Background and purpose: Several P2X 7 receptor antagonists are allosteric inhibitors and exhibit species difference in potency. Furthermore, N 2 -(3,4-difluorophenyl)-N 1 -(2-methyl-5-(1-piperazinylmethyl)phenyl)glycinamide dihydrochloride (GW791343) exhibits negative allosteric effects at the human P2X 7 receptor but is a positive allosteric modulator of the rat P2X 7 receptor. In this study we have identified several regions of the P2X 7 receptor that contribute to the species differences in antagonist effects. Experimental approach: Chimeric human-rat P2X 7 receptors were constructed with regions of the rat receptor being inserted into the human receptor. Antagonist effects at these receptors were measured in ethidium accumulation and radioligand binding studies. Key results: Exchanging regions of the P2X 7 receptor close to transmembrane domain 1 modified the effects of KN62, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and GW791343. Further studies, in which single amino acids were exchanged, identified amino acid 95 as being primarily responsible for the differential allosteric effects of GW791343 and, to varying degrees, the species differences in potency of SB203580 and KN62. The species selectivity of pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulphonic acid was affected by multiple regions of the receptor, with potency being particularly affected by the amino acid 126 but not by amino acid 95. A further region of the rat receptor (amino acids 154-183) was identified that, when inserted into the corresponding position in the human receptor, increased ATP potency 10-fold. Conclusions: This study has identified several key residues responsible for the species differences in antagonist effects at the P2X 7 receptor and also identified a further region of the P2X 7 receptor that can significantly affect agonist potency at the P2X 7 receptor.

No abstract

Background and purpose: The human, rat, and mouse P2X 7 receptors have been previously characterized, and in this study we report the cloning and pharmacological properties of the guinea pig orthologue. Experimental approach: A cDNA encoding for the guinea pig P2X 7 receptor was isolated from a guinea pig brain library. The receptor was expressed in U-2 OS cells using the BacMam viral expression system. A monoclonal antibody was used to confirm high levels of cell surface expression and the functional properties were determined in ethidium bromide accumulation studies. Key results: The predicted guinea pig protein is one amino acid shorter than the human and rat orthologues and over 70% identical to the rat and human receptors. In contrast to human and rat P2X 7 receptors, 2 0 -&3 0 -O-(4benzoylbenzoyl)ATP (BzATP) was a partial agonist of the guinea pig P2X 7 receptor when compared to ATP and acted as an antagonist in some assays. However, as at other species orthologues, BzATP was more potent than ATP. The guinea pig P2X 7 receptor possessed higher affinity for 1-[N,O-bis(5-isoquinoline sulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62), suramin and Coomassie Brilliant Blue than human or rat P2X 7 receptors suggesting that it is pharmacologically different to other rodent or human P2X 7 receptors. Conclusions and implications: The guinea pig recombinant P2X 7 receptor displays a number of unique properties that differentiate it from the human, rat and mouse orthologues and this structural and functional information should aid in our understanding of the interaction of agonists and antagonist with the P2X 7 receptor.

Background and purpose: AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl] phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X7 receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues. Experimental approach: Antagonist effects at recombinant and chimeric P2X7 receptors were assessed by ethidium accumulation and radioligand-binding studies. Key results: AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X7 receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X7 receptors and, for each compound, their potency estimates at human and dog P2X7 receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X7 receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X7 receptor. Conclusions: AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X7 receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X7 receptor at which antagonists can bind and affect receptor function.

No abstract

Background and purpose: Histamine H3 receptor antagonists are currently being evaluated for their potential use in a number of central nervous system disorders including Alzheimer's Disease (AD). To date, little is known about the state of H3 receptors in AD. Experimental approach: In the present study we used the radiolabelled H3 receptor antagonist [ 3 H]GSK189254 to investigate H3 receptor binding in the amyloid over-expressing double mutant APPswe ¥ PSI.MI46V (TASTPM) transgenic mouse model of AD and in post-mortem human AD brain samples. Key results: No significant differences in specific H3 receptor binding were observed between wild type and TASTPM mice in the cortex, hippocampus or hypothalamus. Specific [ 3 H]GSK189254 binding was detected in sections of human medial frontal cortex from AD brains of varying disease severity (Braak stages I-VI). With more quantitative analysis in a larger cohort, we observed that H3 receptor densities were not significantly different between AD and age-matched control brains in both frontal and temporal cortical regions. However, within the AD group, [ 3 H]GSK189254 binding density in frontal cortex was higher in individuals with more severe dementia prior to death. Conclusions and implications:The maintenance of H3 receptor integrity observed in the various stages of AD in this study is important, given the potential use of H3 antagonists as a novel therapeutic approach for the symptomatic treatment of AD.

Background and purpose: Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues. Experimental approach: A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1b release in dog and human whole blood. Key results: The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2′-&3′-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower. Conclusions and implications: Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic.

No abstract