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Nitric oxide…Neurotransmitter?

B.Sc 2002

OOxygen 1s 2s 2p2 2 4

Nitrogen 1s 2s 2p2 2 3

Nitric oxide

Odd number of electrons;free radical

Dinitrogen, molecular nitrogen

Outer shell number = 6





Two electrons needed for stable shell Three electrons needed for stable shell

Each atom now has share of 8 electronsHowever molecular orbital considerations show thattwo electrons are in unpaired orbitals with same spinOxygen is therefore a 'diradical'.

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

Electron number = 8 Electron number = 7: N.

N NO:::

::

N O:: :. N O::: .

:

missing electron pair unpaired electron

..

Oxygen and Nitrogen

Dioxygen, molecular oxygen

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 1980’s 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.

C

NH2

COOHH

(CH2 )3

NH

CNH2

2

NH

C

NH2

COOHH

(CH2 )3

NH

CNH 2 N OH

arginine

citrulline nitric oxide

oxygen N-hydroxy-arginine

C

NH2

COOHH

(CH2 )3

NH

CNH 2 N OH

N-hydroxy-arginine

+

+

N

O

C

NH2

COOHH

(CH2 )3

NH

CNH 2 O

O

Mechanism of arginine oxidation by NOS

?How does this occur

Establishedmechanism

1/2

NOS-a

arginine

oxygen superoxide? nitric oxide

citrullineN-hydroxy-arginine

NOS-b

NO synthesis requires molecular oxygen, NADPH, and tetahydrobiopterinThe 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 (non-adrenergic, 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 synthesisedBinds with soluble guanylate cyclase to form cGMPcGMP acts to sequester intracellular calcium and close calcium channelsDrop in intracellular calcium causes relaxation

How is NO inactivated?

NO is oxidised to nitrite and then nitrateNitrate 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

NO appears to act to prevent hypoxia by causing vasodilation.It forms a good mechanism to counter hypoxia BUTIt needs molecular oxygen to be synthesisedNO is a gas and cannot be stored in the tissueCould 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 conditionsMaybe hydroxy-arginine is a storage form

What is physiological role of NO?

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 hypoxia1) 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 autoregulateFreshly made NO is used to maintain calibre of cerebral capillariesDuring 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”

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.

Blockade of NOS blocks LTP in hippocampal slices

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