Objective: The aim of this study was to characterize adenosine receptors in synovial fibroblasts (SFs) and to investigate the potential link between adenosine pathway and pulsed electromagnetic fields (PEMFs). In particular, we articulated our study and we pointed our efforts to: 1) characterize, by a pharmacological point of view, the presence of adenosine receptors subtypes (A1, A2A, A2B and A3) in two cell models: bovine and human SFs; 2) verify the effect of PEMFs on affinity and density parameters of the adenosine receptors characterized ; 3) investigate the functionality of adenosine receptor subtypes in the presence and in the absence of PEMFs through the analysis of cAMP release; 4) investigate if adenosine receptor agonists and PEMF biophysical stimulation, alone or combined, may modulate pro-inflammatory parameters (PGE-2 and IL-6 release; COX-2 expression) in SFs treated with known inflammatory stimuli. Methods: SFs isolated from bovine synovial fluids or from human synovial OA pannus were cultured in monolayer. Western blotting analysis was done to confirm the expression of adenosine receptors in bovine and human SFs. Moreover, competition binding experiments in the absence and in the presence of PEMFs on the adenosine receptors were also performed. In the adenylate cyclase assays, the cAMP levels modulated by typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs were evaluated in both cell types. Further, bovine SFs were treated with TNF-a (10 ng/ml) to activate inflammatory response. Adenosine analogs (CHA for A1 receptors, NECA non-selective agonist, CGS 21680 for A2A receptors and Cl-IB-MECA for A3 receptors) were added to control and TNF-α-treated bovine cultures both in the absence and in the presence of adenosine deaminase (ADA), which is used to deplete endogenous adenosine. Parallel cultures of bovine SFs were exposed to PEMFs (75 Hz, 1.5 mT) during the period in culture (24 hours). PGE-2 release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. In addition, human SFs were treated with f IL-1β (50 ng/ml) to activate inflammatory response. Parallel cultures of human SFs were exposed to PEMFs (75 Hz, 1.5 mT) during the period in culture (24 hours). PGE-2 and IL-6 release was measured by immunoassays. Results: Bovine and human SFs expressed all adenosine receptors (A1, A2A, A2B, A3). PEMFs evoked an up-regulation of A2A and A3 receptors in both cell types. In both PEMF-treated cell models, cAMP levels modulated by A2A or A3 agonists were significantly increased and decreased respectively, when compared with the untreated cells, both in human and in bovine SFs. Further, TNF-α significantly stimulated PGE-2 release in bovine SFs. All adenosine agonists, except for Cl- IB-MECA, significantly inhibited PGE-2 production. PEMFs inhibited PGE-2 production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE-2 release induced by CHA, CGS 21680 and NECA was stronger than in the absence of ADA and the PEMF-inhibitory effect was lost. Changes in PGE-2 levels were associated to a modification of COX-2 expression. To what concern human SFs, IL-1β strongly increased both PGE-2 and IL-6 release. In parallel experiments, PEMF exposure significantly inhibited PGE-2 and IL-6 release. Conclusions: All adenosine receptors are present and have a similar pharmacological behaviour both in bovine and in human SFs. Further, in these cells PEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities. In addition, this study supports anti-inflammatory activities of A1 and A2A adenosine receptors and PEMFs in bovine SFs. PEMF activity appears to be mediated by a PEMF-induced up-regulation of A2A receptors. Finally, PEMF exposure seems exert anti-inflammatory activities in human SFs. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation, and further may open interesting perspectives to develop new therapeutic approaches in osteoarticular pathologies.

NEW INSIGHTS AND POSSIBLE THERAPEUTIC IMPLICATIONS OF ADENOSINE ANALOGS AND PULSED ELECTROMAGNETIC FIELDS (PEMFs) IN OSTEOARTICULAR PATHOLOGIES

MASIERI, Federica Francesca
2009

Abstract

Objective: The aim of this study was to characterize adenosine receptors in synovial fibroblasts (SFs) and to investigate the potential link between adenosine pathway and pulsed electromagnetic fields (PEMFs). In particular, we articulated our study and we pointed our efforts to: 1) characterize, by a pharmacological point of view, the presence of adenosine receptors subtypes (A1, A2A, A2B and A3) in two cell models: bovine and human SFs; 2) verify the effect of PEMFs on affinity and density parameters of the adenosine receptors characterized ; 3) investigate the functionality of adenosine receptor subtypes in the presence and in the absence of PEMFs through the analysis of cAMP release; 4) investigate if adenosine receptor agonists and PEMF biophysical stimulation, alone or combined, may modulate pro-inflammatory parameters (PGE-2 and IL-6 release; COX-2 expression) in SFs treated with known inflammatory stimuli. Methods: SFs isolated from bovine synovial fluids or from human synovial OA pannus were cultured in monolayer. Western blotting analysis was done to confirm the expression of adenosine receptors in bovine and human SFs. Moreover, competition binding experiments in the absence and in the presence of PEMFs on the adenosine receptors were also performed. In the adenylate cyclase assays, the cAMP levels modulated by typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs were evaluated in both cell types. Further, bovine SFs were treated with TNF-a (10 ng/ml) to activate inflammatory response. Adenosine analogs (CHA for A1 receptors, NECA non-selective agonist, CGS 21680 for A2A receptors and Cl-IB-MECA for A3 receptors) were added to control and TNF-α-treated bovine cultures both in the absence and in the presence of adenosine deaminase (ADA), which is used to deplete endogenous adenosine. Parallel cultures of bovine SFs were exposed to PEMFs (75 Hz, 1.5 mT) during the period in culture (24 hours). PGE-2 release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. In addition, human SFs were treated with f IL-1β (50 ng/ml) to activate inflammatory response. Parallel cultures of human SFs were exposed to PEMFs (75 Hz, 1.5 mT) during the period in culture (24 hours). PGE-2 and IL-6 release was measured by immunoassays. Results: Bovine and human SFs expressed all adenosine receptors (A1, A2A, A2B, A3). PEMFs evoked an up-regulation of A2A and A3 receptors in both cell types. In both PEMF-treated cell models, cAMP levels modulated by A2A or A3 agonists were significantly increased and decreased respectively, when compared with the untreated cells, both in human and in bovine SFs. Further, TNF-α significantly stimulated PGE-2 release in bovine SFs. All adenosine agonists, except for Cl- IB-MECA, significantly inhibited PGE-2 production. PEMFs inhibited PGE-2 production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE-2 release induced by CHA, CGS 21680 and NECA was stronger than in the absence of ADA and the PEMF-inhibitory effect was lost. Changes in PGE-2 levels were associated to a modification of COX-2 expression. To what concern human SFs, IL-1β strongly increased both PGE-2 and IL-6 release. In parallel experiments, PEMF exposure significantly inhibited PGE-2 and IL-6 release. Conclusions: All adenosine receptors are present and have a similar pharmacological behaviour both in bovine and in human SFs. Further, in these cells PEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities. In addition, this study supports anti-inflammatory activities of A1 and A2A adenosine receptors and PEMFs in bovine SFs. PEMF activity appears to be mediated by a PEMF-induced up-regulation of A2A receptors. Finally, PEMF exposure seems exert anti-inflammatory activities in human SFs. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation, and further may open interesting perspectives to develop new therapeutic approaches in osteoarticular pathologies.
CARUSO, Angelo
MARTINI, Alessandro
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2388717
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