Prevention of Hyperoxia‐Induced Alterations in Synaptosomal Membrane‐Associated Proteins by N‐tert‐Butyl‐α‐Phenylnitrone and 4‐Hydroxy‐2,2,6,6‐Tetramethylpiperidin‐1‐oxyl (Tempol)

Abstract

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 to24 h of hyperoxia provided the greatest degree of oxidation of cortical synaptosomal membrane proteins. We reasoned that free radical oxidation was involved in this protein oxidation. In accordance, in the current study we investigated the protective nature of two known free radical scavengers, N-tert-butyl-a-phenylnitrone (PBN) and 4-hydroxy-2,2, 6,6-tetramethylpiperidin-1 -oxyl (Tempol), against 24-h hyperoxia damage. The three techniques used in this study were electron paramagnetic resonance (EPA) protein-specific spin labeling, assay of the activity of the oxidatively sensitive enzyme glutamine synthetase (GS), and measurement of protein carbonyl content. Before hyperoxia, gerbils received intraperitoneal injections of varying concentrations of eitherof the two free radical scavengers. After 30 mm, the gerbils were exposed to 90-100% 02 for 24 h. For the spin labeling experiments, cortical synaptosomes were isolated from gerbils. The membrane proteins were spin labeled with the thiol-specific label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1 -oxyl). As in our earlier study, the EPR spectral parameter of MAL-6--labeled 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 assays, 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) orlempol (p < 0.002). The protein carbonyl 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 potential antiaging agents.

The Lipid Peroxidation Product, 4‐Hydroxy‐2‐trans‐Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

“…Protein-specific spin labeling studies using MAL-6 consistently showed decreased W/S ratios in membranes in conditions of oxidative stress induced either in vitro (Hensley et al, l994b;Trad and Butterfield, 1994;Bellary et al, 1995) or in vivo (Hall et al, 1995a(Hall et al, -c, 1997Howard et al, 1996;Butterfield et al, 1997b). The decreased WIS ratios of MAL-6 in rodent cortical synaptosomal membranes returned toward control values when animals exposed to hyperoxia or IRI or senescent-accelerated animals were previously injected with brain-accessible PBN (Hall et al, 1995b;Howard et al, 1996;Butterfield et al, 1997b).…”

“…lb), which intercalates in the lipid bilayer. The procedures used for spin labeling synaptosomal membranes with MAL-6 or 5-NS were described before (Hensley et al, 1994b;Howard et al, 1996;Butterfield et al, 1997b).…”

“…The 2,4-dinitrophenylhydrazine adducts ofprotein carbonyls were determined spectroscopically as previously described (Howard et al, 1996;Butterfield and Stadtman, 1997;Butterfield et al, l997b).…”

“…Boxed region of spectrum is expanded in the bottom panel showing measurements necessary to calculate the W/S ratio. Samples were spin-labeled as described previously (Hall et al, 1995a;Howard et al, 1996;Butterfield et al, 1997a,b). d: EPR spectrum of 5-NS-labeled synaptosomes.…”

“…The ratio of the EPR spectral amplitudes of the weakly immobilized line (W) to that of the strongly immobilized line (5), in the low-field region of the EPR spectrum of MAL-6-labeled synaptosomal membranes, is the W/S ratio. This parameter is known to be highly sensitive to protein conformational changes and protein-protein interactions (Butterfield, 1982;Trad and Butterfield, 1994;Bellary et al, 1995;Hall et al, 1995aHall et al, -c, 1997Howard et al, 1996;Butterfield et al, 1997b). Increased protein-protein interactions, decreased segmental motion, and/or conformational changes in the spin-labeled proteins lead to decreased molecular motion resulting in a lowering of the W/S ratio (Butterfield, 1982;Wyse and Butterfield, 1988;Trad and Butterfield, 1994;Bellary et al, 1995;Hall et al, 1995aHall et al, -c, 1997Howard et al, 1996;Butterfield et al, l997b).…”