ACTIVATION BY P2X(7) AGONISTS OF 2 PHOSPHOLIPASES-A(2) (PLA(2)) IN DUCTAL CELLS OF RAT SUBMANDIBULAR-GLAND - COUPLING OF THE CALCIUM-INDEPENDENT PLA(2) WITH KALLIKREIN SECRETION

Isolated ductal cells of rat submandibular gland phospholipid pools we re labeled with [H-3]arachidonic acid (AA). The tracer was incorporate d 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'-triph osphate (Bz-ATP) was able to mimic the effect of ATP (EC50 = 15 mu M), without activation of phospholipase C. The purinergic antagonists oxi dized ATP, suramin, and Coomassie Blue partly inhibited the response t o 1 mM ATP and 100 mu M Bz-ATP; the response was also blocked by the a ddition of Mg2+ or Ni2+. Expression of P2X(7) receptor mRNA in these c ells was confirmed by reverse transcription-polymerase chain reaction. In the presence of extracellular calcium, the phospholipase A(2) inhi bitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (a nonspecific in hibitor), arachidonyl trifluoromethylketone (AACOCF(3), an inhibitor o f the calcium-dependent cytosolic PLA(2) (cPLA(2))), and bromoenol lac tone tan inhibitor of the calcium-independent PLA(2) (iPLA(2))) inhibi ted the release of [H-3]AA induced by ATP and Bz-ATP. In the absence o f extracellular calcium, the release of [H-3]AA in response to the pur inergic 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 conclude d that the P2X(7) receptor in ductal cells is coupled to kallikrein se cretion through a calcium-dependent cPLA(2) and a calcium-independent iPLA(2).