Abstract
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+) mobilization or 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 PLA(2) 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.
Highlights
Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini
We found that concentrations of ATP that are able to activate PLD failed to stimulate the generation of IP3 or the mobilization of intracellular Ca2ϩ, protein kinase C (PKC) may still be involved in PLD activation through mechanisms that are independent of Phosphatidylinositol-specific phospholipase C (PI-phospholipase C (PLC))
It was reported [50] that, unlike PLD-1, the activity of PLD-2 is regulated by cytosolic phospholipase A2 (PLA2), and we recently demonstrated that ATP stimulates both calcium-dependent cytosolic phospholipase A2 and calcium-independent cytosolic phospholipase A2 activities [8]
Summary
Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. ATP-stimulated PLD was attenuated by inhibition or downregulation of protein kinase C (PKC). Magnesium, which decreases the concentration of ATP4؊, and nickel, which blocks nonspecific cation channels coupled to purinergic receptors, inhibited PLD activation by ATP. Among various ATP analogs, only the P2Z/P2X7 purinergic receptor agonist benzoyl-benzoyl ATP stimulated PLD-2. It is concluded that in rat SMG acinar cells, PLD-2 is upregulated by exogenous ATP through a mechanism involving Ca2؉ influx, cytosolic PLA2, and PKC. The data suggest an involvement of P2X7 receptors in PLD-2 stimulation by ATP.—PérezAndrés, E., M. Activation of phospholipase D-2 by P2X7 agonists in rat submandibular gland acini.
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