Extracellular ATP released from stimulated and/or damaged cells modulates physiological responses via stimulation of various purinoceptors. We previously showed that ATP potentiated the Ag-induced mast cell (MC) degranulation via purinoceptors pharmacologically similar to the ionotropic P2X4 receptor. In this study, we investigated the role of P2X4 receptor in MC degranulation induced by stimulation of IgE-Fc«RI complex with Ag, using bone marrow-derived MCs (BMMCs) prepared from wild type and P2X4 receptor-deficient (P2rx4 2/2 ) mice. ATP significantly increased Ag-induced degranulation in BMMCs prepared from wild type mice. This effect of ATP was reduced in BMMCs prepared from P2rx4 2/2 mice. The potentiating effect of ATP was restored by expressing P2X4 receptor in P2rx4 2/2 BMMCs. The P2X4 receptor-mediated effects were maintained even after differentiating into the connective tissue-type MCs. P2X4 receptor stimulation did not affect the Ag-induced Ca 2+ response but enhanced Ag-induced early signals, such as tyrosine phosphorylation of Syk and phospholipase C-g. Interestingly, these effects of ATP on Syk phosphorylation were not impaired by pretreatment with Cu 2+ , an inhibitor of the P2X4 receptor channel, or removal of external Ca 2+ , suggesting that a mechanisms other than Ca 2+ influx through ion channel activity may be involved. In vivo experiments revealed that systemic and intradermal passive anaphylaxis responses were significantly alleviated in P2rx4 2/2 mice. Taken together, the present data suggest that the P2X4 receptor plays an essential role in ATP-induced upregulation of MC degranulation in response to Ag, and also contributes to the Ag-induced allergic response in vivo.

Aldosterone induces extracellular signal-regulated kinase (ERK)-dependent cardiac remodeling. Fenofibrate improves cardiac remodeling in adult rat ventricular myocytes (ARVM) partly via inhibition of aldosterone-induced ERK1/2 phosphorylation and inhibition of matrix metalloproteinases. We sought to determine whether aldosterone caused apoptosis in cultured ARVM and whether fenofibrate ameliorated the apoptosis. Aldosterone (1 microM) induced apoptosis by increasing terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive nuclei in ARVM. Spironolactone (100 nM), an aldosterone receptor antagonist, but not RU-486, a glucocorticoid receptor, inhibited aldosterone-mediated apoptosis, indicating that the mineralocorticoid receptor (MR) plays a role. SP-600125 (3 microM)-a selective inhibitor of c-Jun NH(2)-terminal kinase (JNK)-inhibited aldosterone-induced apoptosis in ARVM. Although aldosterone increased the expression of both stress-activated protein kinases, pretreatment with fenofibrate (10 microM) decreased aldosterone-mediated apoptosis by inhibiting only JNK phosphorylation and the aldosterone-induced increases in Bax, p53, and cleaved caspase-3 and decreases in Bcl-2 protein expression in ARVM. In vivo studies demonstrated that chronic fenofibrate (100 mg*kg body wt(-1)*day(-1)) inhibited myocardial Bax and increased Bcl-2 expression in aldosterone-induced cardiac hypertrophy. Similarly, eplerenone, a selective MR inhibitor, used in chronic pressure-overload ascending aortic constriction inhibited myocardial Bax expression but had no effect on Bcl-2 expression. Therefore, involvement of JNK MAPK-dependent mitochondrial death pathway mediates ARVM aldosterone-induced apoptosis and is inhibited by fenofibrate, a peroxisome proliferator-activated receptor (PPAR)alpha ligand. Fenofibrate mediates beneficial effects in cardiac remodeling by inhibiting programmed cell death and the stress-activated kinases.

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