Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

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 ADPsensitive 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.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

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.

���Papel dos recetores purin��rgicos no crescimento e temodela����o do tecido cavernoso���

A adenosina �� um nucle��sido ub��quo envolvido na regula����o de controlo do t��nus vascular do tecido cavernoso, desempenhando um papel importante na fisiopatologia da Disfun����o Er��til (DE) resistente aos f��rmacos relaxantes musculares cl��ssicos. Apesar da import��ncia comprovada dos recetores da adenosina na fisiopatologia da DE no homem, pouca informa����o �� conhecida no que diz respeito �� express��o e localiza����o dos recetores purin��rgicos no Tecido Cavernoso de Ratazana (TCR). Neste trabalho avaliou-se o fen��tipo dos recetores purin��rgicos respons��veis pela regula����o do t��nus do tecido er��til de ratazana por imunofluoresc��ncia indireta aplicada �� microscopia confocal em co-culturas de c��lulas endoteliais e musculares lisas do TCR. Para al��m da caracteriza����o imunofenot��pica, desenvolveu-se uma t��cnica que permite diferenciar funcionalmente em tempo real (por microscopia confocal funcional) c��lulas musculares lisas e c��lulas endoteliais isoladas de TCR em co-cultura marcadas com a sonda fluorescente Fluo-4NW. Esta t��cnica permite distinguir cada um dos subtipos celulares mediante o padr��o e a magnitude das oscila����es dos n��veis intracelulares de Ca2+ ([Ca2+]i) em resposta ao ATP (agonista P2) e �� fenilefrina (PE, agonista ��-adren��rgico). Nas c��lulas musculares lisas, observou-se uma resposta mais acentuada ao agonista ��-adren��rgico, PE, e uma resposta menos significativa ao ATP. O contr��rio foi observado relativamente ��s c��lulas endoteliais. A incuba����o das c��lulas musculares lisas e endoteliais com ATP (300 ��M) causou um aumento dos n��veis de [Ca2+]i. O efeito do ATP (300 ��M) parece envolver a ativa����o de recetores dos subtipos P2X1 e P2X3 sens��veis ao bloqueio com NF023 (3��M) e A317491 (100 nM), respetivamente. J�� o aumento dos n��veis [Ca2+]i produzido pelo ADP (300 ��M) parece envolver a ativa����o de recetores P2Y1, P2Y12 e P2Y13 mediante o antagonismo produzido pelos antagonistas MRS 2179 (0,3��M), AR-C66096 (0,1 ��M) e MRS 2211 (10��M), respetivamente. Os dois tipos celulares expressam imunorreatividade contra recetores A2A, A2B, P2X1, P2X3, P2Y1, P2Y12 e P2Y13.

Data Extraction Tool to Analyse, Transform and Store Real Data from Electricity Markets

The study of electricity markets operation has been gaining an increasing importance in the last years, as result of the new challenges that the restructuring process produced. Currently, lots of information concerning electricity markets is available, as market operators provide, after a period of confidentiality, data regarding market proposals and transactions. These data can be used as source of knowledge to define realistic scenarios, which are essential for understanding and forecast electricity markets behavior. The development of tools able to extract, transform, store and dynamically update data, is of great importance to go a step further into the comprehension of electricity markets and of the behaviour of the involved entities. In this paper an adaptable tool capable of downloading, parsing and storing data from market operators��� websites is presented, assuring constant updating and reliability of the stored data.

Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts

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 membrane-bound P2 purinoceptors 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 -smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca2+]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca2+]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca2+ in the extracellular fluid. The biphasic [Ca2+]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca2+]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.