AMPA receptor phosphorylation is selectively regulated by constitutive phospholipase A₂and 5-lipoxygenase activities

Abstract

The present investigation provides the first indication that constitutive, calcium-independent phospholipase A2 activity (iPLA2) modulates phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of glutamate receptors. Preincubation of frozen-thawed brain sections with two iPLA2 inhibitors, bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACO), produced a dose-dependent enhancement in phosphorylation at both Ser831 and Ser845 sites on the GluR1 subunit of AMPA receptors. This effect was not associated with changes in phosphorylation at the Ser sites of either the GluR2/3 subunits of AMPA receptors or the NR1 subunits of N-methyl-D-aspartate (NMDA) receptors, nor was it reproduced by inhibition of the calcium-dependent form of PLA2 activity. These results suggest that the effects of these inhibitors are selective to GluR1 subunits and that they are dependent on iPLA2 activity. The ability of iPLA2 inhibitors to increase GluR1 phosphorylation was mimicked by the 5-lipoxygenase (5-LO) inhibitor MK-886, but not by blockers of 12-lipoxygenase (12-LO) or cyclooxygenase. Additional experiments indicated that calcium-mediated truncation of GluR1 subunits was reduced by iPLA2 inhibitors, an effect that was not correlated with overall changes in the distribution of AMPA receptors between intracellular and membrane compartments prepared from whole brain sections. However, quantitative autoradiographic analysis indicated enhanced 3H-AMPA binding to the CA1 stratum radiatum of the hippocampus in BEL-treated sections. Saturation kinetics experiments demonstrated that this binding augmentation was due to an increase in the maximal number of AMPA binding sites. Altogether, our results point to the conclusion that basal iPLA2 activity, through the generation of 5-LO metabolites, regulates AMPA receptor phosphorylation of GluR1 subunits, an effect that might selectively influence the number of membrane receptors in area CA1 of the hippocampus.

Glutamate presynaptic vesicular transporter and postsynaptic receptor levels correlate with spatial memory status in aging rat models

“…Briefly, extracts were centrifuged at 1000g for 10 minutes, and the supernatants obtained were then centrifuged at 14,000g for 20 minutes. The resulting pellet was defined as the crude synaptosomal fraction and resuspended in Tris-acetate buffer (50 mM, pH 7.4, 100 mM EGTA, 0.32 M sucrose, and protease inhibitors) (Henley, 1995;Menard and Quirion, 2012b;Menard et al, 2004Menard et al, , 2007. Afterward, crude synaptosomal fraction was diluted in 20 mM Tris-HCl, pH 6.0, 0.1 mM CaCl 2 with 1% Triton X-100, mixed for 20 minutes at 4 C with agitation, and centrifuged at 40,000g for 20 minutes at 4 C (Menard and Quirion, 2012b;Moron et al, 2007).…”

“…To obtain postsynaptic densities (PSD), homogenates were purified by differential centrifugation as previously described (Henley, 1995;Menard and Quirion, 2012b;Menard et al, 2004Menard et al, , 2007. Briefly, extracts were centrifuged at 1000g for 10 minutes, and the supernatants obtained were then centrifuged at 14,000g for 20 minutes.…”

Phospholipases A2 and Inflammatory Responses in the Central Nervous System

“…These results suggest that iPLA2 activates the formation of lysophospholipids in astrocytes, which causes openings of the Ca 2+ channels in the plasma membrane to refill Ca 2+ stores, and thus allows normal Ca 2+ -signaling. Another important role of iPLA2s in synaptic plasticity is the regulation of hippocampal AMPA receptor (Menard et al 2005). Hippocampal injection of BEL attenuated short-term and long-term memory in inhibitory avoidance learning (Schaeffer and Gattaz 2005).…”

Effects of MK-886, a 5-lipoxygenase activating protein (FLAP) inhibitor, and 5-lipoxygenase deficiency on the forced swimming behavior of mice

“…The 5-LOX pathway is also active in the mammalian brain [2,8]. 5-LOX inhibitors, including MK-886, stimulate phosphorylation and membrane insertion of the GluR1 subtype of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) glutamate receptors [12,23]. Drug-induced increases of GluR1 phosphorylation have been linked to antidepressant activity [5,18,21].…”

“…Interestingly, in contrast to the stimulation of GluR1 phosphorylation in wild-type mice [12], repeated injections of MK-886 did not increase brain GluR1 phosphorylation in 5-LOX-deficient mice (unpublished observation). Hence, it is possible that 5-LOX inhibition produces behavioral effects by increasing GluR1 phosphorylation via the dopamineric system and/or through dopamine-independent [12,23] mechanisms.…”