PHARMACOLOGICAL CHARACTERIZATION OF P2X1 AND P2X3 PURINERGIC RECEPTORS IN BOVINE CHONDROCYTES AND FIBROBLAST-LIKE SYNOVIOCYTES

Extracellular nucleotides, acting through purinergic receptors, play a central role in the regulation of bone and cartilage metabolism. Chondrocytes within articular cartilage are responsible for the synthesis and degradation of the extracellular matrix, which in healthy tissue provide to mechanical functionality. Fibroblast-like synoviocytes are closely involved in the pathogenesis of joint destruction by an increased proliferation and by the secretion of a wide range of pro-inflammatory mediators including cytokines, growth factors and lipid mediators. The aim of the present study is that of characterizing from a biochemical, physico chemical and functional point of view P2X1 and P2X3 purinergic receptors in bovine chondrocytes and fibroblast-like synoviocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating cell activities was investigated by analyzing NO and PGE2 release. Saturation, competition binding experiments and western blotting assays on the P2X1 and P2X3 purinergic receptors in bovine chondrocytes and fibroblast-like synoviocytes 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. Bovine chondrocytes and fibroblast-like synoviocytes 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 ATP and ,-methyleneATP were able to increase NO and PGE2 release. These data demonstrate the presence of functional P2X1 and P2X3 purinergic receptors in bovine chondrocytes and in fibroblast-like synoviocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate the functional responses. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases involving the articular cartilage resorption and the joint inflammatory diseases.