CELL COMMUNICATION

BY NITRIC OXIDE

THE IMPACT A TWO-ATOM MOLECULE HAS ON OUR CELLS

PROCESOS BIOQUMICOS Y METABLICOS
UNIVERSIDAD DE SALAMANCA. FACULTAD DE MEDICINA
PROF. MARGARITA GIGLIONE




CONDE URRERO, RODRIGO
MOREIRA DE SOUSA COLMENTE, ANDR TIAGO
MOSQUERA FOUFE, MARIO
MOSTEIRO CADAVAL, ALEJANDRA
NAVAS LVAREZ, SARA

CURSO 2013-2014
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INDEX

1. BRIEF INTRODUCTION TO CELL COMMUNICATION. INTRACELLULAR RECEPTORS

2. NITRIC OXIDE COMMUNICATION DISCOVERY

3. NITRIC OXIDE MOLECULE. NITRIC OXIDE SYNTHESIS
a. eNOS
b. nNOS
c. iNOS

4. CELL COMUNICATION BY NITRIC OXIDE
a. Smooth Muscle
b. Blood vessels. Penis stimulation
c. Macrophages and neutrophils.

5. NITRIC OXIDE PHYSIOLOGICAL PROPERTIES

6. VASCULAR PATHOLOGY RELATED TO NO
a. Hypertension
b. Hyperlipemia
c. Diabetes
d. Arteriosclerosis

7. CLINICAL APPLICATION
a. Vascular Therapy
b. Nitro-glycerine
c. Viagra

8. CONCLUSION

9. BIBLIOGRAPHY


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1. Brief introduction to cell communication. Intracellular receptors
Communication between cells is mediated mainly by extracellular signal molecules. Most
cells in multicellular organisms both emit and receive signals. Reception of the signals
depends on protein receptors, which bind the signal molecule, causing the receptor to
activate. The receptor may now activate one or more intracellular signal pathways, which
transmit the signal to the intracellular targets (generally, effector proteins).
Finally, it turns out as a change in cell behaviour, which includes several different
mechanisms: gene regulation, ion channel to open or close, metabolic pathways, etc.

According to the type of receptor protein involved, cell communication may be divided
into two main categories:
- Communication mediated by extracellular receptors
- Communication mediated by intracellular receptors
In order to understand the effect nitric oxide has on target cells, we first need to
understand how intracellular receptors work.
Intracellular Receptors
Intracellular receptors are designed to bind small and hydrophobic molecules, which can
cross the cellular membrane by simple diffusion.
The receptor protein consists in three different domains:
- Its proper amino acid sequence.
- Specific region where signal binds.
- Region where the inhibitor binds (otherwise, the protein would always be active).
When the signal enters the cell, it binds to the receptor and causes a conformation on it,
therefore the inhibitor is released. This way, the receptor is activated. Most of the
responses, include protein synthesis.
The receptor (linked to the signal) usually enters the nucleus as a dimer. When in the
nucleus, it binds to a specific sequence of the DNA and it activates or restrains actions in
the cell (according to the type of signal).

2. Nitric Oxide Communication Discovery
As it usually happens in science, Nitric Oxide discovery by Furchgott was thanks to two
casual circumstances.
That first one was a change in the way he used to study arteries: from bands to arrows.
The second one, the fact that one of his laboratory technicians had put acetylcholine on an
already contracted artery. Furchgott observed its dilatation (instead of the presumed
contraction).
These evidences made him suggest (in 1980) the existence of something he named as
EDRF (Endothelium-Derived Relaxing Factor).
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After some speculation associating EDRF to a free radical, it was not until 1987 when both
Palmer and Moncada demonstrated simultaneously that EDRF was nothing but Nitric
Oxide.

3. Nitric Oxide Molecule.

Nitric oxide is a gas produced in every combustion process, since a cars motor to the
cigarette smoke. It is part of environmental pollution and until some years ago it was
considered a toxic molecule.
However, recent studies have shown that nitric oxide plays various roles due to its
molecular structure. It consists of a bond between a nitrogen atom to another of oxygen,
being the smallest molecule in our body. Consequently, it can freely diffuse through the
membrane.
Due to its unpaired electron, it is very reactive; its lifetime is just a few seconds but it is an
important cellular signalling molecule, involved in vascular and airway tone, peristalsis,
angiogenesis and insulin secretion.

Nitric Oxide synthesis
Nitric oxide is produced by deamination of L-arginine into citrulline, in a reaction catalysed
by nitric oxide synthases (NOSs). This reaction can be noticed in mammals, fish, birds or
even bacteria. The various isoforms of these enzymes are: eNOS (endothelial NOS), nNOS
(neuronal NOS), iNOS (inducible NOS) and bNOS (bacterial NOS).
Nitric oxide produced by eNOS is involved in regulation of cardiac function and
angiogenesis, it is a potent vasodilator identical to endothelium-derived relaxing factor
(EDRF), dilating blood vesses and relaxing smooth muscle.
NO binds to guanylate cyclase and induces smooth muscle relaxation by increasing
intracellular cGMP that will inhibit calcium entry into the cell or activating potassium
channels leading to hyperpolarization and relaxation.
The neuronal isoform nNOS is involved in the development of nervous system and in
protecting heart against cardiac arrhythmia (tachycardia or bradycardia) induced by
myocardial infarctions.
Inducible isoform iNOS plays a major role on immune defense against pathogens or tumor
growth and may cause a septic shock.
Bacterial NOS can protect bacteria against oxidative stress or antibiotics.

4. Cell communication by Nitric Oxide
Nitric Oxide Gas (NO) signals by directly regulating the activity of specific proteins inside
the target cell.
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NO acts as a signal molecule in both animals and plants. Even some bacteria can detect a
very low concentration of NO.
Because dissolved NO passes readily across membranes, it rapidly diffuses out of the cell
where it is produced and into neighbouring cells. As it has a short life (5-10 seconds, after
words, it is transformed into nitrates and nitrites), it can only act locally.
In some target cells NO action consists on binding to iron in the active site of the enzyme
guanylyl cyclase, stimulating this enzyme to produce cyclic GMP. Thus, guanylyl cyclase
acts both as an intracellular receptor for NO and as an intracellular signalling protein.
NO can increase cyclic GMP in the cytosol within seconds, which has different effects
depending on the cells where NO binds. Some examples are listed below:
- Cyclic GMP on smooth muscle cells causes muscle to relax. It has this role in the walls
of blood vessels, for example.
This effect of NO on blood vessels provides an explanation for the mechanism of
action of nitro-glycerine, which has been used to treat patients with angina (pain
resulting from inadequate blood flow to the heart muscle). (See 4.1).
- NO released by autonomic nerves in the penis, for example, causes the local blood
vessel dilation that is responsible for penile erection.
- NO is also produced by activated macrophages and neutrophils to help them to kill
invading microorganisms.
NO can also signal cells independently of cyclic GMP. It can, for example, alter the activity
of an intracellular protein by covalently nitrosylating thiol (SH) groups on specific
cysteines in the protein.

5. Nitric Oxide Physiological Properties
Nitric Oxide is most probably one of the best drugs a vascular surgeon could use. This is
due to its physiological properties, which include: vasodilatation, platelet antiagregation,
inhibition of smooth muscular division.
Nevertheless, our organism has this molecule as a natural fact. Thus, this molecule is ideal
and its deficit is involved in most of vascular pathologies.
As NO is responsible for arterial dilatation, one of its functions is to regulate arterial
pressure.
Through cGMP synthesis, NO is also capable of avoiding platelet aggregation.
In order to inhibit leucocyte association and smooth muscular proliferation, NO blocks
genetic expression of adhesion proteins (such as P-selectine for platelets and B-integrins
for leucocytes). Thanks to this mechanism, NO inhibits growth factors segregation, those
factors are involved in changing smooth muscular cells activity from contractile to
secretor. It is known that this secretory activity is responsible for atheroma plaques to
appear.
Furthermore, NO acts as a neurotransmitter in the Central Nervous System.

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6. Vascular Pathology related to NO
Under those circumstances in which the endothelium capacity to produce NO decreases,
we are dealing with endothelial dysfunction. Given that levels of NO will descend, all its
physiological properties will be lost, causing a series of well-known illnesses to appear;
such as: arteriosclerosis, thrombosis, vasospasm

6.1. HYPERTENSION
Hypertension may be caused by a diminution on NO levels. Although the exact
mechanism is still unknown, arterial hypertension (AHT) could be brought about by a
lack or diminution of blood-vessel-relaxation endothelium dependent (EDRF,
Endothelium Derived Relaxing Factor).

6.2. HYPERLIPEMIA
In patients suffering from lipid alterations, it has been demonstrated the existence of
an alteration in blood vessel dilatation; even before the apparition of structural
changes in the vessel wall.
In fact, it has been proved that this endothelial dysfunction (and the consequent NO
diminution) could be reverted by hexogen administration of NO precursor (L-arginine);
as well as, decreasing cholesterol levels.

6.3. AUTOINMUNE DIABETES MELLITUS:
Insulin-dependent diabetes mellitus (IDDM or type I) accounts for 10% of all cases of
diabetes. It is believed to be the consequence of specific destruction of insulin
secreting -cells found in islets of Langerhans.
-cells destruction is the result of autoimmune process.
Nitric Oxide has been evidenced to have an active role on this destruction, which
involves two different signal paths: a) NO free radicals may destroy specific cell
proteins b) Cytokine and endotoxin-induced nitric oxide production by macrophages
potently inhibits mitochondria activity.
This evidence suggests NO could be one of the causes for type I diabetes apparition.

6.4. ARTERIOSCLEROSIS:
Those patients who suffer from a peripheral arterial disease (PAD), resemble to have
lower NO levels than those from control groups. This deficit is even bigger as ischemia
levels increase.
On the other hand, it has been evidenced that NO inhibits arteriosclerosis
development.
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Therapeutic interventions over risk factors (such as: tobacco, hyperlipemia) have
demonstrated to be useful (reducing mortality and mobility).
Likewise, among these steps, it has been proved a slight recovery from endothelial
dysfunction, leading to a NO basal levels recuperation.

Thus, how does NO inhibits arteriosclerosis development? This is thanks to an
inhibition of genetic expression towards vascular-adhesion molecules (like V-CAM-1)
and cytokines. This results on a decrease of endothelium monocyte interaction.
Among other multiple mechanisms, NO also inhibits SMC (Smooth Muscular Cells)
proliferation, and consequently, avoids platelet aggregation and growth factors
release. Both platelets and growth factors would contribute to the atheroma
development.


7. Clinical Applications

7.1. Vascular Therapy
Bypass' realization, an angioplasty, one endarterectomy or implant a stent, causes
endothelial damage.
The arteriosclerotic endothelium is not capable of reaction to this aggression. Under
normal conditions, it would release protective substances, among which NO is
included. These vascular procedures promotes the thrombogenic ability of the body
and induce the expression of vasoactive molecules, cytokines, and growth factor,
which can lead to procedure's failure.
In order to avoid this pathological vascular response, research is being made about the
utilisation of NO, due to it endothelium protective properties. Those include:

A) Using NO donors systemically. This would lead to inhibiting platelet adhesion and
the subsequent CML proliferation to a vascular procedure, avoiding restenosis.
B) Increase the endogenous production of NO by the NO precursor, L-arginine.
Preliminary studies using diets enriched with L-arginine, or administered
intravenously, have managed to increase the flow in the calf muscle, increase
claudication distance and inhibit platelet aggregation in patients with PAD
C) Genetic transfers, which increase NO production.


7.2. Nitro-glycerine
Autonomic nerves in the vessel wall release acetylcholine; the acetylcholine acts on
the nearby endothelial cells that line the interior of the vessel; and the endothelial
cells respond by releasing NO, which relaxes the smooth muscle cells in the wall,
allowing the vessel to dilate. This effect of NO on blood vessels provides an
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explanation for the mechanism of action of nitro-glycerine, which has been used for
about 100 years to treat patients with angina (pain resulting from inadequate blood
flow to the heart muscle). The nitro-glycerine is converted to NO, which relaxes blood
vessels. This reduces the workload on the heart and, as a consequence, reduces the
oxygen requirement of the heart muscle. Many types of nerve cells use NO more
directly to signal to their neighbours.
4.2 Viagra
The drug Viagra, and its newer relatives, inhibit the cyclic GMP phosphodiesterase in
the penis, thereby increasing the amount of time that cyclic GMP levels remain
elevated in the smooth muscle cells of penile blood vessels after NO production is
induced by local nerve terminals. The cyclic GMP, in turn, keeps the blood vessels
relaxed and thereby the penis erect.


8. CONCLUSION
Nitric Oxide is a basic molecule to assure a correct vascular activity. It is therefore essential
for a vascular surgeon to understand its properties, as NO acts as an endothelium
protector (NO inhibits growth factors, cytokines).
Research is open to new clinical applications focused on endothelial regeneration after
angioplasty, bypass
To sum up, there is still a lot to discover before NO becomes just as a common element in
daily therapy.

9. BIBLIOGRAPHY

Alberts, B. Molecular Biology of the Cell

Nitric Oxide: Biology and Pathobiology. Louis J. Ignarro

C. Lpez-Espada. xido Ntrico: Puesta al da

W. K. Alderton, C. E. Cooper and R. G. Knowles. Nitric oxide synthases : structure,
function and inhibition