PREVENTION OF HYPEROXIA-INDUCED ALTERATIONS IN SYNAPTOSOMAL MEMBRANE-ASSOCIATED PROTEINS BY N-TERT-BUTYL-ALPHA-PHENYLNITRONE AND 4-HYDROXY-2,2,6,6-TETRAMETHYLPIPERIDIN-1-OXYL (TEMPOL)

Hyperoxia has been considered a model of free radical reactive oxygen species production in aging and age-related disorders. Previously, we studied the membrane protein alterations that occur during hyperoxia; we found that exposure of young animals to 24 h of hyperoxia provided the greatest degree of oxidation of cortical synaptosomal membrane pro teins. We reasoned that free radical oxidation was involved in this pr otein oxidation. In accordance, in the current study we investigated t he protective nature of two known free radical scavengers, N-tert-buty l-alpha-phenylnitrone (PBN) and 4-hydroxy-2,2, 6,6-tetramethylpiperidi n-1-oxyl (Tempol), against 24-h hyperoxia damage. The three techniques used in this study were electron paramagnetic resonance (EPR) protein -specific spin labeling, assay of the activity of the oxidatively sens itive enzyme glutamine synthetase (GS), and measurement of protein car bonyl content. Before hyperoxia, gerbils received intraperitoneal inje ctions of varying concentrations of either of the two free radical sca vengers. After 30 min, the gerbils were exposed to 90-100% O-2 for 24 h. For the spin labeling experiments, cortical synaptosomes were isola ted from gerbils. The membrane proteins were spin labeled with the thi ol-specific label MAL-B (2,2,6,6-tetramethyl-4-malelmidopiperidin-1-ox yl). As in our earlier study, the EPR spectral parameter of MAL-6-labe led membranes, the W/S ratio, decreased with hyperoxia (p < 0.00001). This effect was lessened significantly with administration of PBN (p < 0.0003) or Tempol (p < 0.00003). For the GS and protein carbonyl assa ys, cortical proteins were used. The activity of the GS decreased with hyperoxia (p < 0.000005), and this effect likewise was lessened with administration of PBN (p < 0.004) or Tempol (p < 0.002). The protein c arbonyl content increased with hyperoxia (p < 0.0002), and there was a protective effect found with Tempol (p < 0.000001). The optimum doses for PBN and Tempol were 20 and 5 mg/kg, respectively. The results are discussed with reference to the use of free radical scavengers as pot ential antiaging agents.