Studies on the cytosolic phospholipase A2 in immortalized astrocytes (DITNC) revealed new properties of the calcium ionophore, A23187.
Besides playing an important role in the maintenance of cell membrane phospholipids, phospholipases A2 (PLA2) are responsible for the release of arachidonic acid (AA) which is a precursor for prostaglandin biosynthesis. The cytosolic PLA2 has been the focus of recent studies, probably due to its ability to respond to protein kinases and changes in intracellular calcium levels. In this study, we examined agents for stimulation of the cytosolic phospholipase A2 in immortalized astrocytes (DITNC). Incubation of DITNC cells with [14C]arachidonic acid (AA) resulted in a time-dependent uptake of the label into phospholipids (PL) and neutral glycerides. In prelabeled cells, release of labeled AA could be stimulated by calcium mobilizing agents such as calcium ionophore A23187 (4-20 microM) and thimerosal (100 microM), and by phorbol myristate acetate (PMA, 100 nM), an agent for activation of protein kinase C. The release of AA could also be stimulated by ATP (200 microM), probably through activation of the purinergic receptor but not by glutamate (1 mM). The stimulated release of AA was dependent on extracellular Ca2+ and was inhibited by mepacrine (50 microM), a non-specific PLA2 inhibitor. Western blot analysis further confirmed the presence of an 85 kDa cPLA2 in both membrane and cytosol fractions of these cells and stimulation by A23187 resulted in translocation of this protein to the membrane fraction. Besides labeled fatty acids, A23187 also stimulated the concomitant release of labeled PL into the culture medium and this event was accompanied by the increased release in lactate dehydrogenase (LDH). Results thus revealed that besides activation of cPLA2, the calcium ionophore A23187 is capable of perturbating cell membrane integrity.
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Biochemistry Department and Nutritional Sciences Program, University of Missouri, Columbia 65212, USA.
Xu
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McGuire
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Devitre
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Sun
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D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001253:Astrocytes / Q000187:drug effects* / Q000201:enzymology; D001692:Biological Transport; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D002461:Cell Line, Transformed; D003470:Culture Media; D003600:Cytosol / Q000187:drug effects* / Q000201:enzymology; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D010738:Type C Phospholipases / Q000378:metabolism*
D002250:Carbon Radioisotopes; D003470:Culture Media; D010743:Phospholipids; D016718:Arachidonic Acid; D000001:Calcimycin; D010738:Type C Phospholipases