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Association with actin mediates the EGTA-resistant binding of cytosolic phospholipase A 2-α to the plasma membrane of activated platelets

Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Cell Biology International (Impact Factor: 1.64). 01/2009; 33(1):83-91. DOI: 10.1016/j.cellbi.2008.09.010

ABSTRACT The association of cytosolic phospholipase A2-α (cPLA2α) with intracellular membranes is central to the generation of free arachidonic acid and thromboxane A2 in activated platelets. Despite this, the site and nature of this membrane association has not been fully characterised upon platelet activation. High resolution imaging showed that cPLA2α was distributed in a partly structured manner throughout the resting platelet. Upon glass activation or thrombin stimulation, cPLA2α relocated to a peripheral region corresponding to the platelet plasma membrane. Upon thrombin stimulation of platelets a major pool of cPLA2α was associated with the plasma membrane in an EGTA-resistant manner. EGTA-resistant membrane binding was abolished upon de-polymerisation of actin filaments by DNase I and furthermore, cPLA2α co-immunoprecipitated with actin upon thrombin stimulation of platelets. Immunofluorescence microscopy studies revealed that, upon platelet activation, cPLA2α and actin co-localised at the plasma membrane. Thus we have identified a novel mechanism for the interaction of cPLA2α with its membrane substrate via interaction with actin.

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    ABSTRACT: Our previous works have elucidated that the 12-lipoxygenase pathway is directly implicated in glutamate-induced neural cell death, and that such that toxicity is prevented by nM concentrations of the natural vitamin E alpha-tocotrienol (TCT). In the current study we tested the hypothesis that phospholipase A(2) (PLA(2)) activity is sensitive to glutamate and mobilizes arachidonic acid (AA), a substrate for 12-lipoxygenase. Furthermore, we examined whether TCT regulates glutamate-inducible PLA(2) activity in neural cells. Glutamate challenge induced the release of [(3)H]AA from HT4 neural cells. Such response was attenuated by calcium chelators (EGTA and BAPTA), cytosolic PLA(2) (cPLA(2))-specific inhibitor (AACOCF(3)) as well as TCT at 250 nM. Glutamate also caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of phospholipid-esterified AA in neural cells. Furthermore, glutamate induced a time-dependent translocation and enhanced serine phosphorylation of cPLA(2) in the cells. These effects of glutamate on fatty acid levels and on cPLA(2) were significantly attenuated by nM TCT. The observations that AACOCF(3), transient knock-down of cPLA(2) as well as TCT significantly protected against the glutamate-induced death of neural cells implicate cPLA(2) as a TCT-sensitive mediator of glutamate induced neural cell death. This work presents first evidence recognizing glutamate-induced changes in cPLA(2) as a novel mechanism responsible for neuroprotection observed in response to nanomolar concentrations of TCT.
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