Nitric Oxide

First discovered as a potent vasodilator in 1980 by Furchgott and Zawadski,

nitric oxide was first termed endothelium-derived relaxing factor (EDRF). Studies
showed that acetylcholine caused the secretion of a molecule, which would
instigate relaxation of the smooth muscle in the vasculature, and this was initially
thought to be a result of an arachidonic acid derivative via the lipoxygenase
pathway. It wasn't until 1986 when Furchgott and Ignarro executed extensive
research into the properties of the unknown lignad, that it was independently
identified by them as nitric oxide.
Synthesis of nitric oxide occurs through nitric oxide synthase enzymes,
which can be found in three isoforms, one inducible form and two constitutive
forms. iNOS or NOS-II, is inducible and is produced in response to intruding
pathogen. It can be found on Kupffer cells, macrophages, neutrophils, fibroblasts
and vascular smooth muscle and exploits the oxidative stress of nitric oxide as a
free radical towards the destruction of foreign microogranisms. iNOS is found in
much greater abundance than the other synthases and generally has a higher
activity leading to greater amounts of NO. The two constitutive forms are eNOS
(NOS-III) and nNOS (NOS-I) and these are located mainly on endothelium but
also can be observed on cardiac myocytes, osteoblasts, osteoclasts, platelets,
respiratory epithelium and renal mesangial cells. Mutant cells, which lack the
eNOS coding gene, are hypertensive demonstrating the impact of NO on blood
pressure maintenance.
The number of NOS present in a given cell is often the limiting factor in
nitric oxide levels as a result of profuse amounts of L-Arginine, the vasodilator's
precursor, being present in the cytoplasm of the endothelial cell. However, in
cases such as hypercholesterolaemia, where NO volumes are reduced, high doses
of the ariginine substrate can act as a form of treatment. This is thought to result
from a number of factors, including a possible depletion of ariginine and
competition of NOS with endogenous inhibitors such as asymmetric
dimethylarginine, a molecule found in increased concentration in patients
suffering from hypercholesterolaemia.
Control of nitric oxide synthesis differs between the constitutive and
inducible forms of NOS. iNOS is “induced” by a catalogue of factors, most
relating to an invasion of foreign microogranisms. Bacterial lipopolysaccharide,
found on the pathogen's cell membrane, or the cytokines produced in response to
this molecule such as γ interferon can activate iNOS. One can also a process of
synergy occurring between TNF-α and IL-1, which are not able to induce the
enzyme on their own but have the potential to amplify a response already initiated
by interferons. Conversely, iNOS can be inhibited by other cytokines such as
TGF-ß as well as by glucocorticoids.
Control of the constitutive forms relies upon an intracellular sequence
dependent upon the calcium-calmodulin complex however this convergent step
can be reached through two processes. Firstly, specific regions of the eNOS can be
phosphorylated through a protein kinase and this can alter the activity of the
enzyme depending on the current concentration of the calcium-calmodulin.
Secondly, agonists such as ACh and substance P, which will act upon the
endothelium, will increase the intracellular levels of Ca2+ thus increasing the
concentration of calcium-calmodulin. This complex directly activates eNOS and
nNOS. In terms of human physiology, vasodilation occurs as a result of increased
blood pressure and this is monitored through stretch receptors found on the
vasculature. Transduction of the physical stimulus through a serine-threonine
protein kinase, known as Akt or Protein Kinase B, increases activity of eNOS.
Protein kinase A also causes a rise in nitric oxide synthesis through
phosphorylation of eNOS while protein kinase C phosphorylates regions in the
calcium-calmodulin domain so decreasing eNOS activity. eNOS activity can also
be upregulated by insulin through activation of tyrosine kinase.

Nitric oxide acts rapidly in the human anatomy and is quickly broken down
because of its short half life and probably its ability as a toxin. Its reaction with
the superoxide anion produces the perioxynitrite anion, which is extremely
poisonous.
 These reaction equations show that initially nitric oxide reacts with oxygen

to form N2O4, which eventually reacts with water to form nitric and nitrous acids.
These can be oxidised to nitrate by oxyhaemoglobin. The haem group of
haemoglobin has a very high affinity for NO, 10000 time greater than oxygen.
Unless O2 is present, NO will bind to haem comfortably however it is rapidly
converted to nitrate and the haem iron oxidised to methaemoglobin when O2 is
available. More importantly, NO will bind reversibly with a specific cysteine
residue found on the globin molecule. This newly-formed S-nitrosylated
haemoglobin allows carrying of NO and is involved in a number of aspects such
as control of blood pressure, blood pressure and vascular resistance.
Physiologically, nitric oxide's primary effect is to vasodilate and this occurs
through activation of soluble guanylate cyclase, an enzyme located in vascular
and nervous tissue. NO binds to the haem group of the enzyme thus activating it
and elevating the levels of the second messenger cGMP intracellularly. Evidence
of this mechanism can be seen on the administration of guanylate cyclase
inhibitors such as ODX). The biochemical implications of cGMP have been
exploited clinically through the breakdown of the second messenger. Drugs such
as Viagra, pharmacologically known as sildenafil, inhibit phosphodiesterase V, the
enzyme responsible for degradation of cGMP. This leads to a sustained elevation
of cGMP and thus a prolonged vasodilation of the vasculature, which in this
particular treatment addresses the corpora cavernosa of the penis and aids in
erectile dysfunction. Other functions of NO involve its significance as a
neurotransmitter in the central nervous system, where it has a role in plasticity via
long-term potentiation, the limitation of aggregation and adhesion in platelets and
its use as a weapon in the immune system, specifically in destruction of
pathogens. Its effects can also be seen to act in certain pathologies e.g.
Excitotoxicity in Hungtidon's disease and atherogenesis in individuals with
Diabetes Mellitus.
After NO promotes activity of gunylate cyclase, the increased cGMP
levels result in a rise of activated Protein kinase G as well as affecting
phosphodiesterases, ion channels and other proteins. PKG inhibits smooth muscle
contraction through phosphorylation of myosin light chain kinase (MLCK) thus
reducing contractility. NO will also hyperpolarise the smooth muscle cells through
opening of K+ channels as well as inhibit adhesion and migration of monocytes,
platelets and the proliferation of fibroblasts. Large amounts of NO will be useful
in the defence against pathogens but will pathologically cause destruction of
neurons. Other effects comprise the vasodilation characteristically seen during
pregnancy and in the reduction of peripheral vascular resistance in the
homeostasis of blood pressure.
Due to the versatility of NO's effects in the body, it has often been exploited
clinically as a treatment to a diverse array of conditions. Glyceryl trinitrate is a
nitric oxide donor drug and has been used to treat angina and heart failure. It can
be administered through a patch though as a side effect, it can cause pulmonary
oedema. NO can also be provided as a gas through breathing to relieve neonatal
respiratory distress syndrome. Its infusion into ventilated alveoli dilates blood
vessels, reducing the risk of intrapulmonary shunting and pulmonary
hypertension. The possibility of NOS inhibitors, such as L-NMMA, as
therapeutic agents is still currently being researched in the treatment of conditions
involving NO overproduction such as in neurodegenerative conditions.
Investigations into the mechanisms and biochemistry of nitric oxide have
proved very useful not only in understanding how the gas acts but also in how it
can

be manipulated pharmacologically to encourage the development of relevant

clinical drugs. For example, research demonstrating reduced nitric oxide levels in
those of african descent has given rise to the extremely effective nitrate drug
BiDil, which though perhaps considered unethical because of its racial
characteristic, has proved very useful in the treatment of congestive heart failure.
Further research such has this has to be done for more revolutionary forms of
treatment, which exploit the impact NO has on the body, to be established.