Nitric oxideNeurotransmitter?

B.Sc 2002

Oxygen and Nitrogen

missing electron pair

Oxygen 1s 2 2s 2 2p4

:O

Nitrogen 1s

2s 2 2p3

N.

Electron number = 8
Outer shell number = 6
Two electrons needed for stable shell

Electron number = 7
Outer shell number = 5
Three electrons needed for stable shell

Dioxygen, molecular oxygen

Dinitrogen, molecular nitrogen

. O:

Outer shell number = 12

Each atom now has share of 8 electrons
However molecular orbital considerations show that
two electrons are in unpaired orbitals with same spin
Oxygen is therefore a 'diradical'.

Nitric oxide
Outer shell number = 11

. N : O:
:

unpaired electron

:
:N : N :
:

Outer shell number = 10

Each atom now has share of 8 electrons
molecular orbital considerations show that
triple bond exists (one sigma and two pi bonding orbitals)
dinitrogen is therefore extremely stable

: N :: O .

Odd number of electrons;

free radical

NO is almost same size as dioxygen and can therefore fit in oxygen binding sites

EDRF: endothelium-derived relaxing factor

Furchgott in 1980 showed that Acetylcholine-stimulated
relaxation of arterial smooth muscle required intact
endothelium in the arteries
Palmer & Moncada in late 1980s showed that EDRF was
nitric oxide, NO, synthesised by a specific enzyme, Nitric
Oxide Synthase (NOS) NOS uses arginine and oxygen to
produce NO and citrulline

Different forms of NOS exist. Constitutive forms

and inducible forms. Neuronal NOS is constitutive,
macrophage NOS is induced.

Mechanism of arginine oxidation by NOS

oxygen

arginine

NH

NH 2
1/2

O2

N-hydroxy-arginine

NH 2

Established
mechanism

NH

(CH 2 )

COOH

NH 2

N OH

COOH

NH 2

citrulline

N-hydroxy-arginine

NH 2

C
NH

(CH 2 )
H

N OH

NH

nitric oxide

NH 2

NO

NH

How does this occur

(CH 2 )

(CH 2 )

C
NH 2

COOH

C
NH 2

COOH

oxygen

superoxide?
NOS-a

arginine

nitric oxide
NOS-b

N-hydroxy-arginine

citrulline

NO synthesis requires molecular oxygen,

NADPH, and tetahydrobiopterin
The intermediate N-hydroxyarginine could be a
storage form of NO. Superoxide anion may be an
intermediate in NO synthesis

Brain NOS is present in CNS including specific neurons in

cerebellum, hippocampus and olfactory lobes; and in NANC (nonadrenergic, non-cholinergic) nerves including innervation to
gastrointestinal tract, pelvic organs and trachea.
Vascular NOS is present in platelets and renal mesangial cells in
addition to endothelium.
NO was initially thought to is unstable but is stable at the partial
pressures of oxygen found in the body. It is metabolised to nitrite
and nitrate which are excreted in urine and provide an index of NO
biosynthesis in humans receiving a low-nitrate diet.

How does NO work?

Diffuses to cells other than the one where it was synthesised
Binds with soluble guanylate cyclase to form cGMP
cGMP acts to sequester intracellular calcium and close calcium
channels
Drop in intracellular calcium causes relaxation

How is NO inactivated?
NO is oxidised to nitrite and then nitrate
Nitrate excreted in urine

Functions of NO in body
Dilates blood vessels
Displaces oxygen from oxyhaemoglobin
metabolic factor mediating increased
blood flow to exercising muscle
Appears to prevent hypoxia
Released from organic nitrate and nitrite
vasodilators
Acts to relax bronchial smooth muscle

NO and haemoglobin
Early studies with free haemoglobin
showed that NO converted it irreversibly to
methaemoglobin
Studies with intact erythrocytes have
suggested that NO can bind reversibly to
Hb, forming nitroso-Hb
Nitroso-Hb may be converted back to
oxyHb in lungs

What is physiological role of NO?

NO appears to act to prevent hypoxia by causing
vasodilation.
It forms a good mechanism to counter hypoxia BUT
It needs molecular oxygen to be synthesised
NO is a gas and cannot be stored in the tissue
Could there be a storage form?
Concept that NO is produced during periods of normoxia,
stored as nitrosothiol or nitrite, converted back to NO in
hypoxic conditions
Maybe hydroxy-arginine is a storage form

A sensible model of NO in the body would exist if NO

could be stored in some way and form reversible
complexes with haemoglobin
Then during hypoxia
1) NO would be released from storage to cause local
vasodilation and unloading of oxygen from
haemoglobin, forming nitroso-Hb
2) Nitroso-Hb is carried back to lungs and is converted
back to oxyhaemoglobin. The released NO relaxes
the lungs and increased oxygenation of Hb
Is nitrite the storage form? There MUST be some way to
back-convert nitrite to NO as organic vasodilators
(GTN, amyl nitrite) are effective!

What is role of NO in brain?

NOS (neuronal) is found in scattered
neurones with axons ramifying near
cerebral blood vessels
NOS (endothelial) is found in many
astrocytes with processes on cerebral blood
vessels
Does NO control cerebral blood flow?

Cerebral NO hypothesis:
NOS controls CBF.
During normoxia NO is synthesised and blockade of
this synthesis reduces CBF and ability to autoregulate
Freshly made NO is used to maintain calibre of cerebral
capillaries
During hypoxia NO is released from storage forms; thus
blockade of NOS does not affect NO response to
hypoxia

Hypothesis 2: Role of NO in supporting neuronal activity

NO acts to increase local blood flow to neurones during
increased activity.
NO therefore matches perfusion to oxygen demand.
NO-containing neurones are stimulated by NMDA receptors.
This allows calcium entry, Ca/calmodulin activates NOS and
produces NO which diffuses to local blood vessels and causes
dilation.Co-stored with GABA as GABA acts to reduce
excess activity

The main features of generally accepted

criteria for a neurotransmitter are as follows:
(1) A system for synthesis of the putative transmitter must be
present.
(2) There must be a store of the putative transmitter in the axon
terminals.
(3) The putative transmitter must be released by nerve
stimulation.
(4) Administration of the putative transmitter must produce a
response that mimics that produced by nerve stimulation.
(5) Drugs that modify the responses to the putative transmitter
should have corresponding effects on the responses to nerve
stimulation.

Can NO be classed as a neurotransmitter?

Nerve terminals contain NOS
NO synthesis is calcium dependent
NO is released from certain autonomic neurones after
stimulation
NO cannot be classed as a classical transmitter because (as
a gas) it cannot be stored in nerve terminals
Action Potential that lets Ca into presynaptic axon may
trigger NO synthesis
NO is not released from vesicles by nerve action but
synthesised after each AP

Is NO a retrograde transmitter
Blockade of NOS blocks LTP in hippocampal slices
NO may be made in post-synaptic neurone and diffuse back to
presynaptic neurone. Suggested as a mechanism of synaptic
strengthening.Problem with this idea is that not all neurones
contain NOS.

NO & Pain
There are conflicting reports on the role of NO in the transmission
of pain in the spinal cord.
NOS inhibitors have been reported to reduce responses to pain, but
sometimes increase responses.
There is very good evidence for increased NO synthesis in the
spinal cord in animals with chronic pain.

This increase may reflect more ongoing activity of neurones in

chronic pain states rather than an increase in NO as a pain
transmitter

Toxic effects of NO
NO on its own is not toxic,but it reacts with superoxide to
form peroxynitrite, a toxic compound