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Background and Purpose: Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5'-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptors (A1 R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A1 R agonists neither affect basal osteoclastogenesis nor do they reverse A1 R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A1 R antagonist-mediated inhibition, and, when we saw no effect, determined whether A1 R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation.Experimental Approach: Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand-macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa.Key Results: Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC50 = 20-70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold.Conclusions and Implications: Based on these findings, we hypothesize that the A1 R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A1 R antagonists act as inverse agonists to release A1 R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A1 R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation. [ABSTRACT FROM AUTHOR]

Background and Purpose Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 ( CD39), ecto-5′-nucleotidase ( CD73), nucleotide pyrophosphatase phosphodiesterase 1 ( NPP-1) and tissue non-specific alkaline phosphatase ( TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptors ( A1R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A1R agonists neither affect basal osteoclastogenesis nor do they reverse A1R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A1R antagonist-mediated inhibition, and, when we saw no effect, determined whether A1R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation. Experimental Approach Osteoclast formation derived from wild-type, CD39 knockout ( KO), CD73 KO, NPP- 1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand-macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression ( Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa. Key Results Rolofylline inhibited osteoclast formation in a dose-dependent manner ( IC50 = 20-70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold. Conclusions and Implications Based on these findings, we hypothesize that the A1R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A1R antagonists act as inverse agonists to release A1R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A1R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Boesenbergia rotunda Linn. (Zingiberaceae) is traditionally used in many Asian countries as medicine for stomach pain and discomfort, viral and bacterial infection, inflammation, and as diuretic agent. Aim of the study: The study aimed to identify adenosine A1 receptor binding compounds from Boesenbergia rotunda rhizome extract by using comprehensive extraction coupled to the NMR metabolomics method. Materials and methods: Dried and powdered Boesenbergia rotunda rhizomes were extracted with the comprehensive extraction method to obtain several fractions with different polarity. Each fraction was divided into two: for NMR analysis and for adenosine A1 receptor binding test. Orthogonal projection to the least square analysis (OPLS) was used to study the correlation between metabolites profile and adenosine A1 receptor binding activity of the plant extracts. Based on Y-related coefficient and variable of important (VIP) value, signals in active area of OPLS loading plot were studied and the respective compounds were then elucidated Results and discussions: Based on OPLS Y-related coefficient plot and variable of importance value plot, several characteristic signals were found to positively correlate to the binding activity. By using 1D and 2D NMR spectra of one of the most active fraction, pinocembrine and hydroxy-panduratin were identified as the possible active compounds. Two signals from ring C of pinocembrine flavanone skeleton with negative coefficient correlations possibly overlapped with those of non-active methoxylated flavanones which were also presence in the extract. NMR based metabolomics applied in this study was able to quickly identify bioactive compounds from plant extract without necessity to purify them. Further confirmation by isolating pinocembrine and hydroxy-panduratin and testing their adenosine A1 receptor binding activity to chemically validate the method are required. Conclusion: Two flavonoid derivatives, pinocembrine and hydroxy-panduratin, have been elucidated as possible active compounds bind to adenosine A1 receptor. Flavonoid was reported to be one of natural antagonist ligand for adenosine A1 receptor while antagonistic activity to the receptor is known to associate with diuretic activity. Thus, the result of this research supports the traditional use of Boesenbergia rotunda rhizome extract as diuretic agent. [Copyright &y& Elsevier]

Coffee and tea contain the stimulants caffeine and theophylline. These compounds act as antagonists of adenosine receptors. Adenosine promotes sleep and its extracellular concentration rises in association with prolonged wakefulness, particularly in the basal fore-brain (BF) region involved in activating the cerebral cortex. However, the effect of adenosine on identified BF neurons, especially non-cholinergic neurons, is incompletely understood. Here we used whole-cell patch-clamp recordings in mouse brain slices prepared from two validated transgenic mouse lines with fluorescent proteins expressed in GABAergic or parvalbumin (PV) neurons to determine the effect of adenosine. Whole-cell recordings were made from BF cholinergic neurons and from BF GABAergic and PV neurons with the size (>20μM) and intrinsic membrane properties (prominent H-currents) corresponding to cortically projecting neurons. A brief (2 min) bath application of adenosine (100μM) decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (EPSCs) in all groups of BF cholinergic, GABAergic, and PV neurons we recorded. In addition, adenosine decreased the frequency of miniature EPSCs in BF cholinergic neurons. Adenosine had no effect on the frequency of spontaneous inhibitory postsynaptic currents in cholinergic neurons or GABAergic neurons with large H-currents but reduced them in a group of GABAergic neurons with smaller H-currents. All effects of adenosine were blocked by a selective, adenosine A1 receptor antagonist, cyclopentyltheophylline (CPT, 1μM). Adenosine had no postsynaptic effects. Taken together, our work suggests that adenosine promotes sleep by an A1 receptor-mediated inhibition of glutamatergic inputs to cortically projecting cholinergic and GABA/PV neurons. Conversely, caffeine and theophylline promote attentive wakefulness by inhibiting these A1 receptors in BF thereby promoting the high-frequency oscillations in the cortex required for attention and cognition. [ABSTRACT FROM AUTHOR]

The effect of an allosteric modulator of the adenosine A1 receptors was investigated using an ischaemia-reperfusion protocol in murine isolated hearts. Isolated hearts were perfused with Kreb-Henseleit solution gassed with carbogen gas (95% O2 and 5% CO2) in Langendorff mode and electrically paced at 480 bpm. Following 20 min equilibration and 20 min global normothermic ischaemia, the allosteric modulator VCP333 (1 μM) or the adenosine A1 receptor partial agonist VCP102 (10 μM) were infused after 5 min of reperfusion for 15 min. Upon termination of the drug treatment, reperfusion continued for a further 40 min. At the end of 60 min reperfusion, treatment with VCP333 or VCP102 improved the recovery of the left ventricular developed pressure when compared to control group responses (p < 0.05). Neither compound affected end diastolic pressure, coronary flow rates or dP/dtmax values when compared to control tissues during reperfusion (p > 0.05). The infusion of VCP102 or VCP333 during reperfusion reduced cardiac troponin I efflux to 6.7% and 25% respectively of control heart efflux (p < 0.05). This data indicates that the allosteric modulator of the adenosine A1 receptor (VCP333) has similar characteristics to the adenosine receptor partial agonist VCP102 as it improves cardiac function and reduces myocardial cell death following an ischaemic episode. [ABSTRACT FROM AUTHOR]

The concept of ligand-receptor binding kinetics is emerging as an important parameter in the early phase of drug discovery. Since the currently used kinetic assays are laborious and low throughput, we developed a method that enables fast and large format screening. It is a so-called dual-point competition association assay, which measures radioligand binding at two different time points in the absence or presence of unlabeled competitors. Specifically, this assay yields the kinetic rate index (KRI), which is a measure for the binding kinetics of the unlabeled ligands screened. As a prototypical drug target, the adenosine A1 receptor (A1R) was chosen for assay validation and optimization. A screen with 35 high-affinity A1R antagonists yielded seven compounds with a KRI value above 1.0, which indicated a relatively slow dissociation from the target. All other compounds had a KRI value below or equal to 1.0, predicting a relatively fast dissociation rate. Several compounds were selected for follow-up kinetic quantifications in classical kinetic assays and were shown to have kinetic rates that corresponded to their KRI values. The dual-point assay and KRI value may have general applicability at other G-protein-coupled receptors, as well as at drug targets from other protein families. [ABSTRACT FROM PUBLISHER]

Abstract: The present study explored a link between spinal 5-HT7 and adenosine A1 receptors in antinociception by systemic amitriptyline in normal and adenosine A1 receptor knock-out mice using the 2% formalin test. In normal mice, antinociception by systemic amitriptyline 3mg/kg was blocked by intrathecal administration of the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 10nmol. Blockade was also seen in adenosine A1 receptor +/+ mice, but not in −/− mice lacking these receptors. In both normal and adenosine A1 receptor +/+ mice, the selective 5-HT7 receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride (SB269970) 3μg blocked antinociception by systemic amitriptyline, but it did not prevent antinociception in adenosine A1 receptor −/− mice. In normal mice, flinching was unaltered when the selective 5-HT7 receptor agonist (2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin (AS-19) 20μg was administered alone, but increased when co-administered intrathecally with DPCPX 10nmol or SB269970 3μg. Intrathecal AS-19 decreased flinching in adenosine A1 receptor +/+ mice compared to −/− mice. Systemic amitriptyline appears to reduce nociception by activating spinal adenosine A1 receptors secondarily to 5-HT7 receptors. Spinal actions constitute only one aspect of antinociception by amitriptyline, as intraplantar DPCPX 10nmol blocked antinociception by systemic amitriptyline in normal and adenosine A1 receptor +/+, but not −/− mice. Adenosine A1 receptor interactions are worthy of attention, as chronic oral caffeine (0.1, 0.3g/L, doses considered relevant to human intake levels) blocked antinociception by systemic amitriptyline in normal mice. In conclusion, adenosine A1 receptors contribute to antinociception by systemic amitriptyline in both spinal and peripheral compartments. [Copyright &y& Elsevier]

The interaction between adenosine and soluble epoxide hydrolase (sEH) in vascular response is not known. Therefore, we hypothesized that lack of sEH in mice enhances adenosine-induced relaxation through A2A adenosine receptors (AR) via CYP-epoxygenases and peroxisome proliferator-activated receptor γ (PPARγ). sEH-/- showed an increase in A2A AR, CYP2J, and PPARγ by 31%, 65%, and 36%, respectively, and a decrease in A1AR and PPARγ (30% and 27%, respectively) vs. sEH-/-. 5=-N-ethylcarboxamidoadenosine (NECA, an adenosine receptor agonist), CGS 21680 (A2A AR-agonist), and GW 7647 (PPARγ-agonist)-induced responses were tested with nitro-L-arginine methyl ester (L-NAME) (NO-inhibitor; 10-4 M), ZM-241385, SCH- 58261 (A2A AR-antagonists; 10-6 M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10-5 M), 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; 10 μM) or trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB, sEH-inhibitors; 10-5 M), and T0070907 (PPARγ-antagonist; 10-7 M). In sEH-/- mice, ACh response was not different from sEH-/- (P < 0.05), and L-NAME blocked ACh-responses in both sEH-/- and sEH-/- mice (P < 0.05). NECA (10-6 M)-induced relaxation was higher in sEH-/- (-12.94 × 3.2%) vs. sEH-/- mice (-5.35 × 5.2%); however, it was blocked by ZM-241385 (-22.42 × 1.9%) and SCH-58261(-30.04 × 4.2%). CGS-21680 (10-6 M)- induced relaxation was higher in sEH-/- (-37.4 × 5.4%) vs. sEH-/- (-2.14 × 2.8%). L-NAME (sEH-/-, -30.28 × 4.8%, P < 0.05) did not block CGS-21680-induced response, whereas 14,15- EEZE (-7.1 × 3.7%, P < 0.05) did. Also, AUDA and t-AUCB did not change CGS-21680-induced response in sEH-/- (P > 0.05), but reversed in sEH-/- (from -2.14 × 2.8% to -45.33 × 4.1%, and -63.37 × 7.2, respectively). PPARγ-agonist did not relax as CGS 21680 (-2.48 × 1.1 vs. -37.4 × 5.4%) in sEH-/-, and PPARγ- antagonist blocked (from -37.4 × 5.4% to -9.40 × 3.1) CGS 21680-induced relaxation in sEH-/-. Our data suggest that adenosine- induced relaxation in sEH-/- may depend on the upregulation of A2A AR, CYP2J, and PPARγ, and the downregulation of A1 AR and PPARα. [ABSTRACT FROM AUTHOR]

We report the design, synthesis, and validation of the novel compound photocaged N 6 -cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. G i protein-coupled A 1 receptors (A 1 Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

Given the neuroprotective effects of trans-resveratrol (RV), this study aimed to investigate the involvement of the adenosine A1 receptor (A1R) in RV-mediated neuroprotection in a rat intracerebral hemorrhage (ICH) model induced by intrastriatal injection of collagenase. Rats were divided into 5 groups: (1) control, (2) sham-operated, (3) ICH pretreated with vehicle, (4) ICH pretreated with RV, and (5) ICH pretreated with RV and the A1R antagonist DPCPX. At 48 hours after ICH, the rats were subjected to neurological testing. Brain tissues were assessed for neuronal density and morphological features using routine and immunohistochemical staining. Expression of tumor necrosis factor-α (TNF-α), caspase-3, and RIPK3 proteins was examined using ELISA. A1R, MAPK P38, Hsp90, TrkB, and BDNF genes were examined using RT-qPCR. RV protected against neurological deficits and neuronal depletion, restored the expression of TNF-α, CASP3, RIPK3, A1R, and Hsp90, and increased BDNF/TrkB. DPCPX abolished the effects of RV on neurological outcomes, neuronal density, CASP3, RIPK3, A1R, Hsp90, and BDNF. These data indicate that the neuroprotection by RV involves A1R and inhibits CASP3-dependent apoptosis and RIPK3-dependent necroptosis in the perihematoma region; this is likely to be mediated by crosstalk between A1R and the BDNF/TrkB pathway., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Association of Neuropathologists, Inc. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

ABSTRACT Introduction. Penile erection is a complex neurovascular physiological event controlled by multiple factors and signaling pathways. A considerable amount of evidence indicates that adenosine plays a significant role in cavernosal smooth muscle relaxation. However, the specific role of adenosine and its receptors in erectile physiology and pathology is not fully understood. Aim. To determine the role of the adenosine A1 receptor (ADORA1) in penile erection. Method. Adenosine A1 receptor deficient ( Adora1-/-) mice and aged-matched wild-type (WT) mice were utilized. We evaluated the in vivo erectile function by measuring the intracavernosal pressure (ICP) in response to cavernous nerve stimulation (CNS). Enzyme-linked immunosorbent assay was used to measure the norepinephrine (NE) plasma concentration in the corpus cavernosum and systemic circulation. We also evaluated the myosin light chain phosphorylation ( p-MLC) in penile tissue pre- and post-CNS. Main Outcome Measurement. The main outcome measurement of this research was the evaluation of in vivo erectile response to CNS by measuring the ICP in Adora1-/- mice and WT mice and to identify the localization and specific neuron types of ADORA1 expression by dual immunostaining and immunofluorescence co-localization. Result. In vivo, both the ratio of CNS-induced Maximum ICP to mean arterial pressure and CNS-induced slope in Adora1-/- mice were significantly lower than WT mice. At the cellular level in penile tissue, we determined that ADORA1 was highly abundant in neuronal cells. During penile erection, Adora1-/- mice exhibited a higher level of NE plasma concentration in the penis than WT mice. And WT mice had a significantly greater reduction in p-MLC compared to Adora1-/- mice. Conclusion. Our results show that ADORA1 is enriched on neuron cells where it functions to control NE release. Activation of this receptor during penile erection results in reduced NE release and reduced cavernosal smooth muscle contraction, therefore facilitating penile erection. Ning C, Qi L, Wen J, Zhang Y, Zhang W, Wang W, Blackburn M, Kellems R, and Xia Y. Excessive penile norepinephrine level underlies impaired erectile function in adenosine A1 receptor deficient mice. J Sex Med **;**:**-**. [ABSTRACT FROM AUTHOR]

Abstract: While neonatal caffeine treatment is commonly used to alleviate apnea of prematurity in neonates and to improve neurological outcomes, its effects on adenosine A1 and A2A receptors (A1-R and A2A-R) are poorly known. We hypothesized that the central pharmacological action of caffeine is mediated by modification of the postnatal development of the adenosinergic system during a critical period. On postnatal days 2–6 (P2–P6) two groups of newborn rats were orally administered water plus glucose and/or caffeine at therapeutic doses to mimic the clinical use of caffeine in human neonates. Cardio-respiratory parameters were measured and the presence of A1-R and A2A-R and c-Fos protein was identified immunohistochemically in animals sacrificed from P2 to P11. % Haemoglobin saturation, and hearth and breath rates were significantly increased in caffeine-treated group (P5–P6). Significant differences were identified in the relative gene expression of A1-R and A2A-R, with an increase of A1-R labeling in the anterior hypothalamic area, ventromedial hypothalamic nucleus, parabrachial complex and ventrolateral medulla of the caffeine-treated group at P6. A moderate increase in A2A-R labeling was observed in ponto-medullary nuclei and other hypothalamic areas. An increase in c-Fos-positive labeled cells was found in the caffeine-treated group at P5–P6 within the same areas described above, with the most clear-cut increase seen in the arcuate nucleus. Indeed, increased A1-R and A2A-R gene expression was observed in both the brainstem and hypothalamus at P5. Up-regulation of adenosinergic maturation in central cardio-respiratory areas in caffeine-treated neonatal rats could explain the pharmacological effects of caffeine observed in premature infants. [Copyright &y& Elsevier]

The aim of this study was to investigate the effect of age on the distribution of adenosine A1 receptors (A1Rs) and adenosine A2A receptors (A2ARs) in the striatum of healthy subjects using PET imaging with 8-dicyclopropylmethyl-1-[11C]methyl-3-propylxanthine ([11C]MPDX) and [7-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([11C]TMSX), respectively. We recruited 8 young (22.0 ± 1.7 years) and 10 elderly (65.4 ± 7.6 years) volunteers to undergo [11C]MPDX PET scanning, and 11 young (22.7 ± 2.7 years) and six elderly (60.7 ± 8.5 years) volunteers to undergo [11C]TMSX PET scanning. A dynamic series of decay-corrected PET scans was performed for 60 min following injection of [11C]MPDX or [11C]TMSX. We calculated the binding potential ( BPND) of [11C]MPDX and distribution volume ratio ( DVR) of [11C]TMSX in the striatum. The BPND of [11C]MPDX was significantly lower in elderly than in young subjects, both in the putamen and head of the caudate nucleus. The BPND was negatively correlated with age in both the putamen and the head of the caudate nucleus. However, no difference was found between the DVR of [11C]TMSX in the striata of young and elderly subjects, nor was there a correlation between age and the DVR of [11C]TMSX. The effect of age on the distribution of A1Rs in the human striatum described herein is similar to previous reports of age-related decreases in dopamine D1 and D2 receptors. Unlike A1Rs, however, this study suggests that the distribution of A2ARs does not change with age. Synapse 66:832-839, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Temporal adaptation of tubuloglomerular feedback (TGF) permits readjustment of the relationship of nephron filtration rate [single nephron glomerular filtration rate (SNGFR)] and early distal tubular flow rate (VED) while maintaining TGF responsiveness. We used closed-loop assessment of TGF in hydropenia and after acute saline volume expansion (SE; 10% body wt over 1 h) to determine whether 1) temporal adaptation of TGF occurs, 2) adenosine A1 receptors (A1R) mediate TGF responsiveness, and 3) inhibition of TGF affects SNGFR, VED, or urinary excretion under these conditions. SNGFR was evaluated in Fromter-Wistar rats by micropuncture in 1) early distal tubules (ambient flow at macula densa), 2) recollected from early distal tubules while 12 nl/min isotonic fluid was added to late proximal tubule (increased flow to macula densa), and 3) from proximal tubules of same nephrons (zero flow to macula densa). SE increased both ambient SNGFR and VED compared with hydropenia, whereas TGF responsiveness (proximal-distal difference in SNGFR, distal SNGFR response to adding fluid to proximal tubule) was maintained, demonstrating TGF adaptation. A1R blockade completely inhibited TGF responsiveness during SE and made VED more susceptible to perturbation in proximal tubular flow, but did not alter ambient SNGFR or VED. Greater urinary excretion of fluid and Na+ with A1R blockade may reflect additional effects on the distal nephron in hydropenia and SE. In conclusion, A1R-independent mechanisms adjust SNGFR and VED to higher values after SE, which facilitates fluid and Na+ excretion. Concurrently, TGF adapts and stabilizes early distal delivery at the new setpoint in an A1R-dependent mechanism. [ABSTRACT FROM AUTHOR]

Abstract: The adenosine A1 receptor is a member of the large membrane protein family that signals through G proteins, the G protein-coupled receptors (GPCRs). GPCRs consist of seven transmembrane domains connected by three intracellular and three extracellular loops. Their N-terminus is extracellular, the C-terminal tail is in the cytoplasm. The transmembrane domains in receptor subfamilies that bind the same endogenous ligand, such as dopamine or adenosine, tend to be highly similar. In contrast, the loop regions can vary greatly, both in sequence and in length, and the role these loops have in the activation mechanism of the receptors remains unclear. Here, we investigated the activating role of the second and third extracellular loop of the human adenosine A1 receptor. By means of an (Ala)3 mutagenic scan in which consecutive sets of three amino acids were mutated into alanine residues in EL2 and a classical alanine scan in EL3, we revealed a strong regulatory role for the second extracellular loop (EL2) of the human adenosine A1 receptor. Besides many residues in the second and the third extracellular loops important for adenosine A1 receptor activation, we also identified two residues in EL2, a tryptophan and a glutamate, that affect the influence of the allosteric modulator PD81,723. These results, combined with a comparison of the different receptor loop regions, provide insight in the activation mechanism of this typical class A GPCR and further emphasize the unique pharmacological profile the loops can provide to individual receptors, even within subfamilies of GPCRs. [Copyright &y& Elsevier]

Abstract: The present study aimed to clarify the protective effect of adenosine receptors against the excitotoxicity of cochlear afferent dendrites. The effects of 2-chloro-N6-cyclopentyladenosine (CCPA), an A1 adenosine receptor agonist, and 5′-N-cyclopropyl-carboxamidoadenosine (CPCA), an A2A adenosine receptor agonist, on cochlear excitotoxicity induced by kainic acid (KA) were examined using guinea pigs. KA was applied to the round window membrane at a concentration of 10mM for 30min. CCPA or CPCA was given at the onset of KA application. KA morphologically induced the swelling of cochlear afferent dendrites and significantly elevated the threshold of the compound action potential (CAP) of the cochlea. CCPA inhibited the KA-induced CAP threshold shift and swelling of the cochlear afferent dendrites. However, CPCA did not affect cochlear excitotoxicity induced by KA. The results suggest that adenosine A1 receptor activation could prevent the excitotoxicity of cochlear afferent dendrites. [Copyright &y& Elsevier]

Abstract: Activation of adenosine A1 receptors was reported to promote fatty acid synthesis in AML-12 cells, by increasing the expression of SREBP-1c (sterol regulatory binding protein 1c) and FAS (fatty acid synthase). Since these findings have important therapeutic implications for the discovery of adenosine A1 receptor agonists, further studies were undertaken to determine the expression and functional relevance of adenosine A1 receptor in the liver. To that end, we used two classes of distinct adenosine A1 receptor agonists: CPA (N6-cyclopentyl-adenosine), a full agonist and GS-9667 (2-{6-[((1R,2R)-2-hydroxycyclopentyl)-amino]purin-9-yl}(4S,5S,2R,3R)-5-[(2-fluorophenylthio)methyl]-oxolane-3,4-diol), a partial agonist. Treatment of AML-12 cells, HepG2 cells and primary human hepatocytes with either CPA or GS-9667 did not increase the gene expression of SREBP-1c or FAS. Furthermore, in AML-12 and HepG2 cells, CPA did not antagonize forskolin-stimulated cAMP production, a characteristic of adenosine A1 receptor activation, indicating that these cells lack adenosine A1 receptor function. Consistent with this finding, adenosine A1 receptor gene expression was found to be very low and adenosine A1 receptor protein levels were hardly detectable by radioligand binding assays in hepatic cell lines such as AML-12 and HepG2 as well as in both mouse and human liver tissues. Finally, acute treatment with adenosine A1 receptor agonist GS-9667 had no significant effect on gene expression of both SREBP-1c and FAS in livers of Sprague Dawley rats. Taken together, our data suggest that the expression of adenosine A1 receptor is too low to play a major role in the regulation of lipogenic gene expression in hepatocytes. [Copyright &y& Elsevier]

Adenosine A1 receptor-deficient mice develop a phenotype of insulin resistance and grow fat. Participating pathophysiological pathways are not understood in detail yet, as discussed in our recent manuscript. This commentary further explores possible pathophysiological mechanisms with emphasis on the roles of the adipokines resistin, retinolbinding protein 4, adiponectin and the function of the gastric hormone ghrelin in adenosine mediated central regulation of energy balance. The postulate of an important function of ghrelin/A1AR axis provides a good hypothetical basis for further investigations to clarify the mechanism of A1AR-dependent metabolic homeostasis. [ABSTRACT FROM AUTHOR]

Adenosine, a purine nucleoside, is present in all cells in tightly regulated concentrations. It has many different physiological effects in the whole body and in the heart. Adenosine activates four G protein-coupled receptors A1, A2a, A2b, and A3. Activation of myocardial A1 receptors has been shown to inhibit a variety of myocardial pathologies associated with ischemia and reperfusion injury, including stunning, arrhythmogenesis, coronary and ventricular dysfunction, acute myocardial infarction, apoptosis, and chronic heart failure, implying several options for new cardiovascular therapies for diseases, like angina pectoris, control of cardiac rhythm, ischemic injury during an acute coronary syndrome, or heart failure. However, the main issue of using full A1 receptor agonists in such indications is the broad physiologic spectrum of cardiac and extracardiac effects. Desired A1 receptor-mediated protective and regenerative cardiovascular effects might be counter-regulated by unintended side effects when considering full A1 receptor agonists. These effects can be overcome by partial A1 agonists. Partial A1 agonists can be used to trigger only some of the physiological responses of receptor activation depending on endogenous adenosine levels and on receptor reserve in different tissues. CV-Therapeutics reported the identification of a partial A1 receptor agonist CVT-3619, and recently, another partial A1 receptor agonist VCP28 was published. Both compounds are adenosine derivatives. Adenosine-like A1 receptor agonists often have the drawback of a short half-life and low bioavailability, making them not suitable for chronic oral therapy. We identified the first non-adenosine-like partial A1 receptor agonist(s) with pharmacokinetics optimal for oral once daily treatment and characterized the qualities of the partial character of the A1 receptor agonist(s) in preclinical and clinical studies. [ABSTRACT FROM AUTHOR]

Abstract: Growing evidence indicates that G protein-coupled receptors can form homo- and hetero-oligomers to diversify signal transduction. However, the molecular mechanisms and physiological significance of G protein-coupled receptor-oligomers are not fully understood. Both ADOR1 (adenosine A1 receptor) and TBXA2R (thromboxane A2 receptor α; TPα receptor), members of the G protein-coupled receptor family, act on astrocytes and renal mesangial cells, suggesting certain functional correlations. In this study, we explored the possibility that adenosine A1 and TPα receptors form hetero-oligomers with novel pharmacological profiles. We showed that these receptors hetero-oligomerize by conducting coimmunoprecipitation and bioluminescence resonance energy transfer (BRET2) assays in adenosine A1 receptor and TPα receptor-cotransfected HEK293T cells. Furthermore, coexpression of the receptors affected signal transduction including the accumulation of cyclic AMP and phosphorylation of extracellular signal-regulated kinase-1 and -2 was significantly increased by high and low concentrations of adenosine A1 receptor agonist and TPα agonists, respectively. Our study provides evidence of hetero-oligomerization between adenosine A1 and TPα receptors for the first time, and suggests that this oligomerization affects signal transduction responding to different concentrations of receptor agonists. [Copyright &y& Elsevier]

Summary: Prostaglandin (PG) D2 is the most potent endogenous sleep-promoting substance. PGD2 is produced by lipocalin-type PGD synthase localized in the leptomeninges, choroid plexus, and oligodendrocytes in the brain, and is secreted into the cerebrospinal fluid as a sleep hormone. PGD2 stimulates DP1 receptors localized in the leptomeninges under the basal forebrain and the hypothalamus. As a consequence, adenosine is released as a paracrine sleep-promoting molecule to activate adenosine A2A receptor-expressing sleep-promoting neurons and to inhibit adenosine A1 receptor-possessing arousal neurons. PGD2 activates a center of non-rapid eye movement (NREM) sleep regulation in the ventrolateral preoptic area, probably mediated by adenosine signaling, which activation inhibits the histaminergic arousal center in the tuberomammillary nucleus via descending GABAergic and galaninergic projections. The administration of a lipocalin-type PGD synthase inhibitor (SeCl4), DP1 antagonist (ONO-4127Na) or adenosine A2A receptor antagonist (caffeine) suppresses both NREM and rapid eye movement (REM) sleep, indicating that the PGD2-adenosine system is crucial for the maintenance of physiological sleep. [Copyright &y& Elsevier]

Abstract: Adenosine plays an important neuroprotective role in brain, usually mediated by the activation of adenosine A1 receptors. Prolonged activation of a G-protein-coupled receptor generally leads to the partial loss of the responsiveness of receptor-mediated transduction pathways (desensitization). Rat immature cortical neurons were treated with 100nM (−)N6-phenylisopropyladenosine (R-PIA), a selective A1 receptor agonist, and the effect on adenosine A1 receptor/adenylyl cyclase pathway was studied. Incubation with R-PIA for 6, 12, 24 and 48h elicited a time-dependent decrease in adenosine A1 receptors in plasma membranes (92, 58, 43 and 26% of control, respectively), which was associated with variations in microsomal fraction (21, 56, 124 and 233% of control, respectively), suggesting the internalization and down-regulation of adenosine A1 receptors. Moreover, real-time PCR assays showed a significant increase in mRNA levels coding adenosine A1 receptor after the longest treatment period (48h). In addition, αGi1–2 protein levels detected in microsomes and mRNA levels coding αGi1 protein were increased after 48h of treatment with R-PIA, suggesting the synthesis of new αGi1 proteins. Finally, adenylyl cyclase inhibition elicited by 2-Chloro-N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist, was significantly reduced after 12, 24 and 48h of treatment (37, 24 and 23%, respectively) as compared to controls (54%), suggesting the desensitization of adenosine A1 receptor/adenylyl cyclase pathway. These results suggest that adenosine A1 receptors desensitize slowly after prolonged receptor activation in immature cortical neurons, showing mechanisms of desensitization similar to those described not only in fetal but also in adult rat brain. [Copyright &y& Elsevier]

Background: Both nitric oxide (NO) and adenosine A1 receptor activation mediate microvascular vasodilation during intestinal glucose absorption. Our overall hypothesis is that adenosine triphosphate (ATP) utilization during glucose absorption would increase adenosine metabolite release, which acts on adenosine A1 receptors to alter endothelial production of NO and/or activate ATP-dependent potassium channels (K+ ATP) to dilate intestinal microvessels. Methods: Intravital videomicroscopy of the rat jejunum was used to record the vascular responses of inflow (termed 1A) arterioles, proximal (p3A), and distal (d3A) premucosal arterioles during exposure to isotonic glucose or mannitol solutions alone or in the presence of the selective nitric oxide synthase (NOS) inhibitor (L-NMMA), an adenosine A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine (DPCPX)), or a K+ ATP channel inhibitor (glibenclamide). Results: As expected, glucose exposure caused rapid dilation of both p3A and d3A arterioles, while mannitol exposure had no effect on microvascular diameters. Adenosine A1 receptor blockade completely prevented glucose-induced dilation of the premucosal arterioles. NOS inhibition significantly blunted the glucose-induced vasodilation of the premucosal arterioles, but had little effect in the mannitol group. Simultaneous application of both the NOS inhibitor and the adenosine A1 receptor antagonist gave the same reduction in glucose-induced dilation of the premucosal arterioles as the adenosine A1 receptor antagonist alone. Blockade of K+ ATP channels with glibenclamide did not attenuate glucose-induced vasodilation of the premucosal arterioles. Conclusion: These data suggest that glucose-induced vasodilation of premucosal jejunal arterioles is mediated through adenosine A1 receptors, and NO at least partially mediates the adenosine A1 receptor-induced vasodilation. In addition, K+ ATP channels are not involved in premucosal arteriolar vasodilation during intestinal glucose exposure. [Copyright &y& Elsevier]

Abstract: Adenosine-regulated glutamate signaling in astrocytes is implicated in many neurological and neuropsychiatric disorders. In this study, we examined whether adenosine A1 receptor regulates EAAT2 expression in astrocytes using pharmacological agents and siRNAs. We found that adenosine A1 receptor-specific antagonist DPCPX or PSB36 decreased EAAT2 expression in a dose-dependent manner. Consistently, knockdown of A1 receptor in astrocytes decreased EAAT2 mRNA expression while overexpression of A1 receptor upregulated EAAT2 expression and function. Since A1 receptor activation is mainly coupled to inhibitory G-proteins and inhibits the activity of adenylate cyclase, we investigated the effect of forskolin, which activates adenylate cyclase activity, on EAAT2 mRNA levels. Interestingly, we found that forskolin reduced EAAT2 expression in dose- and time-dependent manners. In contrast, adenylate cyclase inhibitor SQ22536 increased EAAT2 expression in dose- and time-dependent manners. In addition, forskolin blocked ethanol-induced EAAT2 upregulation. Taken together, these results suggest that A1 receptor-mediated signaling regulates EAAT2 expression in astrocytes. [Copyright &y& Elsevier]

Adenosine A2 receptors have been suggested to modulate tubuloglomerular feedback (TGF) responses by counteracting adenosine A1 receptor-mediated vasoconstriction, but the mechanisms are unclear. We tested the hypothesis that A2A receptor activation blunts TGF by release of nitric oxide in the juxtaglomerular apparatus (JGA). Maximal TGF responses were measured in male Sprague-Dawley rats as changes in proximal stop-flow pressure (ΔPSF) in response to increased perfusion of the loop of Henle (0 to 40 nl/min) with artificial tubular fluid (ATF). The maximal TGF response was studied after 5 min intratubular perfusion (10 nl/min) with ATF or ATF + A2A receptor agonist (CGS-21680; 10-7 mol/l). The interaction with nitric oxide synthase (NOS) isoforms was tested by perfusion with a nonselective NOS inhibitor [Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME); 10-3 mol/l] or a selective neuronal NOS (nNOS) inhibitor [Nω-propyl-l-arginine (l-NPA); 10-6 mol/l] alone, and with the A2A agonist. Blood pressure, urine flow, and PSF at 0 nl/min were similar among the groups. The maximal TGF response (ΔPSF) with ATF alone (12.3 ± 0.6 mmHg) was attenuated by selective A2A stimulation (9.5 ± 0.4 mmHg). l-NAME enhanced maximal TGF responses (18.9 ± 0.4 mmHg) significantly more than l-NPA (15.2 ± 0.7 mmHg). Stimulation of A2A receptors did not influence maximal TGF response during nonselective NOS inhibition (19.0 ± 0.4) but attenuated responses during nNOS inhibition (10.3 ± 0.4 mmHg). In conclusion, adenosine A2A receptor activation attenuated TGF responses by stimulation of endothelial NOS (eNOS), presumably in the afferent arteriole. Moreover, NO derived from both eNOS and nNOS in the JGA may blunt TGF responses. [ABSTRACT FROM AUTHOR]

Prostaglandin D (PGD) is the most potent endogenous sleep-promoting substance thus far reported. We have extensively studied its action mechanism at the molecular level. PGD is produced by lipocalin-type PGD synthase, which is dominantly localized in the leptomeninges, choroid plexus, and oligodendrocytes in the brain; and it is secreted into the cerebrospinal fluid and stimulates DP receptors localized in the arachnoid membrane of the ventral surface from the basal forebrain to the hypothalamus to increase the extracellular concentration of adenosine as a paracrine sleep-promoting molecule. Adenosine diffuses into the brain parenchyma, suppresses cholinergic arousal neurons in the basal forebrain via adenosine A receptors, activates sleep-active neurons in the ventrolateral preoptic area via adenosine A receptors, and concomitantly suppresses the histaminergic arousal center in the tuberomammillary nucleus through GABAergic and galaninergic inhibitory projections. Administration of an inhibitor of lipocalin-type PGD synthase (SeCl), an antagonist of DP receptors (ONO-4127Na) or an antagonist of adenosine A receptors (caffeine) results in sleep inhibition in rats and mice, indicating that the PGD-adenosine system is crucial for the maintenance of physiological sleep. [ABSTRACT FROM AUTHOR]

Abstract: Co-administration of caffeine profoundly enhances the acute toxicity of 3,4 methylenedioxymethamphetamine (MDMA) in rats. The aim of this study was to determine the ability of caffeine to impact upon MDMA-induced dopamine release in superfused brain tissue slices as a contributing factor to this drug interaction. MDMA (100 and 300μM) induced a dose-dependent increase in dopamine release in striatal and hypothalamic tissue slices preloaded with [3H] dopamine (1μM). Caffeine (100μM) also induced dopamine release in the striatum and hypothalamus, albeit to a much lesser extent than MDMA. When striatal tissue slices were superfused with MDMA (30μM) in combination with caffeine (30μM), caffeine enhanced MDMA-induced dopamine release, provoking a greater response than that obtained following either caffeine or MDMA applications alone. The synergistic effects in the striatum were not observed in hypothalamic slices. As adenosine A1 receptors are, one of the main pharmacological targets of caffeine, which are known to play an important role in the regulation of dopamine release, their role in the modulation of MDMA-induced dopamine release was investigated. 1μM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A1 antagonist, like caffeine, enhanced MDMA-induced dopamine release from striatal slices while 1μM 2,chloro-N(6)-cyclopentyladenosine (CCPA), a selective adenosine A1 receptor agonist, attenuated this. Treatment with either SCH 58261, a selective A2A receptor antagonist, or rolipram, a selective PDE-4 inhibitor, failed to reproduce a caffeine-like effect on MDMA-induced dopamine release. These results suggest that caffeine regulates MDMA-induced dopamine release in striatal tissue slices, via inhibition of adenosine A1 receptors. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated the effects of N6-cyclopentyladenosine (CPA), a potent and selective adenosine A1 receptor (A1R) agonist in normal and nerve-injured rats and mechanisms of its action by behavioral tests and electrophysiological technique. The results showed: (1) In normal rats, intraperitoneal administration of CPA (1mg/kg) increased paw withdrawal latencies, in a way blocked by a selective A1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 3mg/kg, i.p.), but had no influence on the threshold of mechanical stimulation. (2) In rats with neuropathic pain induced by spinal nerve ligation (SNL), CPA reduced thermal hyperalgesia and mechanical allodynia, which could last 6h and 10h, respectively (n =6/group, P <0.05). Both of the effects could be blocked by pretreatment of DPCPX intraperitoneally. (3) The baseline of C-fiber but not A-fiber evoked field potentials was depressed by spinal application of CPA (0.01mM), and this effect was prevented by application of DPCPX (0.02mM) 30min before CPA. (4) Spinal application of CPA depressed long-term potentiation (LTP) of A- and C-fiber evoked field potentials, and both the depression could be blocked by pretreatment of DPCPX 30min before CPA. These results suggested that the activation of A1R has different influences on normal and neuropathic rats probably due to the absence and presence of central sensitization in spinal dorsal horn. [ABSTRACT FROM AUTHOR]

Abstract: Through activation of the A1 adenosine receptors (A1Rs) at both the central and peripheral level, adenosine produces antinociception in a wide range of tests. However, the mechanisms involved in the peripheral effect are still not fully understood. Therefore, the mechanisms by which peripheral activation of A1Rs reduces inflammatory hypernociception (a decrease in the nociceptive threshold) were addressed in the present study. Immunofluorescence of rat dorsal root ganglion revealed significant expression of A1Rs in primary sensory neurons associated with nociceptive pathways. Functionally, peripheral activation of A1Rs reduced inflammatory hypernociception because intraplantar (i.pl.) administration of an A1R antagonist (DPCPX) enhanced carrageenan-induced hypernociception. On the other hand, local (paw) administration of CPA (a selective A1R agonist) reversed mechanical hypernociception induced by carrageenan or by the directly acting hypernociceptive mediator prostaglandin E2 (PGE2). Down-regulation of A1Rs expression in primary nociceptive neurons by intrathecal treatment with antisense oligodeoxinucleotides significantly reduced peripheral antinociceptive action of CPA. Direct blockade of PGE2 inflammatory hypernociception by the activation of A1Rs depends on the nitric oxide/cGMP/Protein Kinase G/KATP signaling pathway because the peripheral antinociceptive effect of CPA was prevented by pretreatment with inhibitors of neuronal nitric oxide synthase (N-propyl-l-arginine), guanylyl cyclase (ODQ), and Protein Kinase G (KT5823) as well as with a KATP blocker (glibenclamide). However, this effect of CPA was not reduced by naloxone, excluding the participation of endogenous opioids. These results suggest that the peripheral activation of A1R plays a role in the regulation of inflammatory hypernociception by a mechanism that involves the NO/cGMP/PKG/KATP intracellular signaling pathway. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 1667–1677. Neuroprotection is one of the prominent functions of the interleukin (IL)-6-type cytokine family, for which the underlying mechanism(s) are not fully understood. We have previously shown that neuroprotection and neuromodulation mediated by IL-6 require neuronal adenosine A1 receptor (A1R) function. We now have investigated whether two other IL-6-type cytokines [oncostatin M (OSM) and leukemia inhibitory factor (LIF)] use a similar mechanism. It is presented here that OSM but not LIF, enhanced the expression of A1Rs (both mRNA and protein levels) in cultured neurons. Whereas the neuroprotective effect of LIF was unchanged in A1R deficient neurons, OSM failed to protect neurons in the absence of A1R. In addition, OSM pre-treatment for 4 h potentiated the A1R-mediated inhibition of electrically evoked excitatory post-synaptic currents recorded from hippocampal slices either under normal or hypoxic conditions. No such effect was observed after LIF pre-treatment. Our findings thus strongly suggest that, despite known structural and functional similarities, OSM and LIF use different mechanisms to achieve neuroprotection and neuromodulation. [ABSTRACT FROM AUTHOR]

AbstractAim:To define the effect of adenosine A1 receptor (A1R) on delta opioid receptor (DOR)-mediated signal transduction.Methods:CHO cells stably expressing HA-tagged A1R and DOR-CFP fusion protein were used. The localization of receptors was observed using confocal microscope. DOR-mediated inhibition of adenylyl cyclase was measured using cyclic AMP assay. Western blots were employed to detect the phosphorylation of Akt and the DOR. The effect of A1R agonist N6-cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay.Results:CHA 1 μmol/L time-dependently attenuated DOR agonist [D-Pen2,5]enkephalin (DPDPE)-induced inhibition of intracellular cAMP accumulation with a t1/2=2.56 (2.09–3.31) h. Pretreatment with 1 μmol/L CHA for 24 h caused a right shift of the dose-response curve of DPDPE-mediated inhibition of cAMP accumulation, with a significant increase in EC50 but no change in Emax. Pretreatment with 1 μmol/L CHA for 1 h also induced a significant attenuation of DPDPE-stimulated phosphorylation of Akt. Moreover, CHA time-dependently phosphorylated DOR (Ser363), and this effect was inhibited by A1R antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) but not by DOR antagonist naloxone. However, CHA failed to produce the down-regulation of DOR, as neither receptor affinity (Kd) nor receptor density (Bmax) of DOR showed significant change after chronic CHA exposure.Conclusion:Activation of A1R by its agonist caused heterologous desensitization of DOR-mediated inhibition of intracellular cAMP accumulation and phosphorylation of Akt. Activation of A1R by its agonist also induced heterologous phosphorylation but not down-regulation of DOR. [ABSTRACT FROM AUTHOR]

Abstract: A1 adenosine receptors are highly expressed in the central nervous system. Mitochondrial function is a major player in adenosine receptors-mediated effects. Here, by using mice with genetic deletion of the A1 receptor, we addressed the existence of a relationship between mitochondria functions and adenosine A1 receptor. Mitochondrial functions and effects of MPP+ in primary mixed cultures are influenced by the presence of the A1 receptor, demonstrating, for the first time, the mitochondrial localization of the adenosine A1 receptor and suggesting a role for this receptor as a mitochondrial vulnerability factor. [Copyright &y& Elsevier]

Caffeine is the most consumed psychoactive drug in the world. It is a non-selective adenosine receptor antagonist that in the brain targets mainly adenosine A{1} and A{2A} receptors. The same as classical psychostimulants, caffeine produces motor-activating, reinforcing and arousing effects. This depends on the ability of caffeine to counteract multiple effects of adenosine in the central ascending neurotransmitter systems. Motor and reinforcing effects depend on the ability of caffeine to release pre- and postsynaptic brakes that adenosine imposes on the ascending dopaminergic system. By targeting A{1}–A{2A} receptor heteromers in striatal glutamatergic terminals and A{1} receptors in striatal dopaminergic terminals (presynaptic brake), caffeine induces glutamate-dependent and glutamate-independent release of dopamine. These presynaptic effects of caffeine are potentiated by the release of the postsynaptic brake imposed by antagonistic interactions in the striatal A{2A}–D{2} and A{1}–D{1} receptor heteromers. Arousing effects of caffeine depend on the blockade of multiple inhibitory mechanisms that adenosine, as an endogenous sleep-promoting substance, exerts on the multiply interconnected ascending arousal systems. Those mechanisms include a direct A{1}-receptor mediated modulation of the corticopetal basal forebrain system and an indirect A{2A}-receptor mediated modulation of the hypothalamic histaminergic and orexinergic systems. [ABSTRACT FROM AUTHOR]

Caffeine is well known for its complex pharmacological actions, in part reflecting the multiple molecular targets of caffeine. The adenosine receptors are the primary extracellular targets of caffeine. Since caffeine has similar affinity for several adenosine receptors, it has been difficult to determine which receptor subtypes mediate caffeine's effects using pharmacological tools. The development of genetic mutant mice deficient in adenosine receptors and other signaling molecules has allowed targeted inquiry into the molecular targets by which caffeine elicits its biological effects on behavior and gene expression. This review summarizes recent work using genetic knockout models to elucidate the mechanisms of caffeine action in the brain. This review focuses on insights into caffeine action from genetic knockout models on: (1) the molecular basis for caffeine's effects on psychomotor activity; (2) the involvement of adenosine receptors in caffeine-mediated arousal and cognitive effects; and (3) a novel approach using knockout animals coupled with microarray profiling to validate multiple molecular targets of caffeine in striatal gene expression. [ABSTRACT FROM AUTHOR]

Abstract: The perifornical–lateral hypothalamic area (PF-LHA) plays a central role in the regulation of behavioral arousal. The PF-LHA contains several neuronal types including wake-active hypocretin (HCRT) neurons that have been implicated in the promotion and/or maintenance of behavioral arousal. Adenosine is an endogenous sleep factor and recent evidence suggests that activation and blockade of adenosine A1 receptors within the PF-LHA promote and suppress sleep, respectively. Although, an in vitro study indicates that adenosine inhibits HCRT neurons via A1 receptor, the in vivo effects of A1 receptor mediated adenosinergic transmission on PF-LHA neurons including HCRT neurons are not known. First, we determined the effects of N6-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, on the sleep–wake discharge activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe used for its delivery. Second, we determined the effects of CPA and that of an A1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX) into the PF-LHA on cFos-protein immunoreactivity (Fos-IR) in HCRT and non-HCRT neurons around the microdialysis probe used for their delivery. The effect of CPA on Fos-IR was studied in rats that were kept awake during lights-off phase, whereas the effect of CPDX was examined in undisturbed rats during lights-on phase. CPA significantly suppressed the sleep–wake discharge activity of PF-LHA neurons. Doses of CPA (50 μM) and CPDX (50 μM) that suppressed and induced arousal, respectively, in our earlier study [Alam MN, Kumar S, Rai S, Methippara M, Szymusiak R, McGinty D (2009) Brain Res 1304:96–104], significantly suppressed and increased Fos-IR in HCRT and non-HCRT neurons. These findings suggest that wake-promoting PF-LHA system is subject to increased endogenous adenosinergic inhibition and that adenosine acting via A1 receptors, in part, inhibits HCRT neurons to promote sleep. [Copyright &y& Elsevier]

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is a critical response to perinatal hypoxia. Recent data show that adenosine appears to inhibit baseline levels of fetal cortisol and to restrict the increase in ACTH and cortisol during moderate hypoxia. Because adenosine increases substantially during profound asphyxia, it is possible, but untested, that counterintuitively it might restrict the HPA response to more severe insults. It is unclear which receptors mediate the effects of adenosine on the HPA axis; however, adenosine A1 receptor activation is important for adaptation to hypoxia. We therefore investigated whether adenosine i receptor blockade modulates ACTH and cortisol levels in fetal sheep at 118 to 126 days gestation, randomly allocated to receive an intravenous infusion of either vehicle (vehicle-occlusion, n = 7) or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 receptor antagonist, DPCPX-occlusion, n = 7) infused 60 mm before and during 10 min of umbilical cord occlusion, or infusion of DPCPX for 70 mm without occlusion (DPCPX-sham, n = 6). Experiments were terminated after 72 h. Fetal ACTH levels increased significantly (P < 0.01) during occlusion, but not sham occlusion, and returned to baseline values by 60 min after occlusion. In the vehicle-occlusion group, fetal cortisol and cortisone plasma levels increased significantly (P < 0.05) 60 mm after the occlusion and returned to baseline values by 24 h. In contrast, there was a marked increase in both fetal cortisol and cortisone during DPCPX infusion before occlusion to a level greater even than the maximum rise seen after occlusion alone. This increase was sustained after occlusion, with increased cortisol levels compared with occlusion alone up to 72 h. In conclusion, fetal cortisol concentrations are suppressed by adenosine A1 receptor activity, largely though a direct adrenal mechanism. This suppression can be partially overcome by supraphysiological stimuli such as asphyxia. [ABSTRACT FROM AUTHOR]

1. Adenosine A1 receptors (A1R) play an important role in cardioprotection against hypoxic damage and the opening of mitochondrial permeability transition pores (MPTP) is central to the regulation of cell apoptosis and necrosis. However, it is still unclear whether A1R open MPTP in hypoxic cardiomyocytes. 2. The present study used primary cardiomyocyte cultures from neonatal rats to investigate the mechanisms of A1R activation and the effects of A1R on MPTP opening under hypoxic conditions. 3. Hypoxia increased both MPTP opening and the production of reactive oxygen species (ROS), while decreasing cell viability and mitochondrial membrane potential (Δψ). The A1R agonist 2-chloro- N6-cyclopentyladenosine (CCPA; 500 nmol/L) blocked the increase in MPTP opening and ROS production and maintained cell viability and Δψ under hypoxic conditions. 4. The protective effects of CCPA were eliminated by both the protein kinase C (PKC) inhibitor chelerythine (2 μmol/L) and the mitochondrial ATP-sensitive K+ channel (mitoKATP) inhibitor 5-hydroxydecanoate (500 μmol/L). Moreover, CCPA significantly increased the PKC content in both total protein and membrane protein of cardiomyocytes. 5-Hydroxydecanoate did not prevent these CCPA-induced increases in PKC. 5. These results demonstrate that CCPA reduces MPTP opening in hypoxic cardiomyocytes, possibly by activating PKC, stabilizing Δψ and reducing ROS production following the opening of mitoKATP. Consequently, fewer MPTP open. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) is the endogenous ligand of a G-protein-coupled receptor named as NPSR. Behavioral effects have been recently attributed to NPS, i.e. hyperlocomotion, anxiolysis, and wakefulness. However, little is known about the mechanisms by which NPS evokes such biological actions. The present study aimed to investigate the role played by the adenosine A2A and A1 receptors in hyperlocomotion induced by NPS. Spontaneous locomotion was assessed in an activity cage for 30 min in mice acutely treated with caffeine (a nonselective adenosine receptor antagonist), ZM241385 (a selective A2A receptor antagonist), or CPT (a selective A1 receptor antagonist) before NPS challenge (0.1 nmol, i.c.v.), which induce hyperlocomotion in mice. The pretreatment with caffeine (3 mg/kg, i.p.), in an inactive dose per se, prevented the increase in locomotion evoked by NPS. The co-administration of NPS (0.1 nmol, i.c.v.) and ZM241385 (0.1 pmol, i.c.v.) counteracted hyperlocomotion evoked by NPS. The co-administration of NPS and CPT (0.1 pmol, i.c.v.) slightly facilitated the increase in locomotion evoked by NPS alone. In summary, the pharmacological blockade of A2A receptors significantly attenuated the stimulatory effects of NPS. By contrast, the antagonism of A1 receptors facilitated NPS-induced hyperlocomotion in mice, but we cannot rule out a merely additive effect of two stimulatory systems in the brain. Altogether, this is the first evidence of a putative role played by A2A and A1 receptors in modulating hyperlocomotion induced by NPS. [ABSTRACT FROM AUTHOR]

Abstract: Cerebral ischaemia is a common occurrence in a range of pathological conditions, including stroke and traumatic brain injury. Two of the components in ischaemia are tissue hypoxia and the release of pro-inflammatory agents such as TNF-α. The role of TNF-α in an ischaemic/hypoxic episode is still controversial, although deleterious effects of pro-inflammatory cytokines in the area of injury are well documented. One of the prime adaptive mechanisms in response to hypoxia is the cellular activation of adenosine 1 receptors (A1Rs), which inhibits excitatory synaptic transmission. In the present study we have examined the effect of TNF-α application on synaptic transmission during hypoxic exposure and re-oxygenation using extracellular recordings in the CA1 region of the rat hippocampal slice. Hypoxia caused a reversible depression of the field EPSP (29.6±9.7% of control, n =5), which was adenosine A1 receptor-dependent (85.7±4.3%, in the presence of DPCPX (200nM), the adenosine A1 receptor antagonist). DPCPX inhibited the maintenance of long-term potentiation obtained 30min post hypoxia (143.8±8.2% versus 96.4±10.6% respectively, 1h post tetanus; n =5; p <0.005). In TNF-α (150pM) treated slices hypoxic depression was similar to controls but a reduction in fEPSP slope was observed during re-oxygenation (66.8±1.4%, n =5). This effect was reversed by pre-treatment with SB 203580 (1µM), a p38 MAP kinase inhibitor (91.8±6.9%, n =5). These results demonstrate a novel p38 MAPK dependent role for TNF-α in attenuating synaptic transmission after a hypoxic episode. [Copyright &y& Elsevier]

2-(18) F-fluorodeoxy-D-glucose (FDG) is a glucose analog that is taken up by cells and phosphorylated. The amount of FDG accumulated by cells is a measure of the rate of glycolysis, which reflects cellular activity. As the levels and actions of the neuromodulator adenosine are dynamically regulated by neuronal activity, this study was designed to test whether endogenous adenosine affects tissue accumulation of FDG as assessed by positron emission tomography (PET) or by postmortem analysis of tissue radioactivity. Rats were given an intraperitoneal injection of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX, 3 mg/kg), the adenosine kinase inhibitor ABT-702 (3 mg/kg), or vehicle 10 minutes prior to an intravenous injection of FDG (15.4 ± 0.7 MBq per rat). Rats were then subjected to a 15 minute static PET scan. Reconstructed images were normalized to FDG PET template for rats and standard uptake values (SUVs) were calculated. To examine the regional effect of active treatment compared to vehicle, statistical parametric mapping analysis was performed. Whole-brain FDG uptake was not affected by drug treatment. Significant regional hypometabolism was detected, particularly in cerebellum, of DPCPX- and ABT-702 treated rats, relative to vehicle-treated rats. Thus, endogenous adenosine can affect FDG accumulation although this effect is modest in quiescent rats. [ABSTRACT FROM AUTHOR]

Abstract: Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A1 adenosine receptor (A1R) activation promotes an increase on alpha2-adrenoceptor (Alpha2R) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca2+-dependent, IP3 receptor and intracellular calcium were shown to participate in A1R/Alpha2R interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. [Copyright &y& Elsevier]

The human adenosine A1 receptor gene (ADORA1) localizes to chromosome 1q32 is 76.8 kbp in length and contains six exons. ADORA1 is ubiquitously expressed in the central nervous system and clinical and pharmacological evidence suggest the involvement of adenosine neurotransmission in the pathogenesis of schizophrenia. Therefore, we investigated the contribution of genetic variations of ADORA1 to the pathophysiological mechanisms of Japanese schizophrenia patients.We performed genetic analysis of 29 polymorphic markers in 200 schizophrenic patients and 210 healthy controls from the Kyushu region of Japan. In statistical analysis, we performed the univariate analysis with genotypes and allele frequencies, linkage disequilibrium (LD) analyses, multivariate analysis, haplotype analysis, and sliding window haplotype analysis.In univariate analysis, no statistical difference was shown, after Bonferroni correction. By LD analysis, however, we could not find any LD blocks. In haplotype analysis, a total of 359 haplotypes were estimated. In multivariate analysis, we found three statistically different markers. In sliding window haplotype analysis, there were four statistically different haplotypes.This is the first study describing the involvement of ADORA1 polymorphisms in the pathophysiological mechanisms of schizophrenia in a Japanese population. These results corroborate our previous pharmacological and neurochemical studies in the rat that have suggested an association between ADORA1 neurotransmission and the schizophrenic effects of the N-methyl-D-aspartate receptor antagonist phencyclidine. Thus, ADORA1 polymorphisms may represent good candidate markers for schizophrenia research and ADORA1 may be involved in the pathophysiological mechanisms of schizophrenia in Japanese populations. [ABSTRACT FROM AUTHOR]