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* Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain
Department of Animal Biology and Genetics, Faculty of Medicine, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain
Faculty of Sciences, and Department of Neuroscience, Faculty of Medicine, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain
** Laboratoire de Biochimie et de Biologie Cellulaire, C.P. 205/3, Université Libre de Bruxelles, Boulevard du Triomphe, B1050 Bruxelles, Belgium
1 To whom correspondence should be addressed. e-mail: gbpgomua{at}lg.ehu.es
Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. PLD activation was dependent upon extracellular Ca2+ and did not involve intracellular Ca2+ 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 A2 (PLA2) inhibitor ONO-RS-082 and partially attenuated by the selective Ca2+-dependent cytosolic PLA2 inhibitor, arachidonyl trifluoromethylketone (AACOCF3), or by bromoenol lactone, an inhibitor of Ca2+-independent cytosolic PLA2. Magnesium, which decreases the concentration of ATP4-, 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/P2X7 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/P2X7 receptor antagonist.
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. Also, the data suggest an involvement of P2X7 receptors in PLD-2 stimulation by ATP.
Abbreviations: AACOCF3, arachidonyl trifluoromethylketone; BEL, (E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (bromoenol lactone); Bz-ATP, 2',3'-O-(benzoyl-benzoyl)adenosine 5'-triphosphate; cPLA2, calcium-dependent cytosolic phospholipase A2; DAG, diacylglycerol; HBS, HEPES-buffered saline; iPLA2, calcium-independent cytosolic phospholipase A2; PA, phosphatidic acid; PC, phosphatidylcholine; PI-PLC, phosphatidylinositol-specific phospholipase C; PKC, protein kinase C; PLD, phospholipase D; PMA, 4ß-phorbol 12-myristate 13 acetate; RT-PCR, reverse transcription-polymerase chain reaction; SMG, submandibular gland
Supplementary key words ATP calcium ceramides phospholipase A2 phospholipase C phospholipase D protein kinase C purinergic receptors sphingosine 1-phosphate
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