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The Journal of Neuroscience, September 13, 2006, 26(37):9579-9592; doi:10.1523/JNEUROSCI.2604-06.2006

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Cellular/Molecular
Regulation of Store-Operated Calcium Entry by Calcium-Independent Phospholipase A₂ in Rat Cerebellar Astrocytes

Karthika Singaravelu, Christian Lohr, and Joachim W. Deitmer

Abteilung für Allgemeine Zoologie, Fachbereich Biologie, Technische Universität Kaiserslautern, D-67653 Kaiserslautern, Germany

Correspondence should be addressed to Dr. Joachim W. Deitmer, Abteilung für Allgemeine Zoologie, Fachbereich Biologie, Technische Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany. Email: deitmer{at}biologie.uni-kl.de

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 µM) induced SOCE in both types of astrocyte. A similar Ca²⁺ influx was elicited by the calmodulin antagonist calmidazolium (CMZ) (1 µM). The SOCE channel blocker 2-aminoethoxy-diphenylborate (2-APB) (100 µM) and the Ca²⁺ release-activated channel blocker 3,5-bistrifluoromethyl pyrazole derivative (BTP2) (20 µM) 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 µM) 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.

Key words: Fluo-4; cyclopiazonic acid; calmidazolium; lysophospholipids; calcium stores; Bergmann glia

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Received Feb. 27, 2006; revised July 28, 2006; accepted Aug. 6, 2006.

Correspondence should be addressed to Dr. Joachim W. Deitmer, Abteilung für Allgemeine Zoologie, Fachbereich Biologie, Technische Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany. Email: deitmer{at}biologie.uni-kl.de

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