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Nitric Oxide: an endogenous neurotoxin?
Robert Keynes
Neural Signalling
NO in the CNS
Learning and Memory NeurodegenerationCerebral blood flow
NO and Neurodegeneration
• Acute– Stroke– Head Injury
• Chronic– Alzheimer's– Parkinson’s
• Inflammatory– Multiple Sclerosis
L-Arginine
L-CitrullineNO
nNOSeNOS
iNOS
Ca2+/CaM
Glu
Ca2+
Ca2+ Glu
NO
NONO
NO
GC
GTP cGMP
Kinases
Ionchannels
< 20 nM
NONONO
NO
NO
NONO
NO
NO
NO
NO
500 nM – 10 M
NMDAReceptors
Glutamate release
brief
stimulation
Ca2+
A vicious cycle to cell death?
nNOS
delayed celldeath
NO
L-Arg
cell death
but literature conflicting
aim
• dissociated culture paradigms – nNOS +ve cell numbers vary– Media artifacts (light and buffer)– Variations in stimulus intensity / timing
• NO may also be protective
• examine whether NO is toxic in more realistic paradigm– Hippocampal slice cultures (previously characterised)
• NMDA stimulation or direct NO application • (Inhibitors)• cGMP and Propidium Iodide (cell death)
Response to NMDA
0 4 8 12 16 20 24
0
10
20
30
40
50
60
70
80
90
100 NMDA (300 M)
DG
CA3
CA1
% D
eath
Time (hours)
100 M 300 M 1000 M CA1
DG
CA3
0
10
20
30
40
50
60
70
80
90
100
1000300100
NMDA (M)
CA1CA3DG
% D
eath
NMDA antagonist – MK801(during recovery)
0
10
20
30
40
50
60
70
80
90
100
DGCA3
+ MK801 10 M+ MK801 10 M
NMDA 300 MNMDA 100 M
* **
CA1
% D
eath
Inhibition of NOS(throughout expt)
0
10
20
30
40
50
60
70
80
90
100
CA3 DG
L-NNA 300 ML-NNA 300 M
NMDA 300 MNMDA 100 M
CA1
% D
eath
Why no NO-dependent death?
• How much NO was released?
• How much NO is required to cause death?
How much NO is released?
EC50 of NO at GC =2 nMcGMP < 50 % of max (< 2 nM NO)
0 100 300 300 3000
20
40
60
80
100
120
140
160
180 NMDA Alone
*
*
NMDA (M)
+DEA/NO 300 M
+DEA/NO 100 M
Control
cGM
P (
pm
ol/m
g p
rote
in)
How much NO is required for toxicity?
0
10
20
30
40
50
60
70
80
90
100 NOC-12 (3 mM, 24 hours)
DGCA3CA1
% D
eath
10 M NO is toxic
non-toxic levels
DETA/NO 300 M = 1.2 M NONOC-12 0.3 mM = 2.8 M NODETA/NO 3 mM = 4.5 M NONOC-12 1 mM = 6 M NO
1-2 M NO is toxic in dispersed culturesBal Price and Brown (2000) J.Neurosci, 21, 6480-6491
Summary 1
• NMDA induced neurotoxicity NO- independent– NO concentrations very low (< 2 nM)
• 10 M exogenous NO required to kill slices
HYPOTHESIS - slices may have an endogenous NO inactivation mechanism
How is NO inactivated?
• Autoxidation (slow)– enhanced in lipid phase
• Reaction with superoxide (fast)
• Binding to haem proteins– Haemoglobin in red blood cells– Flavohaemoglobins– cytochrome c oxidase?
NO is inactivated by cerebellar cells and homogenates in vitro
0 10 30 40
0
100
200
300
400
500
Homogenate
Cells
Buffer
[NO
] (n
M)
Time (min)
Data from Dr Charmaine Griffiths
Inhibiting NO inactivation
0 5 30 40 50
400
300
200
100
[NO
] (nM
)
Time (min)
Buffer
Hom + Ascorbate Oxidase
Hom
0
Ascorbate Oxidase DTPA – Iron Chelator Trolox – antioxidant
0 5 10 15
0
100
200
300
400
500
CellsCells+ DTPA
Buffer
[NO
] (n
M)
Time (min)
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 + H2O2
0 10 30 40
0
100
200
300
400
500
Homogenate
Cells
Buffer
[NO
] (n
M)
Time (min)
Continuing NO release prevents further peroxidation - inactivation finally saturates
NO is inactivated by reaction with a pool of peroxidising lipid
Summary 2
• In acutely prepared cerebellar cells or brain homogenates, lipid peroxidation inactivates NO– Measured ascorbate and peroxidation products – Inactivation mimicked by peroxidising lipid
• Pathophysiological relevance– Lipid peroxidation and NO are components of many
diseases • Atherosclerosis • Ischaemia
• Peroxidation NOT responsible for NO inactivation in brain slices (C.Hall)
Peroxidation-independent NO inactivation
Buffer
Glia+ DTPA
+ Trolox
0
100
200
300
nsns
stea
dy-s
tate
[NO
] (nM
)
0 2 4 6 8 10
0
100
200
300
400
0.5 x 106 / ml
Control
[NO
] (n
M)
Time (min)
1 x 10 6 / ml
2 x 10 6 / ml
Conclusions
• NO (from nNOS) is not an endogenous neurotoxin
• iNOS?• Lipid peroxidation powerfully inactivates
NO in vitro– Peroxidising lipid could influence
physiologically relevant NO levels in vivo
• There are other mechanisms that inactivate NO in the brain
Acknowledgements
• John Garthwaite– Sophie Duport– Charmaine Griffiths– Catherine Hall
Funding: The Sir Jules Thorn Charitable Trust