PHARMACOLOGICAL CHARACTERIZATION OF PURINE RECEPTORS IN BOVINE AND HUMAN CHONDROCYTES AND FIBROBLAST-LIKE SYNOVIOCYTES.

Chronic inflammation is an important factor in the pathology of different forms of joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Numerous studies indicate the involvement of purines (adenosine and ATP) in several inflammatory processes, suggesting their possible role as mediators in the inflammatory processes involved in joint diseases. This study is based on the pharmacological characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes. The data obtained show that in bovine chondrocytes and fibroblast-like synoviocytes adenosine receptors are all expressed and distributed similarly in both substrates and they have a high affinity and the receptor density varies in the range 30-84 fmol/mg protein. Then we studied the functionality of adenosine receptors by assessing the ability of typical adenosine agonists and antagonists to modulate the release of cAMP. Adenosine agonists (CHA, NECA, CGS 21680 and Cl-IB-MECA) were able to modulate the levels of cAMP in agreement with their values of Ki. In both substrates A2A and A3 receptors were more involved in the modulation of cAMP, as antagonists of these receptors were able to specifically block the effect of the agonists. We subsequently evaluated the ability of adenosine analogues to modulate the release of prostaglandin E2 (PGE2) and expression of cyclooxygenase-2 (COX-2) in bovine fibroblast-like synoviocytes. The results indicate that TNF-α and LPS were able to stimulate the release of PGE2, adenosine agonists (CHA, CGS21680, NECA) with the exception of Cl-IB-MECA inhibits the release of PGE2. Besides the variation in the levels of PGE2 was associated with modification of the expression of COX-2. Finally, the adenosine agonists did not showed a statistically significant modulation of the cellular proliferation in these substrates, even if in fibroblast-like synoviocytes Cl-IB-MECA induced a small reduction in proliferation. We also performed a pharmacological characterization of adenosine receptors in human fibroblast-like synoviocytes. We found that adenosine receptors were present, agonists and antagonists showed affinity in the nanomolar range. In particular A2A and A3 agonist were able to inhibit p38 MAPK and NF-κB pathway and were able to diminish TNF-α and IL-8 production. Thus A2A and A3 receptors were closely associated with a down-regulation of the inflammatory status. To evaluate the role of ATP in chondrocytes and fibroblast-like synoviocytes, we initially evaluated the thermodynamic characteristics (G°, H°, S°) of the binding of human purinergic P2X1 and P2X3 receptors in HEK-293 cells. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonists binding was enthalpy and entropy-driven while antagonists binding was totally entropy-driven. We have also evaluated the same parameters in bovine chondrocytes. Then the capability to modulate the cellular activity, by analyzing the release of nitric oxide (NO) and PGE2 was also performed. The results showed that bovine chondrocytes express both P2X1 and P2X3 receptors. The binding of agonists is enthalpy and entropy-driven and totally entropy-driven for antagonists. Typical agonists such as ATP and αβ-methylene-ATP were able to increase the release of NO and PGE2, the antagonist A317491 was able to block the stimulatory effect mediated by agonists, suggesting the involvement of these receptors in modulating the inflammatory response in bovine chondrocytes. Finally, we evaluated the expression of P2X1 and P2X3 receptors in human fibroblast-like synoviocytes and in SW982 cells (cells derived from human synovial sarcoma) and their possible involvement in modulating the inflammatory response by assessing the activation of NF-kB and the production of TNF -α, IL-6 and PGE2. Interestingly P2X1 and P2X3 receptors were present in both substrates and purinergic agonists and antagonists have a different thermodynamic behavior. From the functional point of view P2X1 receptors exhibit anti-inflammatory effects, by reducing the activation of NF-kB and the release of TNF-α, whereas P2X3 receptors mediate an opposite response. This study characterized for the first time, the presence of adenosine receptors and P2X1 and P2X3 purinergic receptors in chondrocytes and fibroblast-like synoviocytes, the cells most involved in the pathogenesis of several inflammatory joint diseases like OA and RA. This research highlighted the involvement of these receptors in the modulation of the inflammatory response in both cell types. The functionality of these purine receptors suggest the use of selective adenosine and/or purinergic ligands in the treatment of inflammatory diseases. In addition, the expression and the signal transduction system of adenosine and purine receptors in human fibroblast-like synoviocytes highlight the potential pharmacological use of novel purine ligands in disorders associated with inflammation.