Figure 3.

Co-treatment with eNOS gene transfer and dietary BH₄ significantly reduced oxidative stress in the ischemic gastrocnemius muscle. These studies were carried out 4 days after AdeNOS or PBS infusion (14 days after induction of ischemia). (a) The expression of nitrotyrosine in the ischemic gastrocnemius muscle was lower in rats receiving either AdeNOS or BH₄ alone than in rats receiving only PBS; moreover, nitrotyrosine was undetectable by western blotting in the ischemic gastrocnemius from rats receiving co-treatment with AdeNOS and BH₄ (m ± SD, n = 4–5, *P < 0.05 versus [PBS, −BH₄]). (b) These findings were confirmed by immunohistochemistry; thus, less nitrotyrosine was evident in the gastrocnemius muscles from rats receiving either AdeNOS or BH₄, but virtually none was evident in rats receiving both treatments. Representative micrographs are shown (×400); nitrotyrosine is stained brown. (c) The ratio of the reduced:oxidized forms of glutathione (GSH: GSSG) in the ischemic gastrocnemius muscle was greater in rats receiving AdeNOS alone than in rats receiving PBS; in contrast, treatment with BH₄ alone had no effect. Rats receiving co-treatment with AdeNOS and BH₄ had a GSH:GSSG ratio greater than rats receiving AdeNOS alone (m ± SD, n = 4–5, *P < 0.05 versus [PBS, −BH₄], ^†P < 0.05 versus [eNOS, +BH₄]). BH₄, tetrahydrobiopterin; eNOS, endothelial nitric oxide synthase; PBS, phosphate-buffered saline.