Evidence has shown that both sleep loss and daily caffeine intake can induce changes in grey matter (GM). Caffeine is frequently used to combat sleepiness and impaired performance caused by insufficient sleep. It is unclear (1) whether daily use of caffeine could prevent or exacerbate the GM alterations induced by 5-day sleep restriction (i.e. chronic sleep restriction, CSR), and (2) whether the potential impact on GM plasticity depends on individual differences in the availability of adenosine receptors, which are involved in mediating effects of caffeine on sleep and waking function. Thirty-six healthy adults participated in this double-blind, randomized, controlled study (age = 28.9 ± 5.2 y/; F:M = 15:21; habitual level of caffeine intake < 450 mg; 29 homozygous C/C allele carriers of rs5751876 of ADORA2A, an A2A adenosine receptor gene variant). Each participant underwent a 9-day laboratory visit consisting of one adaptation day, 2 baseline days (BL), 5-day sleep restriction (5 h time-in-bed), and a recovery day (REC) after an 8-h sleep opportunity. Nineteen participants received 300 mg caffeine in coffee through the 5 days of CSR (CAFF group), while 17 matched participants received decaffeinated coffee (DECAF group). We examined GM changes on the 2nd BL Day, 5th CSR Day, and REC Day using magnetic resonance imaging and voxel-based morphometry. Moreover, we used positron emission tomography with [18F]-CPFPX to quantify the baseline availability of A1 adenosine receptors (A1R) and its relation to the GM plasticity. The results from the voxel-wise multimodal whole-brain analysis on the Jacobian-modulated T1-weighted images controlled for variances of cerebral blood flow indicated a significant interaction effect between caffeine and CSR in four brain regions: (a) right temporal-occipital region, (b) right dorsomedial prefrontal cortex (DmPFC), (c) left dorsolateral prefrontal cortex (DLPFC), and (d) right thalamus. The post-hoc analyses on the signal intensity of these GM clusters indicated that, compared to BL, GM on the CSR day was increased in the DECAF group in all clusters  but decreased in the thalamus, DmPFC, and DLPFC in the CAFF group. Furthermore, lower baseline subcortical A1R availability predicted a larger GM reduction in the CAFF group after CSR of all brain regions except for the thalamus. In conclusion, our data suggest an adaptive GM upregulation after 5-day CSR, while concomitant use of caffeine instead leads to a GM reduction. The lack of consistent association with individual A1R availability may suggest that CSR and caffeine affect thalamic GM plasticity predominantly by a different mechanism. Future studies on the role of adenosine A2A receptors in CSR-induced GM plasticity are warranted.

We used coarse-grained molecular dynamics (CG MD) simulations to study protein-cholesterol interactions for different activation states of the A2A adenosine receptor (A2AR) and the A1 adenosine receptor (A1R) and predict new cholesterol binding sites indicating amino acid residues with a high residence time in three biologically relevant membranes. Compared to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-cholesterol and POPC-phosphatidylinositol-bisphosphate (PIP2)-cholesterol, the plasma mimetic membrane best described the cholesterol binding sites previously detected for the inactive state of A2AR and revealed the binding sites with long-lasting amino acid residues. We observed that using the plasma mimetic membrane and plotting residues with cholesterol residence time ≥2 μs, our CG MD simulations captured most obviously the cholesterol-protein interactions. For the inactive A2AR, we identified one more binding site in which cholesterol is bound to residues with a long residence time compared to the previously detected, for the active A1R, three binding sites, and for the inactive A1R, two binding sites. We calculated that for the active states, cholesterol binds to residues with a much longer residence time compared to the inactive state for both A2AR and A1R. The stability of the identified binding sites to A1R or A2AR with CG MD simulations was additionally investigated with potential of mean force calculations using umbrella sampling. We observed that the binding sites with residues to which cholesterol has a long residence time in A2AR have shallow binding free energy minima compared to the related binding sites in A1R, suggesting a stronger binding for cholesterol to A1R. The differences in binding sites in which cholesterol is stabilized and interacts with residues with a long residence time between active and inactive states of A1R and A2AR can be important for differences in functional activity and orthosteric agonist or antagonist affinity and can be used for the design of allosteric modulators, which can bind through lipid pathways. We observed a stronger binding for cholesterol to A1R (i.e., generally higher association rates) compared to A2AR, which remains to be demonstrated. For the active states, cholesterol binds to residues with much longer residence times compared to the inactive state for both A2AR and A1R. Taken together, binding sites of active A1R may be considered as promising allosteric targets.

YZG-331 is a synthetic novel derivates of N6-(4-hydroxybenzyl) adenine riboside (NHBA), which has potent sedative and hypnotic effects based on our previous study. We are now aiming to investigate the mechanism of YZG-331. In this research, the behavioral studies showed that YZG-331 (4, 8, 16 mg/kg, i.g.) could reduce the spontaneous locomotor activity in mice, which could be blocked by AM (non-selective adenosine receptor antagonist), DPCPX (adenosine A1 receptor (A1R) antagonist), and SCH58261 (adenosine A2a receptor (A2aR) antagonist). Moreover, YZG-331 no longer exerted sedative effect in A1R or A2aR knockdown mice. YZG-331 (2.5, 5, 10 mg/kg, i.g.) prolonged sleeping time in pentobarbital sodium treated mice, which can be prevented by DPCPX or SCH58261. The above results demonstrated that YZG-331 exerted sedative and hypnotic effects through A1R and A2aR. In addition, it was found that YZG-331 (25, 50, 100 μM) decreased intracellular calcium level and YZG-331 (10 mg/kg, i.g.) decreased CaMKII phosphorylation (pCaMKII) level in mouse hypothalamus and cortex. In summary, this study indicated that activation of A1R/ A2aR and down regulation of Ca2+-CaMKII signaling pathway were involved in the sedative and hypnotic effects of YZG-331.

The adenosine A1 receptor (A1AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A1AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A1AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A1AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A1AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A1AR molecular pharmacology and signaling in living cells.

Among still comparatively few G protein-coupled receptors, the adenosine A2A receptor has been co-crystallized with several ligands, agonists as well as antagonists. It can thus serve as a template with a well-described orthosteric ligand binding region for adenosine receptors. As not all subtypes have been crystallized yet, and in order to investigate the usability of homology models in this context, multiple adenosine A1 receptor (A1AR) homology models had been previously obtained and a library of lead-like compounds had been docked. As a result, a number of potent and one selective ligand toward the intended target have been identified. However, in in vitro experimental verification studies, many ligands also bound to the A2AAR and the A3AR subtypes. In this work we asked the question whether a classification of the ligands according to their selectivity was possible based on docking scores. Therefore, we built an A3AR homology model and docked all previously found ligands to all three receptor subtypes. As a metric, we employed an in vitro/in silico selectivity ranking system based on taxicab geometry and obtained a classification model with reasonable separation. In the next step, the method was validated with an external library of, selective ligands with similarly good performance. This classification system might also be useful in further screens.

Adenosine receptors are widely expressed in the brain, and adenosine is a key bioactive substance for neuroprotection. In this article, we clarify systematically the role of adenosine A1 receptors during a range of timescales and conditions when a significant amount of adenosine is released. Using acute hippocampal slices obtained from mice that were wild type or null mutant for the adenosine A1 receptor, we quantified and characterized the impact of varying durations of experimental ischemia, hypoxia, and hypoglycemia on synaptic transmission in the CA1 subregion. In normal tissue, these three stressors rapidly and markedly reduced synaptic transmission, and only treatment of sufficient duration led to incomplete recovery. In contrast, inactivation of adenosine A1 receptors delayed and/or lessened the reduction in synaptic transmission during all three stressors and reduced the magnitude of the recovery significantly. We reproduced the responses to hypoxia and hypoglycemia by applying an adenosine A1 receptor antagonist, validating the clear effects of genetic receptor inactivation on synaptic transmission. We found activation of adenosine A1 receptor inhibited hippocampal synaptic transmission during the acute phase of ischemia, hypoxia, or hypoglycemia and caused the recovery from synaptic impairment after these three stressors using genetic mutant. These studies quantify the neuroprotective role of the adenosine A1 receptor during a variety of metabolic stresses within the same recording system.NEW & NOTEWORTHY Deprivation of oxygen and/or glucose causes a rapid adenosine A1 receptor-mediated decrease in synaptic transmission in mouse hippocampus. We quantified adenosine A1 receptor-mediated inhibition during and synaptic recovery after ischemia, hypoxia, and hypoglycemia of varying durations using a genetic mutant and confirmed these findings using pharmacology. Overall, using the same recording conditions, we found the acute response and the neuroprotective ability of the adenosine A1 receptor depended on the type and duration of deprivation event.

暂无摘要。

Caffeine, a nonselective antagonist of adenosine receptors, is the most popular psychostimulant worldwide. Recently, a protective role of moderate chronic caffeine consumption against neurodegenerative diseases such as Alzheimer\'s and Parkinson\'s disease has been discussed. Thus, aim of the present study was an in vivo investigation of effects of long-term caffeine consumption on the adenosine A1 receptor (A1AR) in the rat brain.
Sixteen adult, male rats underwent five positron emission tomography (PET) scans with the highly selective A1AR radioligand [18F]CPFPX in order to determine A1AR availability. After the first baseline PET scan, the animals were assigned to two groups: Caffeine treatment and control group. The caffeine-treated animals received caffeinated tap water (30 mg/kg bodyweight/day, corresponding to 4-5 cups of coffee per day in humans) for 12 weeks. Subsequently, caffeine was withdrawn and repeated PET measurements were performed on day 1, 2, 4, and 7 of caffeine withdrawal. The control animals were measured according to the same time schedule.
At day 1, after 4.4 h of caffeine withdrawal, a significant decrease (- 34.5%, p < 0.001) of whole brain A1AR availability was observed. Unlike all other investigated brain regions in caffeine-treated rats, the hypothalamus and nucleus accumbens showed no significant intraindividual differences between baseline and first withdrawal PET scan. After approximately 27 h of caffeine withdrawal, the region- and group-specific effects disappeared and A1AR availability settled around baseline.
The present study provides evidence that chronic caffeine consumption does not lead to persistent changes in functional availability of cerebral A1ARs which have previously been associated with neuroprotective effects of caffeine. The acute and region-specific decrease in cerebral A1AR availability directly after caffeine withdrawal is most likely caused by residual amounts of caffeine metabolites disguising an unchanged A1AR expression at this early time-point.

Small animal positron emission tomography (PET) can be used to detect small changes in neuroreceptor availability. This often requires rapid arterial blood sampling. However, current catheterization procedures do not allow repeated blood sampling. We have developed a procedure which allows arterial sampling on repeated occasions in the same animal.
Eleven male Wistar rats were two times catheterized via a superficial branch of a femoral artery and scanned with [(11)C]MPDX and blood sampling. PET images were co-registered to a magnetic resonance imaging (MRI) template. Regional tracer distribution volumes (V T) in the brain were calculated by the Logan analysis. The procedure was repeated after 1 week.
Surgery was successful in 90 % of the cases, and discomfort was minor. The V T data showed small differences between test and retest, low between subject variability, and a strong agreement between and within subjects.
Repeated quantitative imaging with a high reproducibility is possible with this approach.

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.