Adenosine A1 Receptor Binding Research Articles

A genetic variation in the adenosine A2A receptor gene contributes to variability in oscillatory alpha power in wake and sleep EEG and A1 adenosine receptor availability in the human brain

The EEG alpha rhythm (∼ 8–13 Hz) is one of the most salient human brain activity rhythms, modulated by the level of attention and vigilance and related to cerebral energy metabolism. Spectral power in the alpha range in wakefulness and sleep strongly varies among individuals based on genetic predisposition. Knowledge about the underlying genes is scarce, yet small studies indicated that the variant rs5751876 of the gene encoding A2A adenosine receptors (ADORA2A) may contribute to the inter-individual variation. The neuromodulator adenosine is directly linked to energy metabolism as product of adenosine tri-phosphate breakdown and acts as a sleep promoting molecule by activating A1 and A2A adenosine receptors. We performed sleep and positron emission tomography studies in 59 healthy carriers of different rs5751876 alleles, and quantified EEG oscillatory alpha power in wakefulness and sleep, as well as A1 adenosine receptor availability with 18F-CPFPX. Oscillatory alpha power was higher in homozygous C-allele carriers (n = 27, 11 females) compared to heterozygous and homozygous carriers of the T-allele (n(C/T) = 23, n(T/T) = 5, 13 females) (F(18,37) = 2.35, p = 0.014, Wilk's Λ = 0.487). Furthermore, a modulatory effect of ADORA2A genotype on A1 adenosine receptor binding potential was found across all considered brain regions (F(18,40) = 2.62, p = 0.006, Wilk's Λ = 0.459), which remained significant for circumscribed occipital region of calcarine fissures after correction for multiple comparisons. In female participants, a correlation between individual differences in oscillatory alpha power and A1 receptor availability was observed. In conclusion, we confirmed that a genetic variant of ADORA2A affects individual alpha power, while a direct modulatory effect via A1 adenosine receptors in females is suggested.

Efficacy of Trabodenoson in a Mouse Keratoconjunctivitis Sicca (KCS) Model for Dry-Eye Syndrome.

To determine the efficacy of trabodenoson, an adenosine mimetic with highly selective adenosine A1 receptor binding properties, in a preclinical mouse model for dry-eye disease. Dry-eye disease was induced in adult male C57BL/6 mice using a combination of desiccating environment and transdermal administration of scopolamine. Mice were treated concurrently and twice daily with either vehicle, 6% trabodenoson, or 0.05% cyclosporine (Restasis). Efficacy (P < 0.05 versus vehicle) was determined by clinical assessment of dry-eye symptoms using corneal fluorescein staining and tear volumes and histopathologically by quantifying lacrimal gland pathology and conjunctival goblet cells. Twice-daily topical (ocular) administration of trabodenoson increased tear levels and reduced corneal fluorescein staining (P < 0.05) as compared with vehicle-treated eyes in a mouse model of dry-eye disease. Furthermore, significant infiltration of immune cells in the lacrimal gland and reduced number of mucin-producing conjunctival goblet cells were noted in both untreated and vehicle-treated eyes. Comparatively, trabodenoson treatment significantly reduced lacrimal gland infiltration and increased the number of goblet cells (P < 0.05 for both versus vehicle). These trabodenoson-related effects on lacrimal gland pathology and goblet cells were similar to or better than the effects observed with cyclosporine treatment. Topical ocular delivery of trabodenoson significantly improves the clinical and histopathological signs associated with dry-eye disease in mice. This improvement appears to be related to anti-inflammatory effects from targeting adenosine signaling and represents a novel therapeutic approach to develop for the management of dry-eye disease.

Benzopyrone represents a privilege scaffold to identify novel adenosine A1/A2A receptor antagonists

Adenosine receptor antagonists are under investigation as potential drug candidates for the treatment of certain cancers, neurological disorders, depression and potentially improve tumour immunotherapy. The benzo-γ-pyrone scaffold is well-known in medicinal chemistry with diverse pharmacological activities attributed to them, however, their therapeutic potential as adenosine receptor antagonists have not been investigated in detail. To expand on the structure–activity relationships, the present study explored the adenosine A1 and A2A receptor binding affinities of a selected series of benzo-γ-pyrone analogues. In vitro evaluation led to the identification of 5-hydroxy-2-(3-hydroxyphenyl)-4H-1-benzopyran-4-one with the best adenosine A2A receptor affinity among the test compounds and was found to be non-selective (A1Ki = 0.956 µM; A2AKi = 1.44 µM). Hydroxy substitution on ring A and/or B play a key role in modulating the binding affinity at adenosine A1 and A2A receptors. Adenosine A1 receptor affinity was increased to the nanomolar range with hydroxy substitution on C6 (ring A), while meta-hydroxy substitution on ring B governed adenosine A2A receptor affinity. The double bond between C2 and C3 of ring C as well as C2 phenyl substitution was shown to be imperative for both adenosine A1 and A2A receptor affinity. Selected benzo-γ-pyrone derivatives behaved as adenosine A1 receptor antagonists in the performed GTP shift assays. It may be concluded that benzo-γ-pyrone based derivatives are suitable leads for designing and identifying adenosine receptor antagonists as treatment of various disorders.

Increased Binding Potential of Brain Adenosine A1 Receptor in Chronic Stages of Patients with Diffuse Axonal Injury Measured with [1-methyl-11C] 8-dicyclopropylmethyl-1-methyl-3-propylxanthine Positron Emission Tomography Imaging.

The positron emission tomography (PET) radioligand for adenosine A1 receptor (A1R) [1-methyl-11C] 8-dicyclopropylmethyl-1-methyl-3-propylxanthine (MPDX) has recently been developed for human brain imaging. In the present study, we evaluated the alteration of the A1R in patients with diffuse axonal injury (DAI) in chronic stage in vivo. Ten patients with DAI (7 men and 3 women) were included in this study. Three PET examinations were sequentially performed to measure A1R binding with 11C-MPDX, glucose metabolism with 18F-fluorodeoxyglucose (FDG), and central benzodiazepine receptor binding with 11C-flumazenil (FMZ), and decreases of 11C-FMZ uptake indicate neuronal loss. 11C- MPDX did not depict any lesion with significantly decreased nondisplaceable binding potential (BPND) in comparison to healthy controls (14 men) in region of interest (ROI) analysis. Instead, it showed a significant increase of BPND in the lower frontal and posterior cingulate cortexes and rolandic area (p < 0.05) in ROI analysis. In 18F-FDG PET, the standardized uptake values (SUVs) ratio to the whole brain were decreased in anterior and posterior cingulate gyrus compared to controls (14 men and 9 women; p < 0.01). In 11C-FMZ PET, the SUV ratio to the cerebellum was decreased in anterior cingulate gyrus in ROI analysis (controls, 9 men and 6 women; p < 0.01). The area with significantly increased 11C-MPDX binding, lower frontal cortex, rolandic area, and posterior cingulate gyrus, did not overlap with the areas of neuronal loss detected by decreased 11C-FMZ binding and did not completely overlap with area of reduced18F-FDG uptake. We obtained the first 11C-MPDX PET images reflecting the A1R BPND in human DAI brain in vivo. 11C-MPDX depicted increased A1R BPND in the areas surrounding the injured brain, whereas 18F-FDG demonstrated reduction throughout the brain. The results suggested that A1R might continuously confer neuroprotective or neuromodulatory effects in DAI even in the chronic stage.

5-Substituted 2-benzylidene-1-tetralone analogues as A1 and/or A2A antagonists for the potential treatment of neurological conditions

Adenosine A1 and A2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer’s and Parkinson’s disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A1 and A2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A1 receptor with Ki values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A2A receptor. These substitution patterns led to enhanced adenosine A1 and A2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A1 receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A1 receptors. In conclusion, compounds 3 and 12, showed the best adenosine A1 and A2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.

Evaluation of 2-benzylidene-1-tetralone derivatives as antagonists of A1 and A2A adenosine receptors.

Antagonists of the adenosine receptors (A1 and A2A ) are thought to be beneficial in neurological disorders, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore 2-benzylidene-1-tetralone derivatives as antagonists of A1 and/or A2A adenosine receptors. In general, the test compounds were found to be selective for the A1 adenosine receptor, with only three test compounds possessing affinity for both the A1 and A2A adenosine receptor. The 2-benzylidene-1-tetralones bearing a hydroxyl substituent at either position C5, C6 or C7 of ring A displayed favourable adenosine A1 receptor binding, while C5 hydroxy substitution led to favourable A2A adenosine receptor affinity. Interestingly, para-hydroxy substitution on ring B in combination with ring A bearing a hydroxy at position C6 or C7 provided the 2-benzylidene-1-tetralones with both A1 and A2A adenosine receptor affinity. Compounds 4 and 8 displayed the highest A1 and A2A adenosine receptor affinity with values below 7μm. Both these compounds behaved as A1 adenosine receptor antagonists in the performed GTP shift assays. In conclusion, the 2-benzylidene-1-tetralone derivatives can be considered as lead compounds to design a new class of dual acting adenosine A1 /A2A receptor antagonists that may have potential in treating both dementia and locomotor deficits in Parkinson's disease.

1,3,7-Triethyl-substituted xanthines—possess nanomolar affinity for the adenosine A1 receptor

Adenosine A1 receptors are attracting great interest as drug targets for their role in cognitive deficits. Antagonism of the adenosine A1 receptor may offer therapeutic benefits in complex neurological diseases, such as Alzheimer’s and Parkinson’s disease. The aim of this study was to discover potential selective adenosine A1 receptor antagonists. Several analogs of 8-(3-phenylpropyl)xanthines (3), 8-(2-phenylethyl)xanthines (4) and 8-(phenoxymethyl)xanthines (5) were synthesized and assessed as antagonists of the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that the 1,3,7-triethyl-substituted analogs (3d, 4d, and 5d), among each series, displayed the highest affinity for the adenosine A1 receptor with Ki values in the nanomolar range. This ethyl-substitution pattern was previously unknown to enhance adenosine A1 receptor binding affinity. The 1,3,7-triethyl-substituted analogs (3d, 4d, and 5d) behaved as adenosine A1 receptor antagonists in GTP shift assays performed with either rat cortical or whole brain membranes expressing adenosine A1 receptors. Further, in vivo evaluation of 3d showed reversal of adenosine A1 receptor agonist-induced hypolocomotion. In conclusion, the most potent evaluated compound, 8-(3-phenylpropyl)-1,3,7-triethylxanthine (3d), showed both in vitro and in vivo activity, and therefore represent a novel adenosine A1 receptor antagonist that may have potential as a drug candidate for dementia disorders.

Association of Adenosine Receptor Gene Polymorphisms and In Vivo Adenosine A1 Receptor Binding in The Human Brain

Adenosine A1 receptors (A1ARs) and the interacting adenosine A2A receptors are implicated in neurological and psychiatric disorders. Variants within the corresponding genes ADORA1 and ADORA2A were shown associated with pathophysiologic alterations, particularly increased anxiety. It is unknown so far, if these variants might modulate the A1AR distribution and availability in different brain regions. In this pilot study, the influence of ADORA1 and ADORA2A variants on in vivo A1AR binding was assessed with the A1AR-selective positron emission tomography (PET) radioligand [(18)F]CPFPX in brains of healthy humans. Twenty-eight normal control subjects underwent PET procedures to calculate the binding potential BPND of [(18)F]CPFPX in cerebral regions and to assess ADORA1 and ADORA2A single nucleotide polymorphism (SNP) effects on regional BPND data. Our results revealed SNPs of both genes associated with [(18)F]CPFPX binding to the A1AR. The strongest effects that withstood even Bonferroni correction of multiple SNP testing were found in non-smoking subjects (N=22) for ADORA2A SNPs rs2236624 and rs5751876 (corr. Pall<0.05). SNP alleles previously identified at risk for increased anxiety like the rs5751876 T-allele corresponded to consistently higher A1AR availability in all brain regions. Our data indicate for the first time that variation of A1AR availability was associated with ADORA SNPs. The finding of increased A1AR availability in regions of the fear network, particularly in ADORA2A risk allele carriers, strongly warrants evaluation and replication in further studies including individuals with increased anxiety.

NMR metabolomics for identification of adenosine A1 receptor binding compounds from Boesenbergia rotunda rhizomes extract

Ethnopharmacological relevanceBoesenbergia 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 studyThe 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 methodsDried 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 discussionsBased 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. ConclusionTwo 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.

The effect of unpredictable chronic mild stress on depressive-like behavior and on hippocampal A1 and striatal A2A adenosine receptors

This study examined the effects of two chronic stress regimens upon depressive-like behavior, A1 and A2A adenosine receptor binding and immunocontent. Male rats were subjected to unpredictable chronic mild stress (UCMS) or to chronic restraint stress (CRS) for 40days. Subsequently, depressive-like behaviors (forced swimming and consumption of sucrose) were evaluated, and A1 adenosine or A2A adenosine receptors were examined in the hippocampus or striatum, respectively. UCMS animals demonstrated depressive-related behaviors (decrease in sucrose consumption and increased immobility in the forced swimming test). This group also presented increased A1 adenosine receptor binding and immunoreactivity in hippocampus, as well as increased striatal A2A adenosine receptor binding in the striatum, without alteration in immunoreactivity. Conversely, the chronic restraint stress group displayed only an increase in A1 adenosine receptor binding and no alteration in the other parameters evaluated. We suggest that the alteration in adenosine receptors, particularly the upregulation of striatal A2A adenosine receptors following UCMS, could be associated with depressive-related behavior.

Comprehensive Extraction Method Integrated with NMR Metabolomics: A New Bioactivity Screening Method for Plants, Adenosine A1 Receptor Binding Compounds in Orthosiphon stamineus Benth

A large number of plant metabolites has provided as an incomparable chemical source for drug development. However, the wide range of the polarity of metabolites has been a big obstacle for full use of the chemical diversity. The initial step conventional extraction method by a single solvent does not make use of all the metabolites contained in plants. Also, it takes a long time to confirm the target activity of a single compound because of tedious separation steps. To solve the problem, a new extraction method coupled to NMR-based metabolomics is applied to identify bioactive natural products. A comprehensive extraction method consisting of a continuous flow of solvent mixtures through plant material was developed to provide extracts with a wider chemical variety than those yielded with a single solvent extraction. As the model experiment, (1)H NMR spectra of the extracts obtained from the comprehensive extraction of Orthosiphon stamineus were subjected to multivariate data analysis to find its adenosine A1 binding activity. On the basis of the results, two flavonoids from a large number of chemicals were clearly verified to show the adenosine A1 binding activity without any further purification steps. This method could provide a solution to the major drawbacks of natural products in drug development.

Screening of selected Asian spices for anti obesity-related bioactivities

The potential health effects of 30 spices, commonly used for daily consumption, were submitted to bioactivity screening with several anti-obesity related bioassays: adenosine A1 receptor binding, cannabinoid CB1 receptor binding, TNF-α and 3T3-L1 adipocytes differentiation induction. Sesame seed and red chilli exhibited high binding activity to the adenosine A1 receptor and nutmeg, mace, black pepper and turmeric to the cannabinoid CB1 receptor, while piment and turmeric showed high inhibition of TNF-α accumulation. Black onion seed proved to be the only spice with high 3T3-L1 adipocyte differentiation induction activity. Several well known major compounds found in these active spices were tested with the respective bioassays but did not show activity. Thus, it appears that other minor compounds or the synergistic effects of different constituents are responsible for the observed activity.

N-Substituted Quinazolin- 2,4-diones as Adenosine Receptor Ligands

Six new N-substituted quinazolinones were synthesized and evaluated for their adenosine antagonistic activity using allergic mice model where 1, 3-dibenzyl and 1, 3-dibutyl-quinazolindiones were found to be potent than 1, 3-dimethylanalogues. They were not significant in controlling the neutrophil and lymphocyte count but effective in controlling the eosinophil influx. In adenosine receptor binding studies, 1,3-dimethyl and 1,3-dibutyl derivatives were found to have significant adenosine A1 receptor binding efficiency with Ki values 9nM and 10nM, respectively, while 1,3- dibenzyl-quinazolinone was found to have significant binding to adenosine A2A receptor showing the influence of alkyl and aralkyl groups present in these compounds. Thus, the present work indicates the possibility to explore quinazolindiones as adenosine receptor ligands.

Increased binding of inhibitory neuronal receptors in the hippocampus in kainate-treated rats with spontaneous limbic seizures

To gain a better understanding of the relationship between epileptogenicity and inhibitory neuronal mechanisms, we examined variations in A1 adenosine (A1A) receptor binding in the hippocampi of rats with spontaneous limbic seizures in the chronic phase after systemic kainic acid treatment. Six weeks after kainate treatment, rats with spontaneous limbic seizures were killed for histological and in vitro autoradiographic analyses of the brain. The analyses were performed using [ 3H] 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1A receptor antagonist. Relative to controls, DPCPX binding was increased in the CA3 region and in the molecular layer of the dentate gyrus in the kainate-treated rats. This is the first evidence of upregulation of the A1A receptor in a model of chronic temporal lobe epilepsy. Increased binding of the A1A receptor may contribute to epileptogenesis in the epileptic focus.

Predictive 3D‐Quantitative Structure‐Activity Relationship for A1 and A2A Adenosine Receptor Ligands

AbstractThe use of QSAR applications to develop adenosine receptor (AR) antagonists is not so common. A library of all xanthine derivatives, obtained at the Department of Technology and Biotechnology of Drugs, was created. Sixty‐three active adenosine A1 receptor ligands and one hundred thirty nine active adenosine A2A receptor ligands were used for 3D‐QSAR investigation. The 3D‐QSAR equations with a high predictive power in estimating the binding affinity values of potential A1 and A2A ARs ligands were derived. For the first time, hybrid shape‐property descriptors were used in 3D‐QSAR for xanthine ARs ligands. The obtained models were characterized by a high regression and cross‐validation coefficients. Two types of the model validation were tested – dividing the library into the training set for model development and external set for model validation and increasing the number of library components and checking the model by cross‐validated regression coefficient. The analysis of the results depicts that for the A1 AR binding activity it is important for ligands to possess R1‐propyl substituents along with the phenyl or benzyl substituents bearing halogen atom and phenethyl moiety. For A2A AR affinity it could be favorable to introduce phenethyl or phenyl substituent connected with the tricyclic ring by the alkoxy chain. The nature of R1 group may not significantly affect the A2A AR affinity. High predictive power of the equations suggests their use for further development of adenosine receptor antagonists within xanthine derivatives.

Identification of Possible Compounds Possessing Adenosine A1 Receptor Binding Activity in the Leaves of Orthosiphon stamineus Using TLC and Multivariate Data Analysis

A novel approach to identify compounds possessing adenosine A1 receptor binding activity in the leaves of O. stamineus was developed. O. stamineus extract is one of the components of a functional beverage used in Indonesia for the treatment of kidney stones. In this study, adenosine A1 receptor binding, which is related to the diuretic action in the treatment of kidney stones was tested. A combination of thin layer chromatography of different extracts prepared by extraction with diverse solvents (n-hexane, chloroform, n-butanol and water), and multivariate data analysis based on orthogonal partial least squares proved to be a promising approach to determine these active compounds. Several methoxyflavonoids, fatty acids or terpenoids were estimated to be related to this activity. The results of this study support the traditional use in Indonesia of O. stamineus as a functional drink to treat kidney stones.

On the stability of 2-aminoselenophene-3-carboxylates: potential dual-acting selenium-containing allosteric enhancers of A1 adenosine receptor binding

Ethyl-2-amino-4,5,6,7-tetrahydro-1-benzoselenophene-3-carboxylate (4), has been prepared as a potential dual-acting selenium-containing allosteric enhancer of adenosine A(1)A receptor binding utilising a modified Gewald reaction. While preliminary testing indicated that 4 is a superior enhancer of A(1)AR binding than its thiophene counterpart, its instability under mildly acidic conditions is cause for concern. X-Ray crystallography, together with DFT calculations, provide evidence that the decomposition of 4 involves the ring-opening of selenophenium ion (12b) followed by the loss of elemental selenium through a radical chain process.

Impact of sleep deprivation on cerebral A1 adenosine receptor (A1AR) density: A PET study

There is evidence from animal experiments that adenosine is an important sleep propensity factor which exerts its function mainly via the A1 adenosine receptor (A1AR). Prolonged wakefulness causes a significant increase of extracellular adenosine in distinct brain regions promoting induction of sleep via A1ARs 1. Recently, in vivo investigation of cerebral A1ARs has become feasible by the selective ligand 18F-CPFPX and PET 2. This pilot study was intended to examine whether the A1AR binding of 18F-CPFPX is changed by prolonged wakefulness. Eleven healthy male volunteers participated in a dynamic 18F-CPFPX bolus/infusions-PET study with venous blood sampling 3. Seven subjects were scanned before and after 24 hours of sustained wakefulness and four subjects served as control group being measured twice on subsequent days after normal 8 h night sleep. The realignment and co-registration of dynamic PET data according to the individual MRI and subsequent normalization to the MNI template were performed with SPM2. Regional distribution volume ratios (DVR) (cerebellum as input) using Logan´s non invasive graphical analysis were determined. The apparent binding potential (BPapp = DVR – 1) was calculated as outcome parameter. Comparing the BPapp values obtained before and after sleep deprivation revealed a tendency towards an increase of the BPapp at the second day (mean intraindividual relative change (day2-day1)/day1 in % SD (paired t-test): frontal 3.32 5.0% (p=0.15), parietal 4.03 5.0% (p=0.073), occipital 6.57 4.1% (p=0.006), striatal 7.87 9.1% (p=0.067)). In contrast there was no difference between both days in the control group (frontal 1.36 6.6 %(p=0.96), parietal 0.02 2.0% (p=0.86), occipital 0.63 2.0% (p=0.64), striatal –0.5 4.4% (p=0.73)). These preliminary results show a tendency towards an increase of the BPapp of 18F-CPFPX after sleep deprivation. An increase of A1AR density would be in line with an upregulation of A1AR mRNA which was observed in animals under persistent sleep deprivation 1. To confirm these preliminary observations a larger number of subjects is currently under investigation.

Ageing‐related decline in adenosine A1 receptor binding in the rat brain: An autoradiographic study

Ageing‐related decline in adenosine A1 receptor binding in the rat brain: An autoradiographic study

Allosteric enhancers of A1 adenosine receptors increase receptor-G protein coupling and counteract Guanine nucleotide effects on agonist binding.

Endogenous ligands of G protein-coupled receptors bind to orthosteric sites that are topologically distinct from allosteric sites. Certain aminothiophenes such as (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]-methanone (PD81,723) and 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-biphenyl-4-yl-methanone (ATL525) are positive allosteric regulators, or enhancers, of the human A1 adenosine receptor (A1AR). In equilibrium binding assays, 125I-N6-aminobenzyladenosine (125I-ABA) binds to two affinity states of A1AR with KD-high (0.33 microM) and KD-low ( approximately 10 nM). Enhancers have little effect on KD-high but convert all A1AR binding sites to the high-affinity state. Enhancers decrease the potency of guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) as an inhibitor of agonist binding by 100-fold and increase agonist-stimulated guanine nucleotide exchange. The association of 125I-ABA to high-affinity receptors on Chinese hamster ovary (CHO)-hA1 membranes does not follow theoretical single-site association kinetics but is approximated by a bi-exponential equation with t1/2 values of 1.85 and 12.8 min. Allosteric enhancers selectively increase the number of slow binding sites, possibly by stabilizing newly formed receptor-G protein complexes. A new rapid assay method scores enhancer activity on a scale from 0 to 100 based on their ability to prevent the rapid dissociation of 125I-ABA from A1AR in response to GTPgammaS. Compared with PD81,723, ATL525 (100 microM) scores higher (27 versus 79) and has less antagonist activity. ATL525 functionally enhances A1 signaling to inhibit cAMP accumulation in CHO-hA1 cells. These data suggest that simultaneously binding orthosteric and allosteric enhancer ligands convert the A1AR from partly to fully coupled to G proteins and prevents rapid uncoupling upon binding of GTPgammaS.