title: Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation.
abstract: BACKGROUND
  Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts.
  
  
  RESULTS
  Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor.
  
  
  CONCLUSIONS
  Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.
authors.affiliation: Laboratório de Farmacologia e Neurobiologia, Unidade Multidisciplinar de Investigação Biomédica (UMIB), Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira 228, Edif, 2 Piso 4, Porto 4050-313, Portugal. farmacol@icbas.up.pt.
authors.forename: Ana Rita
authors.initials: AR
authors.lastname: Pinheiro
authors.forename: Diogo
authors.initials: D
authors.lastname: Paramos-de-Carvalho
authors.forename: Mariana
authors.initials: M
authors.lastname: Certal
authors.forename: Cristina
authors.initials: C
authors.lastname: Costa
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Magalhães-Cardoso
authors.forename: Fátima
authors.initials: F
authors.lastname: Ferreirinha
authors.forename: Maria Adelina
authors.initials: MA
authors.lastname: Costa
authors.forename: Paulo
authors.initials: P
authors.lastname: Correia-de-Sá
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001920:Bradykinin / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D017630:Connexins / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism; D043782:Receptor, Bradykinin B2 / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12 / Q000378:metabolism*
chemical_list: D018031:Connexin 43; D017630:Connexins; D009419:Nerve Tissue Proteins; C548576:P2RY12 protein, human; C492764:PANX1 protein, human; D043782:Receptor, Bradykinin B2; D058925:Receptors, Purinergic P2Y12; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001920:Bradykinin
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