Hyperoxidation of NAD(P)H redox state after anoxia and reoxygenation: Effects of nitric oxide and PARP-1 inhibition

Background

A detailed description of acute changes in neuronal mitochondrial metabolism during and after anoxia is essential for understanding how metabolic dysfunction contributes to the pathogenesis of ischemia/reperfusion. Studies with cultured neurons are typically performed under relatively high PO2 values that do not simulate clinical ischemic conditions. We established a fluorescence microscopy perfusion system that induces true metabolic anoxia, as validated by measurements of the autofluorescence of endogenous reduced NAD(P)H ¹ . This system was used to test the hypothesis that metabolic anoxia and reoxygenation is associated with hyperoxidation of NAD(P)H, an event that can limit neuronal energy metabolism and impair detoxification of reactive oxygen species.

Methods

Primary cultures of rat cortical neurons were placed in a closed micro-perfusion chamber ² and subjected to oxygen-glucose deprivation for 30–45 min at 37°. Oxygen was displaced by continuous infusion of ultra-high purity argon in the perfusate and the chamber. The PO2 of the perfusate in the chamber was monitored continuously by a Optronix Oxylite fiber-optic microprobe. When added, DETA-NO (25 uM) was present in the perfusate as a NO donor to increase the Km of cytochrome oxidase for O2. NAD(P)H autofluorescence was measured at 355 nm excitation and 460 nm emission wavelengths using a ORCA-ER cooled digital CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) mounted on a Nikon Eclipse TE2000-S inverted microscope (Nikon Corp., Japan).

Results

Perfusate PO2 values fell to <0.4 mm Hg within 5 min after the chamber was exposed to argon and the argon-flushed perfusate. This level of hypoxia was associated with an increase in NAD(P)H fluorescence to a level equivalent to the maximum obtained in the presence of 1 mM KCN. Fluorescence remained high in the presence of glucose; however, when glucose was substituted with 2-deoxyglucose, fluorescence declined toward the minimum value observed in the presence of the respiratory uncoupler FCCP. While the presence of 25–200 μM DETA-NO alone had no effect on NAD(P)H fluorescence, 200 μM DETA-NO exacerbated the decline observed during chemical anoxia induced with KCN. Subsequent removal of KCN, or reoxygenation of cells after oxygen/glucose deprivation caused a further NAD(P)H hyperoxidation. This hyperoxidation observed in the absence or presence of DETA-NO and the accelerated decline in fluorescence observed during KCN treatment with DETA-NO was diminished by exposure of cells with a specific PARP-1 inhibitor, DPQ.

Conclusions

True metabolic anoxia in vitro, as defined by a maximum reduced shift in NAD(P)H redox state, requires a very low PO2 and (or) a level of NO sufficient to severely limit the consumption of oxygen by cytochrome oxidase.

Maintenance of reduced NAD(P)H during anoxia is dependent on glycolysis.

Hyperoxidation of NAD(P)H is exacerbated by NO, possibly due to PARP-1 dependent degradation of pyridine nucleotides.