Abstract
BackgroundChronic 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.ResultsBradykinin, 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.ConclusionsBradykinin 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.
Highlights
Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin
Taking into consideration that (1) changes in the regulation of connective tissue adenosine 5’triphosphate sodium salt (ATP) signaling may be important in the pathogenesis of chronic inflammatory pain [18] and that (2) algogenic inflammatory mediators, such as bradykinin, may sensitize cells to autocrine and paracrine signals operated by extracellular adenine nucleotides, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts
Characterization of human fibroblast cells in culture Cultured cells obtained from human subcutaneous connective tissue through the explant technique are elongated and exhibit a spindle-shape morphology, which is characteristic of fibroblasts [20]
Summary
Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. We investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Despite its overwhelming size throughout the body, the connective tissue has been generally overlooked or misunderstood. It has been considered as relatively superfluous apart from its supporting role amongst more specialized tissues [1]. The normal response to mechanical stretch may be dampened by disturbance of the viscoelastic properties of the subcutaneous connective tissue as a consequence of fibroblast remodeling promoted by inflammatory mediators, like neurotrophins, cytokines, peptides, protons, free radicals, histamine, bradykinin, serotonin, and prostanoids [5]. Since the subcutaneous connective tissue is richly innervated by sensory nerve endings, inputs arising from affected connective tissue may alter pain perception
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