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Identification of regions of the P2X(7) receptor that contribute to human and rat species differences in antagonist effects.

ABSTRACT: 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.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.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',4'-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.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.

SUBMITTER: Michel AD

PROVIDER: S-EPMC2584934 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Identification of regions of the P2X(7) receptor that contribute to human and rat species differences in antagonist effects.

Michel A D AD Clay W C WC Ng S W SW Roman S S Thompson K K Condreay J P JP Hall M M Holbrook J J Livermore D D Senger S S

British journal of pharmacology 20080728 5

<h4>Background and purpose</h4>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 diffe ...[more]

PMID: 18660826

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Project description:Sepsis is a major clinical problem associated with significant organ dysfunction and high mortality. The ATP-sensitive P2X7 receptor activates the NLRP3 inflammasome and is a key component of the innate immune system. We used a fluid-resuscitated rat model of fecal peritonitis and acute kidney injury (AKI) to investigate the contribution of this purinergic receptor to renal dysfunction in sepsis. Six and 24 h time-points were chosen to represent early and established sepsis, respectively. A selective P2X7 receptor antagonist (A-438079) dissolved in dimethyl sulfoxide (DMSO) was infused 2 h following induction of sepsis. Compared with sham-operated animals, septic animals had significant increases in heart rate (-1(-4 to 8)% vs. 21(12-26)%; P = 0.003), fever (37.4(37.2-37.6)°C vs. 38.6(38.2-39.0)°C; P = 0.0009), and falls in serum albumin (29(27-30)g/L vs. 26(24-28); P = 0.0242). Serum IL-1β (0(0-10)(pg/mL) vs. 1671(1445-33778)(pg/mL); P < 0.001) and renal IL-1β (86(50-102)pg/mg protein vs. 200 (147-248)pg/mg protein; P = 0.0031) were significantly elevated in septic compared with sham-operated animals at 6 h. Serum creatinine was elevated in septic animals compared with sham-operated animals at 24 h (23(22-25) μmol/L vs. 28 (25-30)μmol/L; P = 0.0321). Renal IL-1β levels were significantly lower in A-438079-treated animals compared with untreated animals at 6 h (70(55-128)pg/mg protein vs. 200(147-248)pg/mg protein; P = 0.021). At 24 h, compared with untreated animals, A-438079-treated animals had more rapid resolution of tachycardia (22(13-36)% vs. -1(-6 to 7)%; P = 0.019) and fever (39.0(38.6-39.1)°C vs. 38.2(37.6-38.7)°C; P < 0.024), higher serum albumin (23(21-25)g/L vs. (27(25-28)g/L); P = 0.006), lower arterial lactate (3.2(2.5-4.3)mmol/L vs. 1.4(0.9-1.8)mmol/L; P = 0.037), and lower serum creatinine concentrations (28(25-30)μmol/L vs. 22(17-27)μmol/L; P = 0.019). P2X7 A treatment ameliorates the systemic inflammatory response and renal dysfunction in this clinically relevant model of sepsis-related AKI.

Project description:BackgroundDopamine receptors interact with other receptors to form heterooligomers. One such complex, the D1-D2 heteromer, demonstrated in cultured striatal neurons and rat striatum has been linked to drug addiction, Parkinson's disease, schizophrenia, depression and anhedonia.MethodsD1-D2 heteromer expression was evaluated using in situ proximity ligation assay, in parallel with cellular colocalization of D1 and D2 mRNA using in situ hybridization in 19 different key rat brain regions. Expression in higher species and changes in rat striatum after repeated cocaine administration were evaluated.ResultsDifferences in D1-D2 heteromer expression in striatal subregions are documented in higher species with nonhuman primate and human demonstrating higher density of heteromer-expressing neurons compared to rodents. All species had higher density of D1-D2 neurons in nucleus accumbens compared to dorsal striatum. Multiple other brain regions are identified where D1-D2 heteromer is expressed, prominently in cerebral cortical subregions including piriform, medial prefrontal, orbitofrontal and others; subcortical regions such as claustrum, amygdala and lateral habenula. Three categories of regions are identified: D1-D2 heteromer expressed despite little to no observed D1/D2 mRNA colocalization, likely representing heteromer on neuronal projections from other brain regions; D1-D2 heteromer originating locally with the density of neurons expressing heteromer matching neurons with colocalized D1/D2 mRNA; regions with both a local origin and targeted inputs projecting from other regions. Repeated cocaine administration significantly increased density of neurons expressing D1-D2 heteromer and D1/D2 mRNA colocalization in rat striatum, with changes in both direct and indirect pathway neurons.ConclusionThe dopamine D1-D2 heteromer is expressed in key brain cortical and subcortical regions of all species examined. Species differences in striatum revealed greater abundance in human>nonhuman-primate>rat>mouse, suggesting an evolutionary biologic role for the D1-D2 heteromer in higher CNS function. Its upregulation in rat striatum following cocaine points to regulatory significance with possible relevance for clinical disorders such as drug addiction. The dopamine D1-D2 receptor heteromer may represent a potential target for neuropsychiatric and neurodegenerative disorders, given its distribution in highly relevant brain regions.

Dataset Information

Identification of regions of the P2X(7) receptor that contribute to human and rat species differences in antagonist effects.

Publications

Identification of regions of the P2X(7) receptor that contribute to human and rat species differences in antagonist effects.

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