receptor and G protein activation of selected A1–Giα fusion proteins. Compared to unfused adenosine A1 receptors, the affinity of N6-cyclopentyladenosine (CPA) at wild-type A1–Giα fusion proteins (351Cys) increased more than eightfold, while the affinity of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not change significantly. Furthermore, we showed that the allosteric enhancer of agonist binding, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)-phenyl]methanone), elicited similar effects on ligand binding; i.e. CPA binding to the A1–Giα fusion proteins was enhanced, whereas the affinity of DPCPX was hardly affected. Moreover, sodium ions were unable to decrease agonist binding to the majority of the A1–Giα fusion proteins, presumably because they exhibit their effect through uncoupling of the R–G complex. From [35S]GTPγS binding experiments, we learned that all the A1–Giα fusion proteins tested had a higher basal receptor activity than the unfused adenosine A1 receptor, thereby providing improved conditions to observe inverse agonism. Moreover, the maximal CPA-induced stimulation of basal [35S]GTPγS binding was increased for the five A1–Giα fusion proteins tested, whereas the inhibition induced by 8-cyclopentyltheophylline (CPT) was more pronounced at 351Cys, 351Ile, and 351Val A1–Giα fusion proteins. Thus, the maximal receptor (de)activation depended on the amino acid at position 351 of the Gi α-subunit. In conclusion, A1–Giα fusion proteins, especially with 351Cys and 351Ile, can be used as research tools to investigate inverse agonism, due to their increased readout window in [35S]GTPγS binding experiments. [Copyright &y& Elsevier]

A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at -70 mV [18.8 ± 1.1 pA (n = 98) at 1 mM] in a dose-dependent manner (EC50 = 177 μM). A similar current was induced by A1 agonist N6-cyclopentyladenosine (1 μM), whereas A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 μM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K+, and was attenuated by Ba2+ (100 μM) and 4-aminopyridine (5 mM) but not tetraethylammonium (5 mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 μM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 μM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 μM). We conclude that a Ba2+- and 4-aminopyridine-sensitive K+ channel in SG neurons is opened via the activation of A1 receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs. [Copyright &y& Elsevier]

The present study examined the actions of adenosine on monosynaptic Aδ and C primary-afferent excitatory postsynaptic currents (EPSCs) recorded from substantia gelatinosa (SG) neurons of an adult rat spinal cord slice. In 67% of the neurons examined, adenosine reversibly decreased the amplitude of the Aδ-fiber EPSC, while in 13% of the neurons the amplitude was reduced or unaffected, which was followed by its increase persisting for several minutes after adenosine washout. The remaining neurons did not exhibit a change in the amplitude. The reduction in Aδ-fiber EPSC amplitude by adenosine was dose-dependent with an effective concentration for half-inhibition (EC50) value of 217 μM. When examined by using a paired-pulse stimulus, a ratio of the second to first Aδ-fiber EPSC amplitude under the reduction was larger than that of EPSC amplitude in the control, suggesting a presynaptic action of adenosine. In 69% of the neurons tested, the C-fiber EPSC was reversibly decreased in amplitude by adenosine (100 μM) by an extent comparable to that of Aδ-fiber EPSC; the remaining neurons were without adenosine actions. Similar inhibitory actions of adenosine were also seen in neurons where both Aδ-fiber and C-fiber EPSCs were elicited. Similar reduction in the Aδ-fiber or C-fiber EPSC amplitude was induced by an A1 adenosine-receptor agonist, N6-cyclopentyladenosine (1 μM), and the adenosine-induced reduction was not observed in the presence of an A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (1 μM). An A2a agonist, CGS 21680 (1 μM), did not significantly affect the Aδ-fiber EPSC amplitude. It is concluded that adenosine presynaptically inhibits monosynaptic Aδ-fiber and C-fiber transmission by a similar extent through the activation of the A1 receptor in many but not all SG neurons; this could contribute to at least a part of antinociception by intrathecally administered adenosine analogues and probably by endogenous adenosine. [Copyright &y& Elsevier]

1. The effect of pyrrolidine dithiocarbamate (PDTC) on kainate (KA)-induced neurotoxicity was examined in Sprague-Dawley rats. 2. At 10 mg/kg, i.p., KA produced seizures accompanied by neuronal loss in the hippocampus and increased levels of malondialdehyde (MDA) and protein carbonyl. 3. Pretreatment with PDTC (100 or 200 mg/kg, p.o., every 12 h ×5) blocked KA-induced neurotoxicities (seizures, increases in MDA and protein carbonyl and neuronal losses) in a dose-dependent manner. These effects were counteracted by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (25 or 50 µg/kg, i.p.), but not by the A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (0.5 or 1 mg/kg, i.p.) or the A2B receptor antagonist alloxazine (1.5 or 3.0 mg/kg, i.p.). 4. Our results suggest that the anticonvulsant and neuroprotective effects of PDTC are mediated, at least in part, via adenosine A1 receptor stimulation. [ABSTRACT FROM AUTHOR]

The modulatory effect of adenosine on γ-aminobutyric acid (GABA)-activated whole-cell currents were investigated in the neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage-clamp conditions. The results showed that: (1) GABA acted on GABAA receptor and elicited inward Cl- currents (IGABA) at a holding potential (VH) of -40 mV; (2) adenosine suppressed GABA-induced Cl- current without affecting the reversal potential of IGABA and the apparent affinity of GABA to its receptor; (3) N6-cyclohexyladenosine mimicked the suppression effect of adenosine on IGABA, whereas 8-cyclopentyl-1,3-dipropylxanthine blocked the suppression effect of adenosine; (4) adenosine fails to suppress IGABA on the neurons that were pretreated with bisindolylmaleimide I (BIM), while after pretreatment with H-89, the inhibitory effect of adenosine on IGABA were not affected; (5) the suppression effect of adenosine on IGABA remained in the presence of BAPTA-AM. The present results indicate that the suppression of adenosine on IGABA is mediated by adenosine A1 receptor and through a Ca2+-independent protein kinase C transduction pathway, and that the interactions between adenosine and GABA might participate in the modulation of nociceptive information transmission at the SDCN. [Copyright &y& Elsevier]

1: The coexistence of both inhibitory A1 and facilitatory A2 adenosine receptors in the rat myenteric plexus prompted the question of how adenosine activates each receptor subtype to regulate cholinergic neurotransmission. 2: Exogenously applied adenosine (0.3-300?µM) decreased electrically evoked [3H]acetylcholine ([3H]ACh) release. Blocking A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (10?nM) transformed the inhibitory action of adenosine into a facilitatory effect. Adenosine-induced inhibition was mimicked by the A1 receptor agonist R-N6-phenylisopropyladenosine (0.3?µM), but the A2A agonist CGS 21680C (0.003?µM) produced a contrasting facilitatory effect. 3: Increasing endogenous adenosine levels, by the addition of (1) the adenosine precursor AMP (30-100?µM), (2) the adenosine kinase inhibitor 5'-iodotubercidin (10?µM) or (3) inhibitors of adenosine uptake (dipyridamole, 0.5?µM) and of deamination (erythro-9(2-hydroxy-3-nonyl)adenine, 50?µM), enhanced electrically evoked [3H]ACh release (5?Hz for 40?s). Release facilitation was prevented by adenosine deaminase (ADA, 0.5?U?ml-1) and by the A2A receptor antagonist ZM 241385 (50?nM); these compounds decreased [3H]ACh release by 31±6% (n=7) and 37±10% (n=6), respectively. 4: Although inhibition of ecto-5'-nucleotidase by a,ß-methylene ADP (200?µM) or by concanavalin A (0.1?mg?ml-1) attenuated endogenous adenosine formation from AMP, analysed by HPLC, the corresponding reduction in [3H]ACh release only became evident when stimulation of the myenteric plexus was prolonged to over 250?s. 5: In summary, we found that endogenously generated adenosine plays a predominantly tonic facilitatory effect mediated by prejunctional A2A receptors. Extracellular deamination and cellular uptake may restrict endogenous adenosine actions to the neuro-effector region near the release/production sites.British Journal of Pharmacology (2004) 141, 925-934. doi:10.1038/sj.bjp.0705697 [ABSTRACT FROM AUTHOR]

Some behavioral and biochemical effects of the systemically administered adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680) in rats are potentiated by adenosine A1 receptor agonists and counteracted by dopamine D2 receptor agonists. In the present study we compared potentiating and antagonistic interactions between CGS 21680 and adenosine A1 and dopamine D2 receptor agonists on motor activity and on cardiovascular responses (arterial blood pressure and heart rate). The motor-depressant effects produced by CGS 21680 (0.5 mg/kg, i.p.) were potentiated by the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 0.3 mg/kg, i.p.) and counteracted by the dopamine D2 receptor agonist quinpirole (0.5 mg/kg, i.p.). In contrast, neither CPA nor quinpirole significantly modified the decrease in arterial pressure or the increase in heart rate induced by CGS 21680. However, the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3, 3 mg/kg, i.p.) counteracted both the motor-depressant and cardiovascular effects of CGS 21680. Therefore, the effects of the systemically administered adenosine A2A receptor agonist CGS 21680 on cardiovascular function, in contrast to its effects on motor behavior, appear to be independent of the effects of adenosine A1 and dopamine D2 receptor activity. [Copyright &y& Elsevier]