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Abstract: Whole-cell patch-clamp recordings from cultured rat dorsal root ganglion neurons demonstrated that the P2Y1 receptor agonists adenosine 5′-O-2-thiodiphosphate (ADP-β-S) and 2-methylthio adenosine 5′-diphosphate (2-MeSADP) inhibit the α,β-methylene adenosine 5′-triphosphate (α,β-meATP)-induced P2X3 receptor-currents. This effect could be antagonized by the wide-spectrum G protein blocker GDP-β-S and the P2Y1 receptor antagonist MRS 2179. The P2Y12,13 receptor antagonist AR-C6993MX and pertussis toxin, a blocker of Gαi/o, did not interact with the effect of ADP-β-S. Hence, the results indicate that ADP-sensitive P2Y1 receptors of rat dorsal root ganglion neurons inhibit ionotropic P2X3 receptors via G protein-activation. [Copyright &y& Elsevier]

The effects of the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamidoadenosine (CGS 21680) on currents mediated by excitatory amino acid receptors were examined in rat striatal brain slices. In a Mg2+-free superfusion medium, CGS 21680 decreased the amplitude of excitatory postsynaptic currents (EPSCs) in about 70% of striatal neurons. The inhibitory effect of CGS 21680 disappeared both in the presence of the adenosine A2A receptor antagonist 8-(3-chlorostyryl) caffeine and the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5). NMDA-induced currents were also depressed by CGS 21680 in a subset of striatal cells, whereas α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced currents were not affected. The results suggest that adenosine A2A receptor agonists inhibit the NMDA component of the EPSC. [Copyright &y& Elsevier]

Abstract: Wild-type human (h) P2X3 receptors expressed in HEK293 cells responded to the prototypic agonist α,β-methylene ATP (α,β-meATP) with rapidly desensitizing inward currents and an increase in the intracellular Ca2+ concentration. In contrast to electrophysiological recordings, Ca2+ microfluorimetry showed a lower maximum of the concentration-response curve of α,β-meATP in the transiently than in the permanently transfected HEK293 cells. However, the concentrations causing 50% of the maximum possible effect (EC50 values) were identical, when measured with either method. In order to determine the role of certain conserved, positively charged amino acids in the nucleotide binding domains (NBD-1-4) of hP2X3 receptors for agonist binding, the lysine-63, -65, -176 and -299 as well as the arginine-281 and -295 residues were substituted by the neutral amino acid alanine. We observed no effect of α,β-meATP at the K63A, K176A, R295A, and K299A mutants, and a marked decrease of agonist potency at the K65A and R281A mutants. The P2X3 receptor antagonist 2'',3''-O-trinitrophenyl-ATP (TNP-ATP) blocked the effect of α,β-meATP at the wild-type hP2X3 receptor with lower affinity than at the mutant K65A, indicating an interference of this mutation with the docking of the antagonist with its binding sites. The use of confocal fluorescence microscopy in conjunction with an antibody raised against the extracellular loop of the hP2X3 receptor documented the expression of all mutants in the plasma membrane of HEK293 cells. Eventually, we modelled the possible agonist and antagonist binding sites NBD-1-4 of the hP2X3 subunit by using structural bioinformatics. This model is in complete agreement with the available data and integrates results from mutagenesis studies with geometry optimization of the tertiary structure predictions of the receptor. [Copyright &y& Elsevier]

Immunocytochemical and Co2+ uptake studies revealed that in primary cultures of rat cortical neurones, the majority of neurones are γ-aminobutyric acid (GABA) immunopositive and can express Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. By fura-2 microfluorimetry, it was shown that the stimulation with the selective agonist (S)-AMPA (0.3–300 μM) induced a concentration-dependent but cell-variable increase in intracellular Ca2+ concentration ([Ca2+]i) (EC50 value 7.4 μM) in more than 80% of the medium-sized multipolar neurones studied. The AMPA-induced rise in [Ca2+]i seems to be due to Ca2+ entry through AMPA receptor channels, because the response was abolished in Ca2+-free solution and by AMPA receptor selective antagonists, but was not significantly influenced by cyclopiazonic acid, an inhibitor of the endoplasmatic Ca2+-ATPase, by selective N-methyl-d-aspartic acid (NMDA) receptor antagonists, as well as the Na+ channel blocker tetrodotoxin and the majority of tested Ca2+ channel blockers. In conclusion, the results indicate that the cerebral cortical neurones in culture represent mostly GABAergic interneurone-like cells and the majority of them possess Ca2+-permeable AMPA receptors, important for intracellular signal transduction and neuronal plasticity. [Copyright &y& Elsevier]