Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

Abstract

The pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme that catalyzes the oxidative decarboxylation of pyruvate and represents the sole bridge between anaerobic and aerobic cerebral energy metabolism. Previous studies demonstrating loss of PDHC enzyme activity and immunoreactivity during reperfusion after cerebral ischemia suggest that oxidative modifications are involved. This study tested the hypothesis that hyperoxic reperfusion exacerbates loss of PDHC enzyme activity, possibly due to tyrosine nitration or S-nitrosation. We used a clinically relevant canine ventricular fibrillation cardiac arrest model in which, after resuscitation and ventilation on either 100% O2 (hyperoxic) or 21-30% O2 (normoxic), animals were sacrificed at 2 h reperfusion and the brains removed for enzyme activity and immunoreactivity measurements. Animals resuscitated under hyperoxic conditions exhibited decreased PDHC activity and elevated 3-nitrotyrosine immunoreactivity in the hippocampus but not the cortex, compared to nonischemic controls. These measures were unchanged in normoxic animals. In vitro exposure of purified PDHC to peroxynitrite resulted in a dose-dependent loss of activity and increased nitrotyrosine immunoreactivity. These results support the hypothesis that oxidative stress contributes to loss of hippocampal PDHC activity during cerebral ischemia and reperfusion and suggest that PDHC is a target of peroxynitrite.

Fig. 1

Selective inhibition of hippocampal pyruvate dehydrogenase complex enzyme activity by hyperoxic resuscitation after cardiac arrest. PDHC maximal enzyme activity was measured spectrofluorometrically using tissue homogenates obtained from samples of the (A) hippocampus and (B) frontal cortex of sham-operated (nonischemic) dogs or dogs at 2 h after 10 min cardiac arrest with resuscitation using either hyperoxic or normoxic ventilation. Values represent the means ± SE for n = 5 animals per group. *Significantly different from sham-operated control and normoxic-resuscitated animals groups; one-way ANOVA with Tukey post hoc analysis; p < 0.05.

Fig. 2

Selective elevation of hippocampal 3-nitrotyrosine immunoreactivity by hyperoxic resuscitation after cardiac arrest. 3-Nitrotyrosine immunoreactivity was measured by ELISA using tissue homogenates obtained from samples of the (A) hippocampus and (B) frontal cortex of sham-operated (nonischemic) dogs or dogs at 2 h after 10 min cardiac arrest with resuscitation using either hyperoxic or normoxic ventilation. Values represent the means ± SE for n = 5 animals per group. *Significantly different from sham-operated control and normoxic-resuscitated animals groups; one-way ANOVA with Tukey post hoc analysis; p < 0.05.

Fig. 3

Pyruvate dehydrogenase complex subunit immunoreactivity in anti-nitrotyrosine-immunoprecipitated hippocampal and cortical homogenate proteins. A nitrotyrosine affinity sorbent was used to capture all nitrated proteins in the hippocampal homogenate samples. Western blot analysis was then performed using a monoclonal antibody cocktail to several PDHC subunits. This antibody reacts the strongest with the E2 subunit of the enzyme although immunoreactivity with several other subunits was also observed. (A) Representative immunoblot comparing PDHC E2 immunoreactivity of a sample from a sham-operated (nonischemic) animal and from animals at 2 h reperfusion using either normoxic or hyperoxic resuscitation. (B) Densitometric analysis of PDHC E2 immunoreactivity. Values are reported in arbitrary units of optical density and are normalized for loading differences by optical density of the IgG light chain. Values represent the means ± SE for n = 5 animals per group. *Significantly different from sham-operated control and normoxic-resuscitated animal groups; one-way ANOVA with Tukey post hoc analysis; p < 0.05. (C) Representative immunoblot depicting the immunoreactivity of other PDHC subunits.

Fig. 4

Effects of SIN-1 on purified pyruvate dehydrogenase complex enzyme activity, immunoreactivity and protein tyrosine nitration. Purified PDHC (200 μg) was incubated in the presence of increasing concentrations of the peroxynitrite-generating system 3-morpholinosydnonomine (SIN-1) for 20 min. After the incubation period, the required substrates and cofactors were added to initiate enzyme activity, which was measured spectrophotometrically. (A) 3-Nitrotyrosine immunoblot. (B) PDHC immunoblot. (C) Quantification of PDHC enzyme activity and 3-nitrotyrosine (NT) immunoreactivity after exposure of purified enzyme to SIN-1 in the absence of required substrates and cofactors. (D) Quantification of PDHC enzyme activity and NT immunoreactivity after exposure of purified enzyme to SIN-1 in the presence of all enzyme substrates and cofactors except coenzyme A, which was used to start the enzyme reaction. PDHC activity is reported as % of control activity measured after preincubation in the absence of SIN-1. A regression analysis (test for trend) was performed for the data located in (C) and (D). NT optical density is reported in arbitrary units (A.U.). Values represent the means ± SE for n = 6 experiments per [SIN-1].

Fig. 5

Time-dependent inhibition of pyruvate dehydrogenase complex enzyme activity and 3-nitrotyrosine immunoreactivity by SIN-1. Purified PDHC (200 μg) was incubated in 50 μM SIN-1 for 0–60 min in the absence of enzyme substrates and cofactors before evaluation of nitrotyrosine (NT) immunoreactivity and enzyme activity. PDHC enzyme activity is reported as % of control activity measured after preincubation in the absence of SIN-1. NT optical density is reported in arbitrary units (A.U.). Values represent the mean ± SE for n = 4 experiments per incubation time.

Fig. 6

Confirmation that peroxynitrite mediates inhibition of pyruvate dehydrogenase by SIN-1. (A and C) Purified PDHC (200 μg) was incubated for 20 min with or without 50 μM 3-morpholinosydnonomine (SIN-1). In (A), 60 units of Cu/Zn superoxide dismutase (SOD) or 10 mM DMSO was present during the incubation with SIN-1, as indicated. In (C), the enzyme was incubated in the absence or presence of 10 mM dithiothreitol (DTT). (B) PDHC was incubated in the same buffer as in (A) and (C) except that SIN-1 was replaced with various concentrations of prepared peroxynitrite. Values are reported as % of control activity measured after preincubation in the absence of SIN-1 or peroxynitrite and represent the means ± SE for n = 6 separate determinations. *Significantly different from control activity, and **significantly different from activity in the presence of SIN-1 and in the absence of DTT, based on one-way ANOVA, Dunnett post hoc analysis; p < 0.05.