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