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Journal of Lipid Research, Vol. 43, 1244-1255, August 2002
Copyright © 2002 by Lipid Research, Inc.

Activation of phospholipase D-2 by P2X₇ agonists in rat submandibular gland acini

Encarnación Pérez-Andrés^*, María Fernández-Rodriguez^*, Mónica González^*, Ana Zubiaga, Ainara Vallejo, Itxaso García, Carlos Matute, Stéphanie Pochet^**, Jean Paul Dehaye^**, Miguel Trueba^*, Aida Marino^* and Antonio Gómez-Muñoz^1,*

^* 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 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/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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