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The endothelium is the thin layer of cells that lines the interior
surface of Blood vessels and lymphatic vessels forming an
interface between circulating blood and lymph in the lumen
and the rest of the vessel wall.
The cells that form the endothelium are called endothelial
Endothelial cells in direct contact with blood are called
vascular endothelial cells where as those in direct contact with
lymph are known as lymphatic endothelial cells.
• Endothelial cells release substances acting directly on
vascular smooth muscle cells, causing either contraction
• In 1980 Furchgott & Zawadzki first described endothelium-
dependent relaxation of the blood vessels by acetylcholine.
Further studies in 1984 revealed that other factors such as
bradykinin, histamine and 5-hydroxytryptamine release
endothelium derived relaxing factor (EDRF), which can
modulate vessel tone.
In 1987 Furchgott proposed that EDRF might be nitric oxide
(NO) based on a study of the transient relaxations of
endothelium-denuded rings of rabbit aorta to „acidified‟
inorganic nitrite (NO-) solutions and the observations that
superoxide dismutase (SOD, which removes O2
ENDOTHELIUM DERIVED RELAXING FACTOR
• In 1988 Palmer et al could detect NO production both
biologically and chemically by chemiluminescence. The following
year in 1989 the enzyme responsible for NO production, NO
synthase, was discovered and NO pathway was proposed.
• Neuronal and humoral mediators, e.g. Ach, adrenaline
, noradrenaline , histamine ,5-HT, ATP, adenosine, substance P
, arginine vasopressin (AT/VP), bradykinin, thrombin and Ca++ -
ionophore A 23187 , VEGF , insulin, angiotensin , TNF-α, IL-6
, arginase , asymmetric dimethylarginine
(ADMA), dimethylarginine dimethylaminohydrolase (DDAH), etc.
acting in their corresponding receptors or cellular structures can
affect production and release of NO.
A chemical compound with formula NO is a free radical gas.
It is first identified as endothelial derived releasing factor(E D
R F ).
At high concentration , fight against infectious organism and
At lower concentration helps in regulating the circulatory
and central nervous system.
Nitric oxide differs from other neurotransmitter and
hormones in a way that it is not regulated by
storage, release , or targeted degradation.
No does not require receptor for its action when
synthesized immediately utilized.
Ca++ clamudulin complex is necessary for nitric oxide
The structure and nature of Nitric
Nitric oxide is a di atomic free radical consisting of one atom
of nitrogen and one atom of oxygen.
Lipid soluble and very small for easy passage between cell
Short lived, usually degraded or reacted within a few seconds.
The natural form is a gas.
Synthesis of Nitric Oxide
Nitric oxide is synthesized from L-arginine.
This reaction is catalyzed by nitric oxide synthase, a
1,2,9,4 amino acid enzyme.
H+H3N + NO
When nitric oxide forms in large parts because superoxide
anion has a height affinity for No.
Superoxide anion reduces No bioavailability.
Nitric oxide also binds to the heme moiety of hemoglobin
and heme moiety of enzyme gunayl cyclase , which is
found in smooth muscle cell and most other cells of body.
When NO formed by vascular endothelium it rapidly
diffuses into the blood where it binds to hemoglobin &
subsequently broken down.
It also diffuses into vascular smooth muscle cells adjacent to
the endothelium where it binds to & activate gunyl cyclase .
This enzyme catalyse the dephosprylation of GTP to cGMP
which serve as a second messenger for many important cellular
function , particular for signaling smooth muscle contraction.
cGMP induces smooth muscle relaxation by multiple
increased intracellular cGMP which inhibit ca++ entry into the
cell and decrease intracellular ca++ concentration.
activates k+ channel which leads to hyper polarization &
• Stimulates a cGMP dependent protein kinase that activates
myosin light chain phosphate (MLCK) the enzyme that
dephosphorylate myosin light chain leads to smooth muscle
Types of NOS
NOS I or n NOS
Central and peripheral neuronal cells, brain, spinal cord, platelets.
Ca++ dependent, used for neuronal communication
NOS II or I NOS
Most nucleated cells, particularly macrophages
Independent of intracellular Ca++ and its regulation depend upon de novo
Inducible in presence of inflammatory cytokines, bacterial liposaccharides.
NOS III or e NOS
Present on Vascular endothelial cells and neuronal cells
Nitric Oxide in the human body has many uses which are best
summarized under five categories.
NO in the nervous system
NO in the circulatory system
NO in the muscular system
NO in the immune system
NO in the digestive system
No in the reproductive system
NO in the gene toxicity
No in the apoptosis
Role of nitric oxide
NO is a signaling molecule, but not necessarily a neurotransmitter.
NO signals inhibition of smooth muscle contraction, adaptive
relaxation, and localized vasodilation.
n nos action in C N S have been associated with pain perception in
spinal cord level.
NO diffuses out of the cells making it vescular storage in vesicles and
release by exocytosis
NO does not bind to surface receptors, but instead exits
cytoplasm, enters the target cell, and binds with intracellular guanyl
Present in presynaptic terminal
Nitric oxide in the Nervous system
NO serves in the body as a neurotransmitter, but there are
Role in Neurodegenerative disease
Implicated in :- Alzheimer disease
Amyotrophic leteral sclerosis
All are related to the excessive release of NO & glutamate
But in Parkinson's disease Glial cells produce excessive levels of nitric
oxide, which may be neurotoxic for a sub population of dopaminergic
neurons, especially those not expressing NADPH- diaphorase activity.
The presence of glial cells expressing nitric oxide synthase in the
substantia nigra of patients with Parkinson's disease represents a
consequence of dopaminergic neuronal loss.
Play a role in long term memory
As a retrograde messenger that facilitates long term potentiation
of neurons (memory)
Synthesis mechanism involve Ca2+/Calmodulin
activates Guanyl cyclase
cycle of nerve action potentials
catalyzes cGMP production
Nitric oxide in the circulatory system
NO serves as a vasodilator
Released in response to high blood flow rate and signaling
molecules (Ach and bradykinin)
Highly localized and effects are brief
If NO synthesis is inhibited, blood pressure increases
NO aids in gas exchange between hemoglobin and cells
Hemoglobin is a vasoconstrictor, Fe scavenges NO
NO is protected by cysteine group when O2 binds to
During O2 delivery, NO locally dilates blood vessels to aid
in gas exchange
Nitric oxide in the Muscular system
NO was originally called EDRF (endothelium derived
NO signals inhibition of smooth muscle contraction
Ca 2+ is released from the vascular lumen activating NOS
NO is synthesized from NOS III in vascular endothelial
This causes guanyl cyclase to produce cGMP
A rise in cGMP causes Ca2+ pumps to be activated, thus
reducing Ca2+ concentration in the cell
This causes muscle relaxation
Role in the Immune system
NOS II catalyzes synthesis of NO used in host defense
Activation of NOS II is independent of Ca2+ in the cell
Synthesis of NO happens in most nucleated
cells, particularly macrophages.
NO is a potent inhibitor of viral replication.
NO is a bactericidal agent
NO is created from the nitrates extracted from food near the
This kills bacteria in the mouth that may be harmful to the
Role In the Digestive system
NO is used in adaptive relaxation
NO promotes the stretching of the stomach in
response to filling.
When the stomach gets full, stretch receptors
trigger smooth muscle relaxation through NO
Role In the Reproductive system:
Nos localized in pelvic nerve neuron innervating the
corpora cavrinosa and the neuronal plexuses of the
adventitial layer of the penile arteries – proven most
effective for erectyl dysfunction.
No and its derivatives produced in inflamed tissue
contribute to the carcinogenesis process due to direct
or indirect DNA damage.
Direct DNA damage: DNA deamination , peroxynitrite
induced adult formation single strand break in the
Indirect DNA damage: interaction of NO reactive
species with other molecule like amines , thioles
-NO after reaction with O2/superoxide forms
Role In Genotoxicity:
Role in wound healing & tissue repair
No is powerful stimulator of cell division maturation
and differentiation . Necessary mediator of neuro
vascularization i.e. angiogenesis and lymph ducts to
nourish the healing of tissue.
Role in Apoptosis
Nitric oxide and its reaction products either promotes or
prevent apoptosis .
Pro-apoptosis effect of NO- induction of apoptosis by NO
resulting in the accumulation of tumor suppressor protein
Anti-apoptotic effect of NO- some studies suggest that
endogenous I Nos expression or exposure to low dose of
NO donors inhibits apoptosis.
Role in Inflammation
NO has shown to act as a mediator of inflammatory
processes. This process has enhanced the effect of
cyclooxygenases and stimulates the production of pro-
Prostacyclin (or PGI2) is a prostaglandin member of the
family of lipid molecules known as eicosanoids. It inhibits
platelet activation and is also an effective vasodilator.
In 1960s, ,Professor John Vane, began to explore the role
of prostaglandins in anaphylaxis and respiratory diseases. Sir
John discovered that aspirin and other oral anti-inflammatory
drugs work by inhibiting the synthesis of prostaglandins.
Sir John and a team had identified a lipid mediator they called
“PG-X,” which inhibits platelet aggregation. PG-X, which later
would become known as prostacyclin, is 30 times more potent
than any other then-known anti-aggregatory agent.
E D H F :
In blood vessels Endothelium-Derived Hyperpolarizing
Factor or EDHF is proposed to be a substance or
electrical signal that is generated or synthesized in and
released from the endothelium; its action is to
hyperpolarise vascular smooth muscle cells, causing these
cells to relax, thus allowing the blood vessel to expand in
Both a vascular endothelial cytochrome P450 (CYP450)
product of arachidonic acid metabolism and the potassium
ion (K+) have been identified as endothelium-derived
hyperpolarizing factors (EDHFs) in animal vascular
Pathways Of EDHF:-
There are two general pathways that explain EDHF:-
1. Diffusible factors are endothelium-derived
substances that are able to pass through internal elastic
layer (IEL), reach underlying vascular smooth muscle
cells at a concentration sufficient to activate ion
channels , and initiate smooth muscle
hyperpolarisation and relaxation.
2. Contact-mediated mechanisms bestow endothelial
hyperpolarisation that passively spreads to the smooth
muscle through inter cellular coupling and therefore
EDHF is considered as a solely electrical event.
Although the phenomenon of EDHF has been observed and reported
in scientific literature, to date the chemical identity of the factor(s)
has not been determined.
1) In some cases, members of a class of arachidonic acid
derivatives, the epoxyeicosatrienoic acids (EETs), have been
found to mediate the vasodilatation. These compounds are formed
by epoxidation of any one of four double bonds of the
arachidonic acid carbon backbone by cytochrome p450
Structure of Arachidonic acid
2) In addition, in some cases hydrogen peroxide has
been suggested to function as an EDHF in some
vascular beds; although the validity of this
observation is debated. because the H2O2 candidacy
is questioned by the fact that it may have an
inhibitory action on K+ channels, at least, in some
3) It Has been suggested that EDHF is Potassium Ions
(K+) as the activation of endothelial K-Ca+ channels
causes an efflux of K+ from endothelial cells towards
the extracellular space.
An increase in extracellular K+ has been shown to
activate an ouabain -sensitive electrogenic Na+–K+-
ATPase followed by hyperpolarization and smooth
muscle cell relaxation.
However the involvement of K+ ions into EDHF-
mediated relaxation does not necessarily involve the
activation Na+–K+-ATPase channels. It is more likely
that K+ ions and gap junctions can be involved in
EDHF-mediated relaxation simultaneously, and may
also act synergistically.
4. An alternative explanation for the EDHF
phenomenon is that direct intercellular
communication via gap junctions allows passive
spread of agonist-induced endothelial
hyperpolarization through the vessel wall. In some
arteries, eicosanoids and K+ ions may themselves
initiate a conducted endothelial
hyperpolarization, thus suggesting that electro tonic
signalling may represent a general mechanism through
which the endothelium participates in the regulation
of vascular tone.
INTERACTION B/W NO,EDHF AND
The three main mediators of endothelial vasodilator
function, NO, prostacyclin, and EDHF appear not to be
mutually exclusive and act synergistically in a complex
manner to maintain the health of the vasculature .
In arteries, NO is the predominant endothelium-derived
vasodilator but has relatively less prominent contribution
in the resistance vessels of the microcirculation where
EDHF appears to predominate.
NO may inhibit EDHF responses as some studies could
only demonstrate. EDHF responses once NO production
had been inhibited.
Pharmacological inhibitors Targets Comments
Apamin S KCa
+ Highly specific
+ Can inhibit some Kv channels
+ Highly specific
+ Inhibit other K+channels at
Tetraethybutylammonium S KCa
+ Inhibit other K+channels at
Ouabain Na+/K+ ATPase
Can affect gap junction
activity at >10−4m
KCL K+ currents
Dilates at >10−2m through KIR
and Na+/K+ ATPase activation
18 α-glycyrrhetic acid Gap junctions
effects on membrane currents
Connexin mimetic peptides Gap junctions Highly specific
Catalase Hydrogen peroxide —
EDHF IN DISEASES
Experimental evidence indicates that a shift away from NO-
mediated endothelium-dependent relaxation toward EDHF
dependent relaxation occurs in disease states.
Alteration of EDHF-mediated responses has been reported with
aging, hypertension, atherosclerosis, hypercholesterolemia, heart
failure, angioplasty, eclampsia, diabetes.
EDHF in Hypertension
• Evidence suggests that CYP expression and EET generation are
increased in hypertension and in hypercholesterolemia.
• Polymorphisms within CYP epoxygenases being associated with
an enhanced risk of developing coronary artery disease and
EDHF in diabetes
EDHF-mediated responses are depressed in some models
of type I and type II diabetes with the exception of murine
EDHF in hypercholesterolemia
In hypercholesterolemia, it has been observed that there is
significant contribution of KCa+ channel activation and a
lower Nitric Oxide release with acetylcholine.