1 Rat ventricular myocytes were dissociated and their responses to extracellularly applied ATP were recorded using patch pipettes under the whole cell configuration. 2 ATP initially induced an inward current followed by an outward current at -50 mV. With a Cs-rich pipette solution the late outward current was blocked, leaving a sustained inward current (IATP) suggesting that a K' conductance underlies the late response.3 When the extracellular Cl-concentration was changed, the reversal potential of IATPS corresponded well to the shift of the Cl-equilibrium potential. IATN was reversibly blocked by the chloride channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS).4 The concentration-response curve of IATh had a Hill coefficient of 0.98 and an ECM value of 5.2x 106M. 5 ATP was more potent than ADP, while AMP and adenosine were ineffective, suggesting that P2-purinoceptor activation induced IATPS* 6 The activation of IATPS was depressed by depleting the extracellular Mg2' and increased by adding 7 Our results strongly suggest that P2-purinoceptor activation by ATP induces both a Cl--conductance (IATPS) and a K+-conductance in rat ventricular myocytes.
Proliferation and migration of cardiac fibroblasts are important in early stage of wound-healing after myocardial infarction. The effects of tumstatin, a cleaved fragment of collagen type IV α3 chain, on these functions of cardiac fibroblasts have not been clarified. In this study, we examined it by using T3 peptide, an active fragment of tumstatin. Cardiac fibroblasts were isolated from ventricles of adult male Wistar rats. Proliferation was examined by a cell counting assay. Boyden chamber assay was performed to examine migration. Expression and phosphorylation of proteins were determined by Western blotting. T3 peptide (300 ng/ml, 24 h) significantly increased proliferation and migration of cardiac fibroblasts. T3 peptide (300 ng/ml, 30 min) significantly increased Akt (Ser473) phosphorylation. LY294002 (10 μM, 30 min pretreatment), a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, significantly inhibited the T3 peptide-induced proliferation, migration, and activation of Akt signaling pathway in cardiac fibroblasts. Cilengitide, an inhibitor of integrin αβ/αβ, suppressed Akt phosphorylation and proliferation of cardiac fibroblasts. Expression of tumstatin decreased in the infarcted area of rat model of myocardial infarction. We for the first time demonstrated that T3 peptide stimulates proliferation and migration at least partly through the activation of PI3K/Akt signaling pathway via binding integrin αβ/αβ in adult rat cardiac fibroblasts. These results indicate that tumstatin promotes the initial stage of wound-healing through activation of cardiac fibroblasts after myocardial infarction.
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