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J Biol Chem, Vol. 273, Issue 46, 30208-30217, November 13, 1998

Activation by P2X₇ Agonists of Two Phospholipases A₂ (PLA₂) in Ductal Cells of Rat Submandibular Gland
COUPLING OF THE CALCIUM-INDEPENDENT PLA₂ WITH KALLIKREIN SECRETION

Eduardo Alzola, Arantza Pérez-Etxebarria, Elie Kabré^**, David J. Fogarty^§§, Mourad Métioui^**, Naima Chaïb^**, José M. Macarulla, Carlos Matute^§§, Jean-Paul Dehaye^**, and Aida Marino

From the  Department of Biochemistry and Molecular Biology, Faculty of Sciences, and ^§§ Department of Neurosciences, Faculty of Medicine and Odontology, University of the Basque Country, 48940 Leioa, Spain, and the ^** Laboratoire de Biochimie Générale et Humaine, Institut de Pharmacie C.P. 205/3, Université Libre de Bruxelles, Boulevard du Triomphe, B1050 Bruxelles, Belgium

Isolated ductal cells of rat submandibular gland phospholipid pools were labeled with [³H]arachidonic acid (AA). The tracer was incorporated preferentially to phosphatidylcholine (46% of the lipidic fraction). Extracellular ATP induced the release of [³H]AA to the extracellular medium in a time- and dose-dependent manner (EC₅₀ = 220 µM). Among other agents tested, only 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) was able to mimic the effect of ATP (EC₅₀ = 15 µM), without activation of phospholipase C. The purinergic antagonists oxidized ATP, suramin, and Coomassie Blue partly inhibited the response to 1 mM ATP and 100 µM Bz-ATP; the response was also blocked by the addition of Mg²⁺ or Ni²⁺. Expression of P2X₇ receptor mRNA in these cells was confirmed by reverse transcription-polymerase chain reaction. In the presence of extracellular calcium, the phospholipase A₂ inhibitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (a nonspecific inhibitor), arachidonyl trifluoromethylketone (AACOCF₃, an inhibitor of the calcium-dependent cytosolic PLA₂ (cPLA₂)), and bromoenol lactone (an inhibitor of the calcium-independent PLA₂ (iPLA₂)) inhibited the release of [³H]AA induced by ATP and Bz-ATP. In the absence of extracellular calcium, the release of [³H]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF₃ and completely blocked by bromoenol lactone. ATP and Bz-ATP stimulated a calcium-independent secretion of kallikrein, which could be blocked by BEL but which was enhanced by AACOCF₃. It is concluded that the P2X₇ receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA₂ and a calcium-independent iPLA₂.

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