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

Isolated ductal cells of rat submandibular gland phospholipid pools were labeled with [H-3]arachidonic acid (AA). The tracer was incorporated preferentially to phosphatidylcholine (46% of the lipidic fraction). Extracellular ATP induced the release of [H-3]AA to the extracellular medium in a time- and dose-dependent manner (EC50 = 220 mu M). 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 mu M), without activation of phospholipase C. The purinergic antagonists oxidized ATP, suramin, and Coomassie Blue partly inhibited the response to 1 mM ATP and 100 mu M Bz-ATP; the response was also blocked by the addition of Mg2+ or Ni2+. Expression of P2X(7) receptor mRNA in these cells was confirmed by reverse transcription-polymerase chain reaction. In the presence of extracellular calcium, the phospholipase A(2) inhibitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (a nonspecific in hibitor), arachidonyl trifluoromethylketone (AACOCF(3), an inhibitor of the calcium-dependent cytosolic PLA(2) (cPLA(2))), and bromoenol lactone tan inhibitor of the calcium-independent PLA(2) (iPLA(2))) inhibited the release of [H-3]AA induced by ATP and Bz-ATP. In the absence of extracellular calcium, the release of [H-3]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF(3) and completely blocked by bromoenol lactone. ATP and Bz-ATP stimulated a calcium-independent secretion of kallikuein, which could be blocked by EEL but which was enhanced by AACOCF(3). It is concluded that the P2X(7) receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA(2) and a calcium-independent iPLA(2).