Skip to main content
Springer Nature Link
Log in

Anoxia-Induced Changes in Pyridine Nucleotide Redox State in Cortical Neurons and Astrocytes

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

    We’re sorry, something doesn't seem to be working properly.

    Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Abstract

NAD(P)H autofluorescence was used to verify establishment of metabolic anoxia using primary cultures of cortical neurons and astrocytes. Cells on cover slips were placed in a chamber and O2 was displaced by continuous infusion of argon. Perfusion with medium at PO2 < 0.4 mm Hg caused an increase in NAD(P)H fluorescence, albeit to levels lower than that obtained with cyanide. Addition of the nitric oxide-generating agent DETA-NO to the hypoxic medium further increased fluorescence to the level with cyanide. Fluorescence under anoxia remained high in the presence of glucose, but declined in neurons and not in astrocytes when glucose was substituted with 2-deoxyglucose. Reoxygenation of neurons resulted in a decline in fluorescence and a loss in fluorescent gradient between fully reduced and fully oxidized (plus respiratory uncoupler). We conclude that (1) DETA-NO is useful for generating metabolic anoxia in the presence of argon (2) Exogenous glucose is necessary to maintain NAD(P)H in a reduced state during metabolic anoxia in neurons but not astrocytes (3) Neurons undergo a partially irreversible decline in NAD(P)H fluorescence during metabolic anoxia and reoxygenation that could contribute to prolonged metabolic failure.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. I Kinetics of oxygen utilization. J Biol Chem 217:383–393

    PubMed  CAS  Google Scholar 

  2. Chance B (2004) Mitochondrial NADH redox state, monitoring discovery and deployment in tissue. Methods Enzymol 385:361–370

    Article  PubMed  CAS  Google Scholar 

  3. Williamson JR, Herczeg BE, Coles HS, Cheung WY (1967) Glycolytic control mechanisms. V. Kinetics of high energy phosphate intermediate changes during electrical discharge and recovery in the main organ of Electrophorus electricus. J Biol Chem 242:5119–5124

    PubMed  CAS  Google Scholar 

  4. Schuchmann S, Kovacs R, Kann O, Heinemann U, Buchheim K (2001) Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal-entorhinal cortex slices. Brain Res Brain Res.Protoc 7:267–276

    Article  PubMed  CAS  Google Scholar 

  5. Klaidman LK, Leung AC, Adams Jr. JD (1995) High-performance liquid chromatography analysis of oxidized and reduced pyridine dinucleotides in specific brain regions. Anal Biochem 228:312–317

    Article  PubMed  CAS  Google Scholar 

  6. Vishwasrao HD, Heikal AA, Kasischke KA, Webb WW (2005) Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy. J Biol Chem 280:25119–25126

    Article  PubMed  CAS  Google Scholar 

  7. Mayevsky A, Zarchin N, Kaplan H, Haveri J, Haselgroove J, Chance B (1983) Brain metabolic responses to ischemia in the mongolian gerbil: in vivo and freeze trapped redox scanning. Brain Res 276:95–107

    Article  PubMed  CAS  Google Scholar 

  8. Perez-Pinzon MA, Mumford PL, Carranza V, Sick TJ (1998) Calcium influx from the extracellular space promotes NADH hyperoxidation and electrical dysfunction after anoxia in hippocampal slices. J Cereb Blood Flow Metab 18:215–221

    Article  PubMed  CAS  Google Scholar 

  9. Tanaka K, Dora E, Greenberg JH, Reivich M (1986) Cerebral glucose metabolism during the recovery period after ischemia–its relationship to NADH-fluorescence, blood flow, EcoG and histology. Stroke 17:994–1004

    PubMed  CAS  Google Scholar 

  10. Welsh FA, Marcy VR, Sims RE (1991) NADH fluorescence and regional energy metabolites during focal ischemia and reperfusion of rat brain. J Cereb.Blood Flow Metab 11:459–465

    PubMed  CAS  Google Scholar 

  11. Kasischke KA, Vishwasrao HD, Fisher PJ, Zipfel WR, Webb WW (2004) Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 305:99–103

    Article  PubMed  CAS  Google Scholar 

  12. Brown GC, Cooper CE (1994) Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 356:295–298

    Article  PubMed  CAS  Google Scholar 

  13. Cleeter MW, Cooper JM, Darley-Usmar VM, Moncada S, Schapira AH (1994) Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett 345:50–54

    Article  PubMed  CAS  Google Scholar 

  14. Shiino A, Matsuda M, Handa J, Chance B (1998) Poor recovery of mitochondrial redox state in CA1 after transient forebrain ischemia in gerbils. Stroke 29:2421–2424

    PubMed  CAS  Google Scholar 

  15. Perez-Pinzon MA, Mumford PL, Carranza V, Sick TJ (1998) Calcium influx from the extracellular space promotes NADH hyperoxidation and electrical dysfunction after anoxia in hippocampal slices. J Cereb.Blood Flow Metab 18:215–221

    Article  PubMed  CAS  Google Scholar 

  16. Perez-Pinzon MA, Mumford PL, Rosenthal M, Sick TJ (1997) Antioxidants, mitochondrial hyperoxidation and electrical recovery after anoxia in hippocampal slices. Brain Res 754:163–170

    Article  PubMed  CAS  Google Scholar 

  17. Feng ZC, Sick TJ, Rosenthal M (1998) Oxygen sensitivity of mitochondrial redox status and evoked potential recovery early during reperfusion in post-ischemic rat brain. Resuscitation. 37:33–41

    Article  PubMed  CAS  Google Scholar 

  18. Chance B, Baltscheffsky H (1958) Respiratory enzymes in oxidative phosphorylation. VII. Binding of intramitochondrial reduced pyridine nucleotide. J Biol Chem 233:736–739

    PubMed  CAS  Google Scholar 

  19. Shuttleworth CW, Brennan AM, Connor JA (2003) NAD(P)H fluorescence imaging of postsynaptic neuronal activation in murine hippocampal slices. J Neurosci 23:3196–3208

    PubMed  CAS  Google Scholar 

  20. Blinova K, Carroll S, Bose SA, Smirnov V, Harvey JJ, Knutson JR, Balaban RS (2005) Distribution of mitochondrial NADH fluorescence lifetimes: steady-state kinetics of matrix NADH interactions. Biochemistry 44:2585–2594

    Article  PubMed  CAS  Google Scholar 

  21. Dawson VL, Dawson TM (2004) Deadly conversations: nuclear-mitochondrial cross-talk. J Bioenerg Biomembr 36:287–294

    Article  PubMed  CAS  Google Scholar 

  22. Suh SW, Aoyama K, Chen Y, Garnier P, Matsumori Y, Gum E, Liu J, Swanson RA (2003) Hypoglycemic neuronal death and cognitive impairment are prevented by poly(ADP-ribose) polymerase inhibitors administered after hypoglycemia. J Neurosci 23:10681–10690

    PubMed  CAS  Google Scholar 

  23. Suh SW, Aoyama K, Matsumori Y, Liu J, Swanson RA (2005) Pyruvate administered after severe hypoglycemia reduces neuronal death and cognitive impairment. Diabetes 54:1452–1458

    Article  PubMed  CAS  Google Scholar 

  24. Tanaka S, Takehashi M, Iida S, Kitajima T, Kamanaka Y, Stedeford T, Banasik M, Ueda K (2005) Mitochondrial impairment induced by poly(ADP-ribose) polymerase-1 activation in cortical neurons after oxygen and glucose deprivation. J Neurochem 95:179–190

    Article  PubMed  CAS  Google Scholar 

  25. Mishra OP, Akhter W, Ashraf QM, Delivoria-Papadopoulos M (2003) Hypoxia-induced modification of poly (ADP-ribose) polymerase and dna polymerase beta activity in cerebral cortical nuclei of newborn piglets: role of nitric oxide. Neuroscience 119:1023–1032

    Article  PubMed  CAS  Google Scholar 

  26. Bal-Price A, Brown GC (2000) Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria. J Neurochem 75:1455–1464

    Article  PubMed  CAS  Google Scholar 

  27. Du L, Zhang X, Han YY, Burke NA, Kochanek PM, Watkins SC, Graham SH, Carcillo JA, Szabo C, Clark RS (2003) Intra-mitochondrial poly(ADP-ribosylation) contributes to NAD+ depletion and cell death induced by oxidative stress. J Biol Chem 278:18426–18433

    Article  PubMed  CAS  Google Scholar 

  28. Ignacio PC, Baldwin BA, Vijayan VK, Tait RC, Gorin FA (1990) Brain isozyme of glycogen phosphorylase: immunohistological localization within the central nervous system. Brain Res 529: 42–9

    Article  PubMed  CAS  Google Scholar 

  29. Almeida A, Moncada S, Bolanos JP (2004) Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway. Nat Cell Biol 6:45–51

    Article  PubMed  CAS  Google Scholar 

  30. Swanson RA (1992) Astrocyte glutamate uptake during chemical hypoxia in vitro. Neurosci Lett 147:143–146

    Article  PubMed  CAS  Google Scholar 

  31. Magistretti PJ, Pellerin L, Rothman DL, Shulman RG (1999) Energy on demand. Science 283:496–147

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

These studies were supported by NIH grants NS34152, NS07375 and HD16596.

Author information

Authors and Affiliations

  1. Anesthesiology, Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Sibel Kahraman & Gary Fiskum

  2. Anesthesiology Research Labs., University of Maryland School of Medicine, 685 W Baltimore Street, MSTF 5-34, Baltimore, MD, 21201, USA

    Sibel Kahraman & Gary Fiskum

Authors
  1. Sibel Kahraman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gary Fiskum
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gary Fiskum.

Additional information

Special issue dedicated to John P. Blass.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kahraman, S., Fiskum, G. Anoxia-Induced Changes in Pyridine Nucleotide Redox State in Cortical Neurons and Astrocytes. Neurochem Res 32, 799–806 (2007). https://doi.org/10.1007/s11064-006-9206-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-006-9206-8

Keywords

Search

Navigation