Studies have shown that lipid peroxidation is associated with modifications of the cerebral NMDA receptor, and that these modifications lead to an increase in intracellular calcium. Previous studies have also shown that superoxide and nitric oxide (NO) react to form peroxynitrite, which causes lipid peroxidation in vivo. The present study tests the hypothesis that 3-morpholinosydnonimine (SIN-1), which releases superoxide and NO simultaneously, increases intracellular calcium via the NMDA receptor. Studies were conducted in cortical synaptosomes prepared from 7 term guinea pigs fetuses. Synaptosomes were loaded with Fura-2 and divided into 4 aliquots. One aliquot served as control (C), and the others were incubated with 100 mM SIN-1 alone (SIN), SIN-1 + superoxide dismutase (SOD) and SIN-1 + oxyhemoglobin(OH). After baseline acquisition, measurements of the intrasynaptosomal calcium were performed in a luminescence spectrometer following the addition of 100 μM glutamate in the presence of 100 μM NMDA and glycine. Viability of the synaptosomes was documented by measuring mitochondrial dehydrogenase activity. The release of peroxynitrite following SIN-1 was demonstrated by the oxidation of dihydrorhodamine. The increase in intracellular calcium in C, SIN, SOD, and OH was 29.5±14.1, 82.6±47.9 (p<0.01 vs C), 38.0±30.5 (p<0.05 vs SIN) and 32.0±24.4 nM/min (p<0.05 vs SIN), respectively. The glutamate-induced calcium increase in C, SIN, SOD, and OH was 84.5±66.5, 182± 126, (p<0.01 vs C), 62.0±66.9(p<0.05 vs SIN), 29.5±22.7 nM (p<0.05 vs SIN), respectively. The results suggest that SIN-1 modifies the calcium permeability of the synaptosomal membrane via peroxynitrite production, but not superoxide or nitric oxide alone. We speculate that the peroxynitrite-mediated increase in intrasynaptosomal calcium leads to stimulation of phospholipase A2 and NO synthase and may further initiate peroxynitrite production and alteration of membrane function. (Funded by NIH#HD-20337).
