Keynes Symp1

354 views
275 views

Published on

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
354
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
2
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Keynes Symp1

  1. 1. Nitric Oxide: an endogenous neurotoxin? Robert Keynes Neural Signalling
  2. 2. NO in the CNS Learning and Memory Neurodegeneration Cerebral blood flow
  3. 3. <ul><li>Acute </li></ul><ul><ul><li>Stroke </li></ul></ul><ul><ul><li>Head Injury </li></ul></ul><ul><li>Chronic </li></ul><ul><ul><li>Alzheimer's </li></ul></ul><ul><ul><li>Parkinson’s </li></ul></ul><ul><li>Inflammatory </li></ul><ul><ul><li>Multiple Sclerosis </li></ul></ul>NO and Neurodegeneration L-Arginine L-Citrulline NO nNOS eNOS iNOS Ca 2+ /CaM Glu Ca 2+ Ca 2+ Glu NO NO NO NO GC GTP cGMP Kinases Ion channels < 20 nM NO NO NO NO NO NO NO NO NO NO NO 500 nM – 10  M
  4. 4. A vicious cycle to cell death? NMDA Receptors Glutamate release brief stimulation Ca 2+ nNOS delayed cell death NO L-Arg cell death
  5. 5. but literature conflicting <ul><li>dissociated culture paradigms </li></ul><ul><ul><li>nNOS +ve cell numbers vary </li></ul></ul><ul><ul><li>Media artifacts (light and buffer) </li></ul></ul><ul><ul><li>Variations in stimulus intensity / timing </li></ul></ul><ul><li>NO may also be protective </li></ul>aim <ul><li>examine whether NO is toxic in more realistic paradigm </li></ul><ul><ul><li>Hippocampal slice cultures (previously characterised) </li></ul></ul><ul><ul><ul><li>NMDA stimulation or direct NO application </li></ul></ul></ul><ul><ul><ul><li>(Inhibitors) </li></ul></ul></ul><ul><ul><ul><li>cGMP and Propidium Iodide (cell death) </li></ul></ul></ul>
  6. 6. Response to NMDA 100  M 300  M 1000  M CA1 DG CA3
  7. 7. NMDA antagonist – MK801 (during recovery)
  8. 8. Inhibition of NOS (throughout expt)
  9. 9. Why no NO-dependent death? <ul><li>How much NO was released? </li></ul><ul><li>How much NO is required to cause death? </li></ul>
  10. 10. How much NO is released? EC 50 of NO at GC =2 nM cGMP < 50 % of max (< 2 nM NO)
  11. 11. How much NO is required for toxicity? non-toxic levels DETA/NO 300  M = 1.2  M NO NOC-12 0.3 mM = 2.8  M NO DETA/NO 3 mM = 4.5  M NO NOC-12 1 mM = 6  M NO 1-2  M NO is toxic in dispersed cultures Bal Price and Brown (2000) J.Neurosci, 21 , 6480-6491 10  M NO is toxic
  12. 12. Summary 1 <ul><li>NMDA induced neurotoxicity NO- independent </li></ul><ul><ul><li>NO concentrations very low (< 2 nM) </li></ul></ul><ul><li>10  M exogenous NO required to kill slices </li></ul>HYPOTHESIS - slices may have an endogenous NO inactivation mechanism
  13. 13. How is NO inactivated? <ul><li>Autoxidation (slow) </li></ul><ul><ul><li>enhanced in lipid phase </li></ul></ul><ul><li>Reaction with superoxide (fast) </li></ul><ul><li>Binding to haem proteins </li></ul><ul><ul><li>Haemoglobin in red blood cells </li></ul></ul><ul><ul><li>Flavohaemoglobins </li></ul></ul><ul><ul><li>cytochrome c oxidase? </li></ul></ul>
  14. 14. NO is inactivated by cerebellar cells and homogenates in vitro 0 10 30 40 0 100 200 300 400 500 Homogenate Cells Buffer [NO] (nM) Time (min) Data from Dr Charmaine Griffiths
  15. 15. Inhibiting NO inactivation Ascorbate Oxidase DTPA – Iron Chelator Trolox – antioxidant 0 5 30 40 50 400 300 200 100 [NO] (nM) Time (min) Buffer Hom + A scorbate O xidase Hom 0 0 5 10 15 0 100 200 300 400 500 Cells Cells + DTPA Buffer [NO] (nM) Time (min)
  16. 16. Ascorbate and Iron (Leaks from cells) (contaminant) + NO NO consumed Padmaja and Huie., (1993) Biochem.Biophys.Res.Commun. 195, 539-544 Ascorbate Oxidase DTPA Trolox Peroxidation inhibited Goss et al. (1997) J. Biol. Chem. 272, 21647-21653 Lipid peroxidation LOO ● Ascorbate (Fenton reaction) OH ● Iron + H 2 O 2
  17. 17. 0 10 30 40 0 100 200 300 400 500 Homogenate Cells Buffer [NO] (nM) Time (min) NO is inactivated by reaction with a pool of peroxidising lipid Continuing NO release prevents further peroxidation - inactivation finally saturates
  18. 18. Summary 2 <ul><li>In acutely prepared cerebellar cells or brain homogenates, lipid peroxidation inactivates NO </li></ul><ul><ul><li>Measured ascorbate and peroxidation products </li></ul></ul><ul><ul><li>Inactivation mimicked by peroxidising lipid </li></ul></ul><ul><li>Pathophysiological relevance </li></ul><ul><ul><li>Lipid peroxidation and NO are components of many diseases </li></ul></ul><ul><ul><ul><li>Atherosclerosis </li></ul></ul></ul><ul><ul><ul><li>Ischaemia </li></ul></ul></ul><ul><li>Peroxidation NOT responsible for NO inactivation in brain slices (C.Hall) </li></ul>
  19. 19. Peroxidation-independent NO inactivation 0 2 4 6 8 10 0 100 200 300 400 Control [NO] (nM) Time (min) 0.5 x 10 6 / ml 1 x 10 6 / ml 2 x 10 6 / ml
  20. 20. Conclusions <ul><li>NO (from nNOS) is not an endogenous neurotoxin </li></ul><ul><li>iNOS? </li></ul><ul><li>Lipid peroxidation powerfully inactivates NO in vitro </li></ul><ul><ul><li>Peroxidising lipid could influence physiologically relevant NO levels in vivo </li></ul></ul><ul><li>There are other mechanisms that inactivate NO in the brain </li></ul>
  21. 21. Acknowledgements <ul><li>John Garthwaite </li></ul><ul><ul><li>Sophie Duport </li></ul></ul><ul><ul><li>Charmaine Griffiths </li></ul></ul><ul><ul><li>Catherine Hall </li></ul></ul><ul><li>Funding: The Sir Jules Thorn Charitable Trust </li></ul>

×