Extracellular ATP-mediated phospholipase a2 activation in rat thyroid FRTL-5 cells: Regulation by a Gi/Go protein, Ca2+, and mitogen-activated protein kinase

Abstract

We investigated the mechanism of phospholipase A2 (PLA2) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA2 activity was determined by measuring the release of [3H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a Gi/Go protein. The AA release was also diminished by chelating extracellular Ca2+ with EGTA or by inhibiting influx 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 release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA2 activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA2 by a Gi/Go protein-dependent mechanism. Moreover, Ca2+, PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process. J. Cell. Physiol. 183:155–162, 2000. © 2000 Wiley-Liss, Inc.