NOS38: NO can also attenuate NOS activity, serving as a negative feedback to

control NO production. NO binds the haem group associated with NOS and prevents

the binding of oxygen to the active site and thus inhibits the oxidation of arginine.

nNOS and eNOS are more sensitive than iNOS to the inhibitory action of NO

2. Reaction with oxygen (O2) and superoxide anion (O2 )9: NO reacts with O2 and O2' to form reactive nitrogen oxide species (RNOS).

Oxygen- In aqueous solutions NO can undergo autoxidation (i.e. reaction with

oxygen) to produce N2O3. N2O3 is the predominant RNOS formed from the

autoxidation of NO in biological systems. N2O3 is rapidly hydrolysed to nitrite with a

half-life of 1ms. The resultant nitrite is taken up by RBCs where it is further oxidised

to nitrate and released back to plasma39.

Superoxide anion (O2' ) 40 -In physiological conditions O2" concentrations are kept low by its neutralisation by superoxide dismutase (SOD), antioxidants, and by its

extremely rapid reaction with NO (almost diffusion limited). However, under

pathological conditions (e.g. atherosclerosis, myocardial ischaemia, sepsis, distressed

lung, inflammatory bowel disease, and amyotrophic lateral sclerosis) when the levels

of 0 2' may be very high, NO combines rapidly with O2" to form peroxynitrite.

Peroxynitrite (ONOO) is itself toxic and acts as a selective oxidant and nitrating agent

to modify proteins (e.g. tyrosine—> nitrotyrosine), lipids, and nucleic acids.

Two major sources of ONOO' formation in our body are mitochondria and immune

cells. In mitochondria, ONOO' is produced as a result of aerobic respiration. The

generation of ONOO' in the mitochondria is intensely controlled by several regulatory

mechanisms including manganese superoxide dismutase (MnSOD). The primary

source for large amounts of ONOO' is immune cells through either NADPH oxidase

or xanthine oxidase. Neither enzyme is directly inhibited by NO. Therefore as NO

migrates near the source of O2', it reacts to form peroxynitrite. However, as

peroxynitrite moves from its source, it is converted by excess NO to N2O3. Thus, the

primary chemistry of ONOO' would be within close proximity of the superoxide

source.

Nitrotyrosine40- Nitrotyrosine is frequently used as a marker of in vivo production of

ONOO'. It is formed by the nitration of tyrosine by ONOO". Nitrotyrosine levels are

increased in conditions associated with increased oxidative stress. Nitrotyrosine has

been found in atherosclerotic plaques, motor neurons of patients with ALS, rejected

renal allografts, inflammatory bowel disease, the synovial fluid of arthritis patients

and the placental tissues from pre-eclamptic pregnancies. Animal studies have

demonstrated nitrotyrosine formation in ischaemia-reperfusion injury in the heart.

Nitration of prostacyclin synthase41-Nitration of endothelial prostacyclin synthase

by ONOO' inhibits its activity and impairs vasorelaxation.

3. Interaction with thiol (-SH) groups9*25: Nitrosation of thiols in proteins such as

albumin results in long-lived S-nitrosothiols (RSNO) with a variety of different

effects in biological systems. Peptides with thiol groups have strong affinity for N2O3.

This makes thiols the primary target for reactive nitrogen oxide species (RNOS) in

biological aqueous solutions.

The amino acid cysteine, which is found in most proteins, contains a thiol group. S-

nitrosylation of cysteine residues resulting from the addition of a NO+ group has been

shown to modify the activity of several proteins. Although it is unlikely that NO acts

directly on the cysteine residue, NO interacts with O2 or 0 2’ to produce RNOS capable

of nitrosylating cysteine residues. Nitrosylation is a non-enzymatic chemical reaction.

In recent years RSNOs have attracted increasing attention as possible preservers of

NO bioactivity in the circulation. They prevent loss of NO from oxidative degradation

nitrosoglutathione (GSNO) which demonstrate vasodilator properties that are equal to

native NO. RSNOs provide a reservoir of NO bioactivity that might be utilised in

states of NO deficiency.

96% of plasma RSNOs are S-nitrosoproteins of which 82% is S-nitroso-albumin11.

4. Interaction with amine groups9: RNOS can also nitrosate the amine group of

proteins to form nitrosamines (R2NNO). Nitrosamines have a stable structure and are

well known for their carcinogenic and mutagenic properties.

5. NO modifications of lipids: NO derivatives may react with unsaturated lipids to

oxidise or nitrate them. One example is the low density lipoprotein (LDL) which is

converted to its atherogenic form by oxidation and nitration.

The reaction between nitric oxide (NO) and lipid peroxyl radicals (LOO*) has been

proposed to account for the potent inhibitory properties of NO toward lipid

peroxidation processes42.

6. NO damage to DNA9: NO is not reactive enough to damage DNA directly, but its

derivatives (i.e. RNOS) can reach the nucleus to oxidise, nitrate, or deaminate

genomic DNA, resulting in strand breaking and mutations. One apparent suspect is

ONOO'. ONOO' can travel up to 9pm and easily pass through biological membranes

to reach the nucleus and modify DNA- preferentially reacting with guanine. Another

powerful reactive nitrogen species is the nitrosating agent N2O3 which can damage

DNA through reactions with its amines.

Chemical modification of DNA by RNOS may be an important contributor to the age-

and inflammatory-related development of cancer or other diseases.

7. Activation of adenosine diphosphate- ribosyltransferases25: This leads to ribosylation of ADP which may have a role in the control of vascular tone via a

mechanism independent of cGMP.

It has been suggested that reduced NO bioavailability increases vascular tone by two

mechanisms. An acute decrease in NO levels leads to vasoconstriction due to a

decrease in cGMP production. If NO levels continue to decrease for a longer time,

ADP-ribosylation by NO is altered which leads to an increased sensitivity to

vasoconstrictor agents such as endothelin.