Real time measurement of hydrogen peroxide (H2O2)and nitric oxide (NO) release in femoral vein ischemia and reperfusion (I/R): The effects of tetrahydrobiopterin (BH4)/dihydrobiopterin (BH2) and the effects of Protein Kinase C (PKC) epsilon activation (ε+)/inhibition (ε−)

Abstract

Endothelial nitric oxide synthase (eNOS) is a possible source of oxidative stress (i.e., superoxide (SO) and H2O2) during reperfusion in which BH4 is oxidized to BH2. Moreover, PKC ε positively regulates NO and SO release from eNOS. The effects of BH4/BH2 or PKC ε +/ε− on NO and H2O2 release have not been directly estimated by real‐time measurement in blood vessels during I/R in vivo. NO and H2O2 were directly measured by inserting either NO or H2O2 microsensors (100 μm diameter) into both femoral veins in the anesthetized rat. One femoral vein was subjected to I/R, which was induced by clamping the femoral artery and vein for 20 min ischemia followed by removing the clamp for 45 min reperfusion. The other non‐ischemic femoral vein served as a sham control in the same animal. H2O2 release increased throughout the first 20 min of reperfusion compared to the sham control. PKC ε− (0.4 mg/kg) or BH4 (6.5 mg/kg) significantly reduced H2O2 release while PKC ε+ (0.9mg/kg) or BH2 (2mg/kg) showed a trend to increase H2O2 release throughout reperfusion. BH4 significantly increased NO release while BH2 showed a trend to decrease NO release. The preliminary data thus far support our hypothesis that BH4 or PKC ε− decrease H2O2 release during reperfusion whereas the opposite effect is observed with BH2 or PKC ε+.This study was supported by NHLBI Grant 1R15HL‐76235‐01 and Center For Chronic Disorders of Aging at PCOM