Nitric oxideNeurotransmitter?B.Sc 2002

EDRF: endothelium-derived relaxing factor

Furchgott in 1980 showed that Acetylcholine-stimulated relaxation of arterial smooth muscle required intact endothelium in the arteriesPalmer & 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 citrullineDifferent forms of NOS exist. Constitutive forms and inducible forms. Neuronal NOS is constitutive, macrophage NOS is induced.

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 relaxationHow is NO inactivated?NO is oxidised to nitrite and then nitrateNitrate excreted in urine

Functions of NO in body

Dilates blood vesselsDisplaces oxygen from oxyhaemoglobinmetabolic factor mediating increased blood flow to exercising muscleAppears to prevent hypoxiaReleased from organic nitrate and nitrite vasodilatorsActs to relax bronchial smooth muscle

NO and haemoglobinEarly studies with free haemoglobin showed that NO converted it irreversibly to methaemoglobinStudies with intact erythrocytes have suggested that NO can bind reversibly to Hb, forming nitroso-HbNitroso-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 formWhat 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 hypoxiaNO would be released from storage to cause local vasodilation and unloading of oxygen from haemoglobin, forming nitroso-HbNitroso-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 vesselsNOS (endothelial) is found in many astrocytes with processes on cerebral blood vesselsDoes 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 dependentNO is released from certain autonomic neurones after stimulationNO cannot be classed as a classical transmitter because (as a gas) it cannot be stored in nerve terminalsAction Potential that lets Ca into presynaptic axon may trigger NO synthesisNO is not released from vesicles by nerve action but synthesised after each AP

Is NO a retrograde transmitterNO 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 & PainThere 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 NONO on its own is not toxic,but it reacts with superoxide to form peroxynitrite, a toxic compound