title: Regulation of store-operated calcium entry by calcium-independent phospholipase A2 in rat cerebellar astrocytes.
abstract: We have studied store-operated Ca2+ entry (SOCE) in Bergmann glia and granule cell layer astrocytes in acute brain slices of the rat cerebellum, using the Ca2+-sensitive fluorescent dye Fluo-4 and confocal laser scanning microscopy. Astrocytes were identified by their morphology, location, and their Ca2+ response in K+-free solution. Depletion of Ca2+ stores by cyclopiazonic acid (CPA) (20 microM) induced SOCE in both types of astrocyte. A similar Ca2+ influx was elicited by the calmodulin antagonist calmidazolium (CMZ) (1 microM). The SOCE channel blocker 2-aminoethoxy-diphenylborate (2-APB) (100 microM) and the Ca2+ release-activated channel blocker 3,5-bistrifluoromethyl pyrazole derivative (BTP2) (20 microM) suppressed the CPA- and the CMZ-induced Ca2+ influx. Pretreatment of acute slices with the specific Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL) (25 microM) blocked the CPA- and the CMZ-induced Ca2+ influx. The lysophospholipid products of iPLA2, lysophosphatidylcholine (250 nM) and lysophosphatidylinositol (250 nM), but not lysophosphatidic acid (250 nM), induced a BTP2- and 2-APB-sensitive, but BEL-insensitive, Ca2+ influx. CPA or CMZ enhanced the BEL-sensitive enzymatic activity of iPLA2 in cerebellar astrocyte culture. Inhibition of iPLA2 expression by specific antisense oligodeoxynucleotide of iPLA2 reduced the SOCE and the Ca2+ store refilling in cultured astrocytes. Spontaneous Ca2+ oscillations in astrocytes in situ were reduced after inhibiting SOCE channels or iPLA2 activity. The results suggest that the depletion of Ca2+ stores activates iPLA2 to open Ca2+ channels in the plasma membrane by the formation of lysophospholipids in astrocytes, presumably to refill the stores and allow normal Ca2+ signaling.
authors.affiliation: Abteilung für Allgemeine Zoologie, Fachbereich Biologie, Technische Universität Kaiserslautern, D-67653 Kaiserslautern, Germany.
authors.forename: Karthika
authors.initials: K
authors.lastname: Singaravelu
authors.forename: Christian
authors.initials: C
authors.lastname: Lohr
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Deitmer
mesh_terms: D000814:Aniline Compounds; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008246:Lysophospholipids / Q000378:metabolism / Q000494:pharmacology; D018613:Microscopy, Confocal; D009924:Organ Culture Techniques; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D014966:Xanthenes
chemical_list: D000814:Aniline Compounds; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004791:Enzyme Inhibitors; C409648:Fluo 4; D008246:Lysophospholipids; D014966:Xanthenes; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium