Anales RANF

P.93 ADENOSINE A2A RECEPTOR ACTIVATION PROMOTES FIBROSIS BY FAVORING PANNEXIN-1 OVEREXPRESSION IN HUMAN SUBCUTANEOUS FIBROBLASTS C. Herman-de-Sousa 1 , A.R. Pinheiro 1,2 , D. Paramos-de-Carvalho 1 , M.A. Costa 1 , M.T. Magalhães-Cardoso 1 , F. Ferreirinha 1 , S Ribeiro 3 & Paulo Correia-de-Sá 1,2 1 Laboratório de Farmacologia e Neurobiologia, MedInUP, Instituto de Ciências Biomédicas Abel Salazar – Universidade do Porto (ICBAS-UP), 2 Área Técnico- Científica de Fisioterapia, Escola Superior de Tecnologia da Saúde do Instituto Politécnico do Porto (ESTSP-IPP), and 3 Serviço de Urologia, Centro Hospitalar do Porto (CHP), Porto, Portugal. Hemichannels containing pannexin-1 (Panx1) and connexin43 (Cx43) have an important role in fibrogenesis associated with chronic diseases (Cronstein & Sitkovsky, 2017, Nature Rev Rheumatol. 13:41; Cogliati et al., 2016, J Membr Biol. 249:199). The adenosine A 2A receptor (A2AR) has a pro-fibrotic role in human subcutaneous tissue fibroblasts (Pinheiro, 2014, PhD Thesis (ICBAS) University of Porto). Here, we set out to investigate if there is a link between A2AR activation and the expression of hemichannels containing Panx1 and Cx3 in human subcutaneous fibroblasts. Human fibroblasts were isolated from the subcutaneous tissue of organ donors with no clinical history of connective tissue disorders after appropriate Ethical approval. We analyzed the expression of A2AR, Panx-1 and Cx43 in human subcutaneous fibroblasts by immunofluorescence confocal microscopy and investigated their role in cell proliferation (MTT test) and type I collagen production (Sirius Red assay) by cells kept in culture for 28 days (first subculture). Prolonged exposure (during 28 days) of human subcutaneous fibroblasts to adenosine or its stable analogues, NECA (300 nM) and CGS 21680 (10 nM), significantly increased type I collagen production. Blockade of Panx1 hemichannels with probenecid (100µM) or carbenoxolone (300 µM) decreased type I collagen production by 30-60% compared to the control situation. Panx1 immunoreactivity of cultured human subcutaneous fibroblasts increased significantly in the presence of NECA (300 nM); an effect that was fully prevented by the selective A2AR antagonist, SCH 442416 (10 nM). Conversely, NECA (300 nM) reduced Cx43 immunoreactivity in these cells. Data show here for the first time that adenosine A2AR activation causes Panx1 overexpression in human subcutaneous fibroblasts, while decreasing the amount of Cx43 hemichannels. Work supported by FCT (FEDER funding, project UID/BIM/4308/2016). CHS is in receipt of a PhD Studentship from NORTE2020 (NORTE-69-2015-15).