Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membranebound P2purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and α-smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca 2+ ] i ) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y 4 agonist, MRS4062, caused a fast desensitizing [Ca 2+ ] i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca 2+ in the extracellular fluid. The biphasic [Ca 2+ ] i response to UTP was attenuated respectively by P2Y 4 blockers, like reactive blue-2 and suramin, and by the P2Y 11 antagonist, NF340. UTP and the P2Y 2 receptor agonist MRS2768 increased, whereas the selective P2Y 11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y 11 receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y 2 , P2Y 4 and P2Y 11 receptors. Data indicate that besides P2Y 4 and P2Y 2 receptors which are responsible for UTP-induced [Ca 2+ ] i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y 11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.

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.

Nucleotides released during heart injury affect myocardium electrophysiology and remodeling through P2 purinoceptors activation in cardiac myofibroblasts. ATP and UTP endorse [Ca ] accumulation and growth of DDR-2/α-SMA-expressing myofibroblasts from adult rat ventricles via P2Y and P2Y receptors activation, respectively. Ventricular myofibroblasts also express ADP-sensitive P2Y , P2Y , and P2Y receptors as demonstrated by immunofluorescence confocal microscopy and western blot analysis, but little information exists on ADP effects in these cells. ADP (0.003-3 mM) and its stable analogue, ADPßS (100 μM), caused fast [Ca ] transients originated from thapsigargin-sensitive internal stores, which partially declined to a plateau sustained by capacitative Ca entry through transient receptor potential (TRP) channels inhibited by 2-APB (50 μM) and flufenamic acid (100 μM). Hydrophobic interactions between G -coupled P2Y purinoceptors and TRP channels were suggested by prevention of the ADP-induced [Ca ] plateau following PIP depletion with LiCl (10 mM) and cholesterol removal from lipid rafts with methyl-ß-cyclodextrin (2 mM). ADP [Ca ] transients were insensitive to P2Y , P2Y , and P2Y receptor antagonists, MRS2179 (10μM), AR-C66096 (0.1 μM), and MRS2211 (10μM), respectively, but were attenuated by suramin and reactive blue-2 (100 μM) which also blocked P2Y receptors activation by UTP. Cardiac myofibroblasts growth and type I collagen production were favored upon activation of MRS2179-sensitive P2Y receptors with ADP or ADPßS (30 μM). In conclusion, ADP exerts a dual role on ventricular myofibroblasts: [Ca ] transients are mediated by fast-desensitizing P2Y receptors, whereas the pro-fibrotic effect of ADP involves the P2Y receptor activation. Data also show that ADP-induced capacitative Ca influx depends on phospholipase C-linked TRP channels opening in lipid raft microdomains. J. Cell. Physiol. 232: 1511-1526, 2017. © 2016 Wiley Periodicals, Inc.

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