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 alpha-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 P2Y(4) 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 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 [Ca2+](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. (C) 2015 Elsevier Ltd. All rights reserved.