Activation of phospholipase D-2 by P2X(7) agonists in rat submandibular gland acini.

Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. PLD activation was dependent upon extracellular Ca 2 + and did not involve intracellular Ca 2 + mobilizationor phosphoinositide-specific phospholipase C activation. ATP-stimulated PLD was attenuated by inhibition or downregulation of protein kinase C (PKC). PLD activation was fully blocked by the cytosolic phospholipase A 2 (PLA 2 ) inhibitor ONO-RS-082 and partially attenuated by the selective Ca 2 + -dependent cytosolic PLAT inhibitor, arachidonyl trifluoromethylketone (AACOCF 3 ), or by bromoenol lactone, an inhibitor of Ca 2 + -independent cytosolic PLA 2 . Magnesium, which decreases the concentration of ATP 4 - , and nickel, which blocks nonspecific cation channels coupled to purinergic receptors, inhibited PLD activation by ATP. Using reverse transcription-polymerase chain reaction and Northern blotting techniques, we demonstrated that the PLD isoform stimulated by ATP was PLD-2. Among various ATP analogs, only the P2Z/P2X 7 purinergic receptor agonist benzoyl-benzoyl ATP stimulated PLD-2. The response to ATP was inhibited by the nonselective P2X purinergic antagonist suramin and by oxidized ATP, a potent P2Z/P2X 7 receptor antagonist. It is concluded that in rat SMG acinar cells, PLD-2 is upregulated by exogenous ATP through a mechanism involving Ca 2 + influx, cytosolic PLA 2 , and PKC. Also, the data suggest an involvement of P2X 7 receptors in PLD-2 stimulation by ATP.