SUSTAINED ACTIVATION OF PHOSPHOLIPASE-D VIA ADENOSINE A(3) RECEPTORS IS ASSOCIATED WITH ENHANCEMENT OF ANTIGEN-IONOPHORE-INDUCED AND CA2-IONOPHORE-INDUCED SECRETION IN A RAT MAST-CELL LINE()

The adenosine analog, N-ethylcarboxamidoadenosine (NECA), causes trans ient activation of phospholipase C and an enhancement of antigen-induc ed secretion in a rat mast cell (RBL-2H3) line via adenosine A(3)-rece ptors (Ramkumar et al., J. Biol. Chem. 268:16887, 1993) by a mechanism that is inhibited by bacterial toxins and potentiated by dexamethason e (Ali et al., J. Biol. Chem. 265:745-753, 1990). Here we show that NE CA synergizes the secretory response to Ca2+-ionophore as well as to a ntigen. The ability of NECA to synergize the secretory responses persi sted for 10 to 20 min, long after the early phospholipase C-mediated r eactions to NECA had subsided. NECA caused, however, a dose-dependent sustained activation of phospholipase D, as indicated by the formation of [H-3]phosphatidic acid, or in the presence of 0.3% ethanol, [H-3]p hosphatidylethanol. This activation was associated with a sustained in crease in diglycerides, in protein kinase C activity and in the phosph orylation of myosin light chains by protein kinase C. The generation o f diglycerides was enhanced in dexamethasone-treated cells and suppres sed in cells that had been treated with cholera toxin or pertussis tox in. Collectively, the studies suggested that the generation of diglyce rides via phospholipase D and the associated activation of protein kin ase C were, by themselves, insufficient signals for secretion in RBL-2 H3 cells, but that these reactions synergized responses to stimulants such as antigen or A23187 that caused substantial increases in [Ca2+]( i).