Nitric Oxides

Presented by Group D

Faculty of Medicine and Health Science.

Department of Medicine and General

Surgery.
Group Names
1-Hawa Osman Jimale
2-
3-
4-
5-
6-
7-
8-
9-
10-
 Presentation outline
 Introduction
 Discovery
 Synthesis
 Signalling mechanisms
 Inactivation
 Effects of NO
 Potentiation of NO synthesis
 Inhibition of NO synthesis
 Side effects
 Summary
 Nitric oxide is a gaseous signalling molecule
- readily diffuses across cell membranes
- regulates a wide range of physiologic & pathophysiologic proc
esses (CV, inflammatory & neuronal)

 Endogenous activator of soluble Guanylyl cyclase,

↓
Cyclic
GMP (cGMP),
‘second messenger’
• 1772: 1st identified as a gas by Joseph
Priestly. (atmospheric pollutant)

• 1980s: Nitroglycerin given for patients with

angina. (MOA not known)

Robert Furchgott investigated vasorelaxants (Ach)

 relaxation of blood vessels only occurred if luminal
endothelial cells were present & intact.
↓
Endothelial Derived Relaxing Factor (EDRF)
• 1977: Ferid Murad investigated how
nitroglycerin works  releases NO 
relaxation of smooth muscle cells

• 1986: Louis Ignarro – identified EDRF.

• Comparison of biochemical & pharmacological

properties of EDRF & NO
 NO - major bioactive component of
EDRF.
Nobel prize - 1998
  by NO synthase
isoenzymes: 3 types –

nNOS eNOS iNOS

Neurons Endothelium Macrophages

cardiac Neutrophils
myocytes airway fibroblasts
epithelium vascular
platelets smooth muscle
Constitutive forms cells
 Cont…

•These isoforms generate NO from the amino

acid L -arginine in an O2 and NADPH-dependent
reaction . This enzymatic reaction involves
enzyme-bound cofactors, including heme,
tetrahydrobiopterin, and flavin adenine
dinucleotide (FAD).
Heme
THB
FAD
• nNOS & eNOS – activated
by calcium-calmodulin.

• Factors ↑ eNOS activity

- shear stress
(physiological)
-Protein kinase A-
mediated phosphorylation.
(eg. Β2 agonists)
- tyrosine kinase
activation (eg. Insulin)

• Protein kinase C ↓ eNOS

activity
 iNOS
• Not regulated by Ca.

• Inflammatory mediators (IFN-ϒ)

↓
activation of iNOS gene
↓
accumulation of iNOS
↓
↑ synthesis of NO.

• Inhibited by glucocorticoids & cytokines (transforming

growth factor-β.)
 1.Metalloproteins –
• NO interacts with metals, esp iron in heme.
• Interaction with other metalloproteins  Cytotoxic effects.

Eg.
- NO inhibits metalloproteins involved in cellular respiration
such as citric acid cycle enzyme aconitase and electron
transport chain protein cytochrome oxidase.

- Inhibition of heme-containing cytochrome P450

enzymes (inflammatory liver disease.)
 2. Thiols –
• NO reacts with thiols ( –SH group)  Nitrosothiols.
‘S-nitrosylation’
- Major targets : H-ras, (regulator of cell
proliferation)

• Glutathione can also be S-nitrosylated

 S-nitrosoglutathione.
- endogenous stabilized form of NO, or
- as carrier of NO.

Vascular glutathione ↓ in diabetes mellitus &

atherosclerosis  ↓ S-nitrosoglutathione
3. Tyrosine nitration –
• Addition of nitrate to tyrosines.
• Marker of excessive NO production

• NO + superoxide  Peroxynitrite (ONOO–),

 DNA damage, tyrosine nitration,
& oxidation of cysteine to disulfides.

• Enzymes involved in superoxide formation

- ↑ in inflammatory and degenerative diseases,
 ↑ peroxynitrite levels.
• Intracellular glutathione - scavenges peroxynitrite,
protective.
 S-nitrosylation
NO + HbO  NO− + Hb of Hb
↓
Carries NO in
circulation

Exhaled
via
NO
lungs 2NO + O2  N2O4

NO + O−  ONOO−
 •NO is also inactivated by reaction with O 2 to form nitrogen dioxide. NO
reacts with superoxide, which results in the formation of the highly reactive
oxidizing species, peroxynitrite. Scavengers of superoxide anion such as
superoxide dismutase may protect NO, enhancing its potency and prolonging
its duration of activation.
1) NO  vascular smooth muscle 
vasorelaxation. (Mutant mice lacking eNOS gene
- hypertensive,

2) Also has antithrombotic effects –

Both endothelial cells & platelets contain eNOS,

NO-cGMP pathway

inhibits platelet activation.

Endothelial dysfunction  ↓
3) Protective against atherosclerosis
Mech:
a) Inhibition of proliferation & migration of vascular smooth
muscle cells.

b) ↓ endothelial adhesion of monocytes & leukocytes

(d/t inhibition of expression of adhesion molecules)

c) As anti-oxidant - ↓ formation of foam cells in vessel

wall.

d) ↓ endothelial cell permeability to lipoproteins

* Animals treated with eNOS inhibitors  ↑

2. Septic shock –
• Bacterial endotoxins + cytokines (TNF-α)
↓
macrophages,
Synthesis of neutrophils, T
cells, hepatocytes,
iNOS smooth muscle
cells, endothelial
cells
↓
Exaggerated hypotension
↓
Shock (& death)

• Reversed by iNOS inhibitors (but no overall improvement in

survival in G –ve sepsis)
3. Infection & Inflammation –
• NO has both beneficial and detrimental roles.
• Inflammatory mediators (TNF & IL-1)
↓
Induction of iNOS in leukocytes,
↓
NO synthesis (+ ONOO−) -
important microbicide.
Vasodilation Activates COX-2 Other mech

Acute inflammation
• Both acute & chronic inflammatory conditions –
prolonged or excessive NO production
 exacerbate tissue injury.

• Eg. psoriasis lesions, airway epithelium in asthma,

& inflammatory bowel lesions
- ↑ levels of NO and iNOS,

Persistent iNOS induction  disease pathogenesis.

• Thus inhibition of NO pathway - beneficial effect

4. CNS –

•NO synthesized postsynaptically may function as a retrograde messenger and

diffuse to the presynaptic terminal to enhance the efficiency of neurotransmitter
release and enhances the process of synapse strengthening that underlies
learning and memory
•NO – important role as NT
•Not stored
•Induced at postsynaptic sites in neurons: activation of NMDA receptors,
↓
Ca2+ influx and activation of nNOS.
•Excessive NO synthesis  excitotoxic neuronal death in several neurologic
diseases
(stroke, ALS & Parkinson’s disease)
•NOS inhibitors - ↓ neuronal damage.
5. PNS –
• Nonadrenergic, noncholinergic (NANC) neurons
(GIT & reproductive tracts)
• NO (NANC neurons)  relaxation of the smooth
muscle
in the corpora cavernosa
↓
penile erection

• Phosphodiesterase (PDE5) breakdown of cGMP

PDE-5Is  ↑ NO signalling
(sildenafil, tadalafil etc) - Erectile dysfunction
GI effects –

• Protective effect.
• Preclinical studies –
- maintain gastric mucosal integrity (inhibiting gastric
acid secretion & ↑ gastric blood flow)
- inhibits leukocyte adherence to endothelium,
- repair NSAID-induced damage.

• Epidemiological studies –
use of NO-donating agents with
NSAIDs (CINODs) 
↓ risk for GI bleeding.
• Large conc  deleterious
effects.
6. Respiratory disorders –
• NO inhalation  dilates pulmonary vessels,
↓
↓ pulmonary vascular resistance & ↓ pul artery
pressure.

 also improves oxygenation

(by ↓ ventilation-perfusion mismatch)

- administered to newborns with hypoxic resp failure

• also shown to ↑ cardio-pul functions in adults with PAH.

 Replacement Rx with NO donors

 Dietary supplementation with L-arginine

 Antioxidants - ↓ reactive O2 species

 Drugs that restore endothelial function in patients with

metabolic risk factors for vascular disease
- ACE inhibitors, statins, insulin, oestrogens
  β2-adrenoceptor agonists & related
drugs
↓
activates L-arginine/NO pathway

 PDE inhibition  potentiate NO actions

(PDE degrade cGMP)

Eg. PDE-5Is (sildenafil)

↓
 Release NO or related species
- used clinically to elicit smooth muscle
relaxation.

 Different classes -
1) Organic nitrates
2) Organic nitrites
3) Sodium nitroprusside
 1.Organic nitrates –
• Nitroglycerin  dilates veins and coronary arteries
↓
↓ preload (‘antianginal effect’)

metabolized to NO - by mitochondrial aldehyde reductase,

(venous smooth muscle)

• Others – isosorbide mono/dinitrate

• less significant effects on aggregation of platelets

• Rapid tolerance during continuous administration.
( d/t generation of reactive oxygen species)
2. Organic nitrites –
• Eg. Amyl nitrite
• Arterial vasodilators
• No rapid tolerance seen

• Amyl nitrite (abuse potential)

+
PDE-5Is
↓
Lethal hypotension

Therefore replaced by
nitroglycerin
3. Sodium nitroprusside –
• Dilates both arterioles & venules
• Used for rapid pressure ↓ in arterial hypertension.

• Sodium nitroprusside

Light/chemicals/enzymes

5 CN− + NO
 4.NO gas –
• Inhalation of NO  ↓ pul artery pressure
↑ lung perfusion

• Uses –
1) PAH
2) Acute hypoxemia
3) CPR

• Short-term improvements in pulmonary function

• N2O3 & methemoglobin levels monitored during inhaled

NO Rx.
Inhibitor Mechanism

NGmonomethyl-L-arginine Non-selective, competitive

(L-NMMA) inhibitor
- binds arginine-binding site
in NOS
NG-nitro-L arginine
methyl ester (L-NAME)

ADMA
Inhibitor Mechanism

N-iminoethyl-L- Selective iNOS Rx of inflammatory

lysine, inhibitor conditions (asthma)

Glucocorticoids.
7-Nitroindazole, Selective nNOS Neurodegenerative
inhibitor conditions
S-methyl-L-
thiocitrulline

NO Feedback
inhibition
 L-arginine/ NO pathway – imp role in disease pathogenesis

 Evidence: Following indirect approaches –

1) Analysing nitrate &/or cGMP in urine

2) Administer [15N]-arginine & use mass spectrometry

(to measure the enrichment of 15N over naturally
abundant [14N]-nitrate in urine)
3) Measuring NO in exhaled air

4) Measuring effects of NOS inhibitors (e.g. L-NMMA)

5) Comparing responses to endothelium-dependent

agonists (e.g. acetylcholine)
& endothelium-independent agonists
(e.g.nitroprusside)

6) Measuring responses to ↑ blood flow (‘flowmediated

dilatation’), - largely mediated by NO
7) Studying histochemical appearances &
pharmacological responses in vitro of tissue obtained
at operation
(e.g. coronary artery surgery).

 All these methods have limitations.

 L-arginine/ NO pathway  New therapeutic

approaches
1) Hematological:
Methemoglobinemia
2) CV: Hypotension
3) Respiratory: dyspnoea, stridor
4) Renal: Hematuria
5) Metabolic: Hyperglycemia
6) Others: withdrawal,
sepsis,
cellulitis,
chest discomfort,
 Nitric oxide (NO) is synthesised from L-arginine &
molecular O2 by Nitric oxide synthase (NOS).

 3 isoforms:
Constitutive forms
nNOS,
(Ca2+ - dependent)
eNOS,

iNOS - Ca2+ independent

 Signalling mechanisms –
1) Metalloproteins -
Soluble guanylyl cyclase: main target
2) S-nitrosylation (thiols)
3) Tyrosine nitration

 Inactivation:
Reaction with - haem,
O2,
O−
 In host immune response, acute & chronic inflammatory
conditions.
 Both ↑/↓ production  disease.


NO donors (e.g. nitroprusside &
organic nitrovasodilators) are well
established

 Type V phosphodiesterase inhibitors (e.g. sildenafil,

tadalafil) potentiate the action of NO
- used to treat erectile dysfunction
 Therapeutic uses (NO/NO donors) –
1) Cardio-
vascular:
Angina, HTN
2) Respiratory:
a) Neonatal
hypoxic resp
failure
b) PAH
3) GIT:
NSAID-induced
4) Erectile dysfunction: PDE-5Is

5) Heart & lung surgery

6) Sickle cell disease

7) Potential anti-cancer agent

- refractory cancers
- chemo-/radio-/immune-sensitizer
- eg. NTG
 8) NO supplements –

-muscle gain, enhanced stamina and vigor.

Thank you