Nanomolar vitamin E alpha-tocotrienol inhibits glutamate-induced activation of phospholipase A2 and causes neuroprotection

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.

Figures

Fig. 1. Glutamate induces release of arachidonic acid (AA) and formation of free polyunsaturated fatty acids (PUFA)

After labeling with [3H]AA (0.5 μCi/dish) overnight, cells were treated with L-glutamate (10 mM; closed bars) or not (open bars) in DMEM for 30–120 min, following which release of [3H]AA into medium was measured [A]. Free saturated and monounsaturated fatty acid levels [B] and free PUFA levels [C] in cells following 30min of glutamate exposure were determined by GC as described in Materials & Methods. Experiments were conducted in triplicates and data represent means ± S.D. of three independent experiments. *, p < 0.05 as compared to the untreated control cells at 30min of treatment; †, p < 0.05 as compared to the untreated control cells at 30min of treatment. §, p<0.05 as compared to glutamate-treated cells at 30min of treatment. Statistical significance was determined by ANOVA.

Fig. 2. Calcium chelators attenuate glutamate-induced arachidonic acid release

After labeling with [3H]AA (0.5 μCi/dish) overnight cells were pre-treated with BAPTA (100 nM, closed bars) and then treated with L-glutamate (10 mM) [A] for 30 min or treated with EGTA (1 mM, closed bars) and L-glutamate (10 mM) [B] for 30 min in DMEM. At the end of incubation, release of [3H]AA into medium was measured as described in Materials & Methods. Experiments were conducted in triplicates and data represent means ± S.D. of threeindependent experiments. *, p < 0.05 as compared to the untreated control cells at 30 min of treatment; †, p < 0.05 as compared to the cells treated with L-glutamate alone for 30 min. Statistical significance was determined by ANOVA.

Fig. 3. cPLA2-specific inhibitors and α-tocotrienol attenuate glutamate-induced arachidonic acid release

Cells, after labeling with [3H]AA (0.5 μCi/dish) overnight, were pre-treated with [A] AACOCF3 (5 μM) or cPLA2α inhibitor (5 μM) for 1 h or [C] α-tocotrienol (250 nM) for10 min and then treated with L-glutamate (10 mM) for 30 min in DMEM. At the end of incubation, release of [3H]AA into medium was measured as described in Materials & Methods. The cPLA2 inhibitor AACOCF3 does not prevent glutamate induced GSH loss in HT4 cells [B]. Cells (1 × 106 cells/100 mm dish) were pre-treated with AACOCF3 (5 μM) for 2 h and then treated with L-glutamate (10 mM) for 8 h, following which GSH levels were measured. Experiments were conducted in triplicates and data represent means ± S.D. of three independent experiments. *, p < 0.05 as compared to the untreated control cells at 30 min of treatment; †, p < 0.05 as compared to the cells treated with L-glutamate alone for 30 min. Statistical significance was determined by ANOVA.

Fig. 4. α-Tocotrienol attenuates glutamate-induced formation of free polyunsaturated fatty acids (PUFA) and loss of arachidonic acid from cellular phospholipids

Cells were pre-treated with α-tocotrienol (250 nM, closed bars) or not (open bars) for 10 min and then treated with L-glutamate (10 mM) in DMEM for 30 min, following which free PUFA levels (arachidonic acid [A], docosahexaenoic acid [B]) in the cells or arachidonic acid levels in cellular phospholipids [C] were determined by GC as described in Materials & Methods. Experiments were conducted in triplicates and data represent means ± S.D. of three independent experiments. *, p < 0.05 as compared to the untreated control cells at 30 min of treatment; †, p < 0.05 as compared to the cells treated with L-glutamate alone for 30 min. Statistical significance was determined by ANOVA.

Fig. 5. Glutamate induces translocation and serine phosphorylation of cPLA2 in a tocotrienol-sensitive manner

cPLA2 translocation in subcellular fractions (cytosol, nucleus & plasma membrane) was detected by Western blots [A] and normalized against house keeping proteins as described in Materials & Methods. Cells were treated with L-glutamate (10 mM) for 0–60 min [B] or were pre-treatedwith α-tocotrienol (250 nM) for 10 min and then were exposed to glutamate (10 mM) for 30 min [C], following which cPLA2 and phosphoserine-cPLA2 were detected in the cellular proteins by Western blots [B & C] and in situ by immunofluorescence microscopy [D] as described in Materials & Methods. *, p < 0.05 as compared to the untreated control cells at 30 min of treatment; †, p < 0.05 as compared to the cells treated with L-glutamate alone for 30 min. Magnification 20×.

Fig. 6. cPLA2-specific inhibitor AACOCF3 and α-tocotrienol protect against glutamate-induced neurotoxicity

Cells (0.1 × 106 cells/well in 12 well plate) without or with pretreatment with AACOCF3 (10 μM) or α-tocotrienol (250 nM) for 2 h were challenged with L-glutamate (10 mM) for 24 h. At the end of the treatment, cell viability was determined by assaying the release of LDH from cells [A], LDH content in the cells [B] and examination of cell morphology [C] as described in Materials & Methods. Experiments were conducted in triplicates and datarepresent means ± S.D. of three independent experiments. †, p < 0.05 as compared to the untreated control cells at 24 h of treatment; *, p < 0.05 as compared to the cells treated with L-glutamate alone for 24 h. Statistical significance was determined by Student’s t test. Magnification 10×.

Fig. 7. cPLA2 knock-down attenuates glutamate-induced neurotoxicity

Cells (0.1 × 106 cells/well in 12 well plates) were transfected with 100 nM siControl non-targeting siRNA or cPLA2 siRNA for 72 h. After achieving >90% transfection efficiency, cPLA2 mRNA expression [A], cPLA2 protein expression [B], and glutamate-induced cytotoxicity [C] were determined as described in Materials & Methods. Experiments were conducted in triplicates and data represent means ± S.D. of three independent experiments. *, p < 0.05 as compared to the siControl non-targeting siRNA-transfected cells [A], [B] and [C]; †, p< 0.05 in cPLA2 siRNA-transfected cells as compared to the siControl non-targeting siRNA-transfected cells treated with L-glutamate alone for 24 h [C]. Statistical significance was determined by Student’s t test.