THAPSIGARGIN-INDUCED SECRETION IS DEPENDENT ON ACTIVATION OF A CHOLERA TOXIN-SENSITIVE AND PHOSPHATIDYLINOSITOL-3-KINASE-REGULATED PHOSPHOLIPASE-D IN A MAST-CELL LINE

Release of secretory granules by rat RBL-2H3 mast cells is mediated pr imarily through activation of protein kinase C (PKC) and elevation of cytosolic free calcium ([Ca++](i)). Here, we show that secretion was a lso dependent on the activation of a cholera toxin-sensitive phospholi pase (PL) D in cells stimulated with thapsigargin. Wortmannin, LY29400 2, butanol, propranolol and Ro31-7549 inhibited responses to variety o f secretagogues in a manner consistent with the notion that secretion was regulated by both PLD and PKC in a phosphatidylinositol-3-kinase-d ependent manner. The effects of these inhibitors, however, were especi ally pronounced in cells activated by thapsigargin. This stimulant ind uced minimal stimulation of PLC but measurable activation of PLD, as a ssessed by formation of phosphatidylethanol in the presence of ethanol . The activation of PLD was suppressed by inhibitors of phosphatidylin ositol-3-kinase and was dependent on a rise in [Ca++](i) because thaps igargin failed to activate PLD and secretion when elevation of [Ca++]( i) was blocked. Treatment of cells with cholera toxin resulted in sele ctive and similar enhancements in the activation of PLD and secretion by thapsigargin, whereas stimulation of PLC and PLA, was unaffected. A role for PKC was indicated by the blockade of secretory response to t hapsigargin by the PKC inhibitor Ro31-7549 and by the ability of the P KC agonist phorbol-12-myristate-13-acetate to reverse the inhibition o f secretion by inhibitors of PLD. Such results suggested that thapsiga rgin, by causing substantial increases in [Ca++](i), induced secondary signals via PLD and PKC that synergized a calcium signal for secretio n.