Arachidonic acid cycloxygenase and lipoxygenase pathways are differently activated by platelet activating factor and the calcium-ionophore A23187 in a primary culture of astroglial cells

The aim of our study has been to investigate the metabolism of endogenous arachidonic acid or that of radiolabeled arachidonate in astroglial cells, stimulated with platelet activating factor (PAF) and with the calcium-ionphore A23187. Primary cultures of astroglial cells were obtained from brain cortex of one-day-old rats and were characterized by immunofluorescent staining vs glial fibrillary acidic protein. In labeled cells, diacylglycerol was formed after stimulation with platelet activating factor, whereas mainly the release of labeled arachidonic acid from phospholipids was observed after stimulation with calcium-ionophore. Both PAF and the calcium-ionophore A23187 actively stimulated the formation of the cycloxygenase products PGD2, TXB2 and 6-keto-PGF1 alpha, measured by radio- or enzyme-immunoassay. Differences were observed, instead, in the formation of the lipoxygenase metabolites, the hydroxyeicosateraenoic acids, which were measured by high pressure liquid chromatography (HPLC) with on line radiodetection for the labeled products, and Leukotriene C4, measured by radioimmunoassay. The formation of hydroxyacids by stimulated cells was confirmed by gas chromatography-mass spectrometry (GC-MS). In labeled cells, both agonists induced the formation of 12- and 15-hydroxyeicosatetraenoic acids, whereas stimulation of unlabeled cells with calcium ionophore resulted in formation of 12-hydroxyeicosatetraenoic acid and Leukotriene C4. Our results suggest that in astroglial cells, PAF, a compound which is produced in several tissues including brain, mobilizes a selected arachidonic acid pool, possibly associated with diacylglycerol production, from phospholipids, thus activating the conversion of the released fatty acid via the cyclo and the 12-lipoxygenase pathways.