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1. Nitric oxide

2. NO Signaling in Cardiomyocytes
• Nitric oxide (NO) is a ubiquitous, naturally occurring molecule
found in a variety of cell types and organ systems.
• In the cardiovascular system, NO is an important determinant of
basal vascular tone, prevents platelet activation, limits leukocyte
adhesion to the endothelium, and regulates myocardial
contractility

3. • NO (molecular weight = 30) is small but plays a big role in physiological
regulation, in the vasculature where its effects were first seen.
• Endothelium derived relaxation factor (EDRF) was discovered its ability to cause
dilatation of vessels by relaxing the arterial muscle layer.
• Only much later was EDRF discovered to be a gas, nitric oxide. More recent
interest in NO is based on the evidence that it is antiatherogenic.
• The pathogenesis of atherosclerosis is complex but many of the known effects of
NO can be implicated in this common and serious condition.

4. • All three forms of NOS-nitric oxide synthases (nNOS or NOS-1, iNOS or
NOS-2, eNOS or NOS-3) found in cardiomyocytes produce NO involved
with cGMP-dependent and cGMP-independent signaling.

5. As shown in Figure nitric oxide is synthesized from the amino acid
arginine under the influence of nitric oxide synthase (NOS). The
term NOS is used to denote a family of three related but distinct
isoenzymes: neuronal NOS (nNOS); endothelial NOS (eNOS,
endothelium and platelets) and inducible NOS (iNOS,
endothelium, vascular smooth muscle and macrophage). In
addition to reduced nicotinamide adenine dinucleotide
phosphate (NADPH) shown in Figure, NOS enzymes also require
flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN)
and tetrahydrobiopterin (BH4) as coenzymes.

7. The nitric oxide synthase reaction .
Nitric oxide is synthesized intracellularly by nitric oxide synthase (NOS). This
reaction is rather complex and involves two successive monooxygenase steps.
In the first step, arginine is converted to N-hydroxyarginine (NOHA), which is
cleaved in the second step to NO and citrulline.

9. • Endothelial NOS (eNOS) and neuronal NOS (nNOS) are found in
the cell types indicated by their names.
• Inducible NOS (iNOS) is found mainly in inflammatory cells. All
these enzymes are homologous and perform the same
reaction, but they differ in their regulatory properties.

10. • Although all three NOS isoforms have been located in the vasculature,
nNOS is the least important in regulating vessel physiology.
• Endothelial NOS is constitutive (always active), producing NO in
nanomolar (10-9 mol/l) quantities, having ‘housekeeping’ roles
maintaining vascular homeostasis.
• However, when iNOS expression is upregulated by the effects of, for
example, cytokines or bacterial toxins causing increased transcription
of the gene, NO production rises in to the micromolar (10-6 mol/l)
concentration range and NO becomes cytotoxic.

11. • Being a gas, NO is freely diffusible and penetrates cell membranes easily. NO
is produced by and acts within the endothelium and platelets but is also a
paracrine hormone targeting vascular smooth muscle cells (VSMC) and white
blood cells.
• A number of the actions of NO in target cells may be explained by its binding
to the haem-containing protein soluble guanylyl cyclase (sGC), an enzyme
which generates a second messenger, cyclic GMP (cGMP) from GTP.
• The heme-protein soluble guanylyl cyclase (sGC) is the intracellular receptor
for nitric oxide (NO). sGC is a heterodimeric enzyme with α and β subunits
and contains a heme moiety essential for binding of NO and activation of the
enzyme. Stimulation of sGC mediates physiologic responses including smooth
muscle relaxation, inhibition of inflammation

13. Action of NO
NO stimulates sGC (soluble Ganylate cyclase) , which produces cGMP. cGMP
activates Protein Kinase G (PKG), which activates multiple targets including
Troponin I (TnI) and L-type calcium channels .

15. Intracellular Mechanisms
• NO also avidly binds to the heme moiety of hemoglobin (in red blood
cells) and the heme moiety of the enzyme guanylyl cyclase, which is
found in vascular smooth muscle cells and most other cells of the
body.
• Therefore, when NO is formed by vascular endothelium, it rapidly
diffuses into the blood where it binds to hemoglobin and is
subsequently broken down. It also diffuses into the vascular smooth
muscle cells adjacent to the endothelium where it binds to and
activates guanylyl cyclase. This enzyme catalyzes the
dephosphorylation of GTP to cGMP, which serves as a second
messenger for many important cellular functions, particularly for
signalling smooth muscle relaxation.

17. • Cyclic GMP induces smooth muscle relaxation by multiple
mechanisms including:
• increased intracellular cGMP, which inhibits calcium entry into the cell,
and decreases intracellular calcium concentrations
• activates K+ channels, which leads to hyperpolarization and relaxation
• stimulates a cGMP-dependent protein kinase that activates myosin
light chain phosphatase, the enzyme that dephosphorylates myosin
light chains, which leads to smooth muscle relaxation.

18. NO-induced production of cGMP is fully or partly responsible for:
• vasorelaxation (the EDRF (Endothelium derived
relaxation factor) effect) in VSMC (vascular smooth muscle cells);
• inhibition of proliferation of VSMC and endothelial cells;
• inhibition of platelet adhesion and aggregation on endothelium;
• inhibition of endothelial apopotosis (at low NO concentration).

19. • NO also exerts its actions independently of the cGMP pathway by the
nitrosylation of sulfhydryl groups on proteins.
• Under physiological conditions,this posttranslational modification affects
the function a wide array of cell proteins, ranging from ion channels to
nuclear regulatory proteins.
Nitrosylation is the general term for covalent incorporation of a nitric oxide "nitrosyl"
moiety into another (usually organic) molecule

20. Significance of nitric oxide (NO)
• Powerful vasodilator—NO-releasing drugs are used in the treatment of
cardiovascular disease
• Inflammatory mediator—inhibition of NO synthesis of interest as a therapeutic
strategy in infection and chronic inflammation
• Neurotransmitter—signaling in the CNS and the autonomic nervous system

21. Vascular Effects of NO
• Vascular actions of NO include the following:
• Direct vasodilation (flow dependent and receptor mediated)
• Indirect vasodilation by inhibiting vasoconstrictor influences (e.g.,
inhibits angiotensin II and sympathetic vasoconstriction)
• Anti-thrombotic effect - inhibits platelet adhesion to the vascular
endothelium
• Anti-inflammatory effect - inhibits leukocyte adhesion to vascular
endothelium; scavenges superoxide anion
• Anti-proliferative effect - inhibits smooth muscle hyperplasia

22. • In its turn,cGMP regulates the cytosolic Ca2+ concentration by controlling
the entry of Ca2+ via ion-specific channels or by preventing the release of
Ca2+ into the cytosol from reservoirs such as the sarcoplasmic reticulum
in muscle cells or the endoplasmic reticulum in many other cell types.
• Examples of cellular effects by NO which are not related to cGMP
include:

23. Examples of cellular effects by NO which are not related to cGMP
include:
1. Inhibition of leucocyte adhesion to endothelium. By acting on the
gene transcription factor called nuclear factor kappa B (NFkB), NO
limits the expression by endothelial cells of monocyte chemotactic
protein-1 (MCP-1) and VCAM-1. As both MCP-1 and VCAM-1 are
involved with pro-inflammatory responses,NOis in this manner an
anti-inflammatory agent;

24. Examples of cellular effects by NO which are not related to
cGMP include:
2. Antioxidant effects. Superoxide (O2
.- ) is a free radical which
contributes to ‘oxidative stress', a process which causes
significant damage of the molecular structure of cells. NO induces
the synthesis of endothelial cell superoxide dismutase (ecSOD)
the enzyme which forms hydrogen peroxide H2O2 from O2
.- . A
positive feedback loop is set up as H2O2 induces eNOS which
produces more NO and so on. This is a protective action as
superoxide which is potentially damaging to cells is ‘buffered' by
NO;

26. Examples of cellular effects by NO which are not related to cGMP
include:
3. Pro-oxidant effects. The antioxidant effect described above occurs
when NO concentrations are low. If the concentration of NO rises,
rather than reducing the damaging effects of O2
.- , the two may
combine to form an even more powerful oxidant called peroxynitrite
(ONOO-);

27. • In addition NO produced by each isoform of NOS can react with superoxide
(O2
−) to form peroxynitrite (ONOO −). Peroxynitrite interacts with lipids,
DNA, and proteins via direct oxidative reactions or via indirect, radical-
mediated mechanisms.
• In vivo, peroxynitrite generation represents a crucial pathogenic mechanism
in conditions such as stroke, myocardial infarction, chronic heart failure,
diabetes, circulatory shock, chronic inflammatory diseases, cancer, and
neurodegenerative disorders.
• NOS1 and NOS3 are constitutively expressed under immunopathological
conditions (e.g. cardiac ishemia aging, cardiac failure) that often result in an
inflammatory response.

29. Examples of cellular effects by NO which are not related to cGMP
include:
4. Mitochondrial function. NO is able to react with transition
metals such as iron, including those contained within haem
groups. Even at low NO concentrations there is competition
between oxygen and NO for reversible binding to cytochrome
c oxidase. If mitochondrial O2 is low, respiration slows, which
may confer antiapoptotic benefit to the cell. As NO
concentration rises and peroxynitrite is formed, electron
transport is irreversibly inhibited, there is increased
production of superoxide and other reactive oxygen species
and apoptosis occurs.