Group II metabotropic glutamate receptor activation prevents apoptotic-like death of cultured astrocytes induced by nitric oxide

DURAND DANIELA; CARUSO CARLA; LASAGA MERCEDES

Buzios, RJ, Brasil

Congreso; I IBRO/LARC CONGRESS OF NEUROSCIENCES OF LATIN AMERICA, CARIBBEAN AND IBERIAN PENINSULA; 2008

Sociedad Argentina de Neurociencias

We investigated the effect of (-)2-oxa-4-aminocyclo-3.1.0 hexane-4,6-dicarboxylic acid (Ly379268) and (2S,1´S,2´S)-2-(carboxycyclopropyl)glycine (L-CCG-I), selective agonists of group II metabotropic glutamate receptors (mGluRs), on nitric oxide (NO)-induced cell death of rat astrocytes in culture. Astrocytes were incubated for 48 h. with diethylenetriamine nitric oxide adduct (DETA/NO) 1 mM in presence or absence of Ly 379268 100 uM or L-CCG-I 1 mM. Receptor selectivity was verified with a specific group II mGluR antagonist, (2S)- -ethylglutamic acid (EGLU) at 300 uM. Astrocytes viability was reduced significantly by DETA/NO by 35% (p0.01 vs. control), as measured by MTT method, while both Ly 379268 and L-CCG-I prevented this effect (p0.05 vs. DETA/NO). DETA/NO increased the percentage of TUNEL positive cells by 5 fold (p0.01) and both Ly 379268 and L-CCG-I decreased this effect by 35% (p0.01 vs. DETA/NO). Preincubation with EGLU blocked the protective effects of the agonists. However, neither DETA/NO nor Ly 379268 modified Caspase-3 activity (Assay Designs Inc.). Moreover, a broad spectrum caspases inhibitor, Z-VAD-FMK, 50 uM did not revert the decrease in cell viability induced by NO. Both p53 and phospho-p53 protein levels (detected by Western-blot) were increased in presence of DETA/NO by 3.5 fold and this increase was reduced by Ly 379268 by 12% and 20% (p0.05), respectively. DETA/NO increased Bax (10 fold, p0.05) and decreased Bcl-2 (by 85%, p0.001) protein levels, while Ly 379268 had no effect on Bax levels but slightly incremented Bcl-2 protein (by 18%, p0.05 vs. DETA/NO). However, we detected by Western-blot an additional band at 100 kDa, which may correspond to active Bax oligomers as described previously1. Putative Bax oligomers were induced by DETA/NO by 2.5 fold (p0.05) and Ly 379268 completely reverted this effect. Moreover, we observed by immunocytochemistry a transition from a punctuate to a diffuse staining pattern of cytochrome c when astrocytes were incubated with DETA/NO. This was partially reverted by Ly 379268, indicating a decrease of the NO-induced-release of cytochrome c from the mitochondria. In summary, group II mGluR activation prevents astrocytic death triggered by NO, by modulating p53 activation, Bcl-2 protein levels, Bax oligomerization, and cytochrome c release from mitochondria. 1) The EMBO Journal 17:3878, 1998.