Study on the activation of phospholipases A(2) by purinergic agonists in rat submandibular ductal cells

Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of [H-3]ar achidonic acid ([H-3]AA) from prelabeled rat submandibular gland (RSMG) duc tal cells respectively two- and threefold. Both agonists also increased the release of [H-3]AA from acini but at a lower level (+50% and +100% respect ively). Carbachol had no significant effect on either cellular population. In ductal cells phorbol myristate acetate, an activator of protein kinase C , slightly increased the basal release of [H-3]AA but did not affect the re lease of [H-3]AA in response to ATP. Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines. The removal of calcium fro m the extracellular medium decreased the response to ATP and Bz-ATP. Only b arium could partly substitute for calcium to restore the purinergic respons e. Zinc inhibited the release of [H-3]AA. Permeabilization of the cells wit h streptolysin O (SLO) activated the calcium-independent phospholipase A(2) activity (iPLA(2)). The iPLA(2), not the calcium-dependent PLA(2) (cPLA(2) ), released [H-3]oleic acid ([H-3]OA) from RSMG ductal cells. It is conclud ed that RSMG ducts have a higher PLA2 activity when compared to acini. This activity is accounted for by iPLA(2) and cPLA(2). Both enzymes are activat ed by P2X agonists by a staurosporine-sensitive mechanism. Cells permeabili zed with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes. In intact RSMG ductal cel ls the two activities can be distinguished by rather specific inhibitors, b y different ionic conditions and also by the fatty acid used to label the c ells. (C) 1999 Elsevier Science B.V. All rights reserved.