Extracellular ATP-mediated phospholipase A(2) activation in rat thyroid FRTL-5 cells: Regulation by a G(i)/G(o) protein, Ca2+, and mitogen-activated protein kinase

We investigated the mechanism of phospholipase A(2) (PLA(2)) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PL A(2) activity was determined by measuring the release of [H-3]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA r elease. This release was totally inhibited by pertussis toxin (PTX) treatme nt, indicating the involvement of a G(i)/C-o protein. The AA release was al so diminished by chelating extracellular Ca2+ with EGTA or by inhibiting in flux of Ca2+ using Ni2+. Although the activation of protein kinase C (PKC) by 12-phorbol 13-myristate acetate (PMA) alone did not induce any AA releas e, the ATP-evoked AA release was significantly reduced when PKC was inhibit ed by CF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kin ase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibito r PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-media ted activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results sugg est that these kinases are involved in the regulation of MAP kinase and PLA (2) activation. Elevation of intracellular cAMP by TSH or by dBucAMP did no t induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evok ed AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activat ion of PLA(2) by a G(i)/G(o) protein-dependent mechanism. Moreover, Ca2+, P KC, MAP kinase; and Src-like kinases are also involved in this regulatory p rocess. (C) 2000 Wiiey-Liss, Inc.