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Objective: The aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE(2)) release.Methods: Saturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X(1) and P2X(3) purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE(2) release) the potency of purinergic agonists and antagonists was evaluated.Results: Bovine chondrocytes expressed P2X(1) and P2X(3) purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5'-triphosphate (ATP) and alpha,beta-methyleneATP were able to increase NO and PGE(2) release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists.Conclusions: These data demonstrate for the first time the presence of functional P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE(2) release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption. [ABSTRACT FROM AUTHOR]

Background and Purpose: Among several pharmacological properties, analgesia is the most common feature shared by either opioid or cannabinoid systems. Cannabinoids and opioids are distinct drug classes that have been historically used separately or in combination to treat different pain states. In the present study, we characterized the signal transduction pathways mediated by cannabinoid CB(2) and µ-opioid receptors in quiescent and LPS-stimulated murine microglial cells.Experimental Approach: We examined the effects of µ-opioid and CB(2) receptor stimulation on phosphorylation of MAPKs and Akt and on IL-1β, TNF-α, IL-6 and NO production in primary mouse microglial cells.Key Results: Morphine enhanced release of the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and of NO via µ-opioid receptor in activated microglial cells. In contrast, CB(2) receptor stimulation attenuated morphine-induced microglial proinflammatory mediator increases, interfering with morphine action by acting on the Akt-ERK1/2 signalling pathway.Conclusions and Implications: Because glial activation opposes opioid analgesia and enhances opioid tolerance and dependence, we suggest that CB(2) receptors, by inhibiting microglial activity, may be potential targets to increase clinical efficacy of opioids. [ABSTRACT FROM AUTHOR]

Summary: Objective: The aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X1 and P2X3 purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE2) release. Methods: Saturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X1 and P2X3 purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X1 and P2X3 purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE2 release) the potency of purinergic agonists and antagonists was evaluated. Results: Bovine chondrocytes expressed P2X1 and P2X3 purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5′-triphosphate (ATP) and α,β-methyleneATP were able to increase NO and PGE2 release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists. Conclusions: These data demonstrate for the first time the presence of functional P2X1 and P2X3 purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE2 release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption. [Copyright &y& Elsevier]

RATIONALE: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality worldwide. Adenosine is an inflammatory regulator that acts through four distinct receptors to mediate pro- and antiinflammatory effects. OBJECTIVES: The primary aim of this study was to investigate the expression, affinity, and density of adenosine receptors in peripheral lung parenchyma from age-matched smokers with COPD (n = 14) and smokers with normal lung function (control group; n = 20). METHODS: Adenosine receptors were analyzed by immunohistochemistry and saturation binding assays using typical antagonist radioligands. RESULTS: A(1), A(2A), A(2B), and A(3) receptors were expressed in different cells in peripheral lung parenchyma. The affinity of A(1), A(2A), and A(3) receptors was significantly decreased in patients with COPD compared with the control group (K(D)[A(1)] = 3.15 +/- 0.19 vs. 1.70 +/- 0.14 nM; K(D)[A(2A)] = 7.88 +/- 0.68 vs. 1.87 +/- 0.09 nM; K(D)[A(3)] = 9.34 +/- 0.27 vs. 4.41 +/- 0.25 nM; p < 0.01), whereas their density was increased (Bmax[A(1)] = 53 +/- 4 vs. 32 +/- 3 fmol/mg protein; Bmax[A(2A)] = 852 +/- 50 vs. 302 +/- 12 fmol/mg protein; Bmax[A(3)] = 2,078 +/- 108 vs. 770 +/- 34 fmol/mg protein; p < 0.01). The affinity of A(2B) receptors was not altered, but the density was significantly decreased in patients with COPD compared with the control group (Bmax = 66 +/- 5 vs. 189 +/- 16 fmol/mg protein; p < 0.01). A significant correlation was found between the affinity and density of the adenosine receptors and the FEV(1)/FVC ratio. CONCLUSIONS: This is the first report showing the presence of adenosine receptors in lung parenchyma in subjects with COPD compared with control smokers. These novel findings strengthen the hypothesis of a potential role played by adenosine receptors in the pathogenesis of COPD. [ABSTRACT FROM AUTHOR]

Caffeine is the most widely used drug in the world and acts mainly through antagonism of the effects mediated by the adenosine receptor subtypes A1, A2A, A2B and A3. We determined whether repeated caffeine administration at different doses and for different periods of time (400 or 600 mg/day for 1 week and 400 mg/day for 2 weeks) alters human neutrophil A2A adenosine receptor density and function. Saturation binding assays showed an increase in affinity (KD) and density (Bmax) of A2A adenosine receptors after caffeine intake. These changes were accompanied by increases in cAMP accumulation and decreases in superoxide anion production after stimulation of the A2A receptor subtype using the agonist 5′-N-ethylcarboxamidoadenosine (NECA). Binding and functional changes of A2A receptors returned to baseline after 48 h of caffeine withdrawal. The findings are consistent with a potential anti-inflammatory effect of caffeine mediated by neutrophil A2A receptors. [ABSTRACT FROM AUTHOR]

Two series of nociceptin (NC)-related peptides with or without replacement of the N-terminal Phe by Tyr have been investigated in an attempt to obtain compounds that interact with the NC receptor (ORL1) and classic opioid receptors. When tested for their ability to displace [3H]NCNH2 ([3H]nociceptin amide; ORL1 sites) or the selective opioid receptor ligands [3H]DAMGO (µ), [3H]deltorphin II (δ) and [3H]U69593 (κ) from their respective binding sites in guinea-pig brain membranes, [Tyr1]NCNH2 and [Tyr1]NC(1–13)NH2 showed high affinities ( K i 2 nM and 5 nM, respectively) for ORL1 and approximately tenfold lower potency for µ (32 nM and 44 nM) and κ sites (42 nM and 48 nM). They also interacted, but with low potency ( K i 410 nM and 310 nM) with δ sites. Shorter fragments as [Tyr1]NC(1–9)NH2 and [Tyr1]NC(1–5)NH2 were found to be inactive on ORL1, δ and κ sites, and extremely weak ( K i 2224 nM and 4228 nM, respectively) on µ. Results of bioassays performed on the guinea-pig ileum (ORL1 and µ receptors), mouse vas deferens (ORL1 and δ receptors), and rabbit vas deferens (κ receptor) confirmed (at least partially) the data of the binding by showing that [Tyr1]NC analogs interact with functional ORL1 as well as with classic opioid receptors. [Tyr1]NCNH2 and [Tyr1]NC(1–13)NH2 behaved as mixed ORL1/opioid receptor agonists showing similar affinities as the control NC sequence while [Tyr1]NC(1–9)NH2 and [Tyr1]NC(1–5)NH2 were inactive on ORL1 receptors but maintained some activities on opioid receptors: their effects were prevented by naloxone. The results of this study indicate that the replacement of Phe1 by Tyr in NC leads to compounds which bind both the ORL1 and µ/κ receptors and may represent new promising agents for use in peripheral organs. [ABSTRACT FROM AUTHOR]