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MIRACLE Molecule(NO)&The SILENT killer (CO)!!!

As signalling molecules

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Nitric oxide(NO)

Joseph Priestly

Discovered in 1772 by Joseph Priestly

He referred to it “nitrous air”.

A colorless and a toxic gas

Since then, it has received the label of being a toxic gas and an air pollutant until over two hundred years

He had also discovered “Oxygen”

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THE NOBEL ASSEMBLY AT KAROLINSKA INSTITUTE

The Nobel Assembly at Karolinska Institutet awarded Nobel Prize in Physiology & Medicine in October 12,1998 jointly to Robert F. Furchgott, Louis J. Ignarro and Ferid Murad for their discoveries concerning“nitric oxide as a signalling molecule in the cardiovascular system".

Robert F Furchgott

Louis J Ignarro

Ferid Murad

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5 What is Nitric Oxide?

First described in 1979 as a potent relaxant of peripheral vascular smooth muscle.

Used by the body as a signaling molecule. Serves different functions depending on

body system. i.e. neurotransmitter, vasodilator, bactericide.

Environmental Pollutant First gas known to act as a biological

messenger

6 The structure and nature of Nitric Oxide

Nitric oxide is a diatomic free radical consisting of one atom of nitrogen and one atom of oxygen

Lipid soluble and very small for easy passage between cell membranes

Short lived, usually degraded or reacted within a few seconds

The natural form is gas The nitrogen atom in NO is derived from the terminal guanidino group of L-

arginine

N O

7 Synthesis of Nitric Oxide

Nitric oxide is synthesized from L-arginine This reaction is catalyzed by nitric oxide

synthase, a 1,294 aa enzymeCOO-

C

(CH2)3NH

C

H2N

H

NH2+

+H3N

Arginine

NOS

NADPH+ O2

NAD+

COO-

C

(CH2)3NH

C

H+H3N

N+

H2NH

OH

N-w-Hydroxyarginine

COO-

C

(CH2)3NH

H+H3N

+ NONOS

CO NH2

Citrulline

8 Types of NOS

NOS I / nNOS Central and peripheral neuronal cells Ca+2 dependent, used for neuronal communication

NOS II / iNOS Most nucleated cells, particularly macrophages Independent of intracellular Ca+2 Inducible in presence of inflammatory cytokines

NOS III / eNOS Vascular endothelial cells Ca+2 dependent Vascular regulation

9 Properties of NOS

NOS l & NOS lll are called as constitutive forms of NOS They produce less NO in the body

On the other hand NOS ll / iNOS produces more NO in the body because

1. Its high concentration & Activity in the body2. Pathological conditions are associated with the cytokines

10 Properties of NOS All three NOS are isoenzymes and are dimers Similar/homologous with cytochrome P450 Each isoform contains iron protoporphyrin ix (heam) , flavin

adenin dinucleotide (FAD) , flavin mononucleotide (FMN) & thetrahydrobiopterin (H4B) as bound prosthetic group

These prosthetic group and the ligand which is going to bind to the enzyme in the presence of the reduced NADPH control the assembly of the enzyme into “The Active Dimer form”

11 Properties of NOS NOS enzymes are functionally BIMODAL in nature which is

associated with distinct structural domains The oxygease domain binds to Heam while reductase bind

to calcium calmodulin , FMN , FAD , NADPH NOS enzymes are the only Flavo Heam enzymes that use

H4B as a redox cofactor The crystal nature of the NOS heam (oxygenase) domain in

iNOS & eNOS has revealed how L arginine heam & H4B bind in the active site

12 Regulation L-Arginine is usually present in excess in endothelial cell cytoplasm, so the

rate of production of NO is determined by the activity of the enzyme rather than by substrate availability.

Nevertheless, very high doses of L-arginine can restore endothelial NO biosynthesis in some pathological states (e.g. hypercholesterolaemia) in which endothelial function is impaired. Possible explanations for this paradox include:

compartmentation: i.e. existence of a distinct pool of substrate in a cell compartment with access to the synthase enzyme, which can become depleted despite apparently plentiful total cytoplasmic arginine concentrations

competition with endogenous inhibitors of NOS such as asymmetric dimethylarginine (ADMA), which is elevated in plasma from patients with hypercholesterolaemia

reassembly/reactivation of enzyme in which transfer of electrons has become uncoupled from L-arginine

relative depletion of arginine, which can inhibit NOS activity by inhibiting translation of iNOS mRNA

13 Activation of NOS

Glutamate neurotransmitter binds to NMDA receptors

Ca++ channels open causing Ca influx into cell Activation of calmodulin, which activates NOS Mechanism for start of synthesis dependent on

body system NO synthesis takes place in endothelial cells,

lung cells, and neuronal cells

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Http://www.kumc.edu/research/medicine/biochemistry/bioc800/sig02-06.htm

15 What is the role of Nitric Oxide in the human body? 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

16 Nitric Oxide in the Nervous System

Nitric oxide as a neurotransmitter NO is a signaling molecule, but not necessarily a

neurotransmitter NO signals inhibition of smooth muscle contraction, adaptive

relaxation, and localized vasodilation Nitric oxide believed to play a role in long term memory

Memory mechanism proposed is a retrograde messenger that facilitates long term potentiation of neurons (memory)

Synthesis mechanism involving Ca/Calmodulin activates NOS-I NO travels from postsynaptic neuron back to presynaptic neuron

which activates guanylyl cyclase, the enzyme that catalyzes cGMP production

This starts a cycle of nerve action potentials driven by NO

17 Is Nitric Oxide a “neurotransmitter?”

NO serves in the body as a neurotransmitter, but there are definite differences between other neurotransmitters used commonly in the body NO is synthesized on demand vs. constant synthesis NO diffuses out of the cells making it vs. storage in vesicles and

release by exocytosis NO does not bind to surface receptors, but instead exits in

cytoplasm, enters the target cell, and binds with intracellular guanylyl cyclase

Similarities to normal NTs Present in presynaptic terminal Natural removal from synaptic junction

18 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 shoots

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

hemoglobin During O2 delivery, NO locally dilates blood vessels to aid

in gas exchange Excess NO is picked up by HGB with CO2

19 Nitric Oxide in the Muscular System

NO was orginally called EDRF (endothelium derived relaxation factor)

NO signals inhibition of smooth muscle contraction Ca2+ is released from the vascular lumen activating NOS NO is synthesized from NOS III in vascular endothelial

cells This causes guanylyl cyclase to produce cGMP A rise in cGMP causes Ca+2 pumps to be activated, thus

reducing Ca2+ concentration in the cell This causes muscle relaxation

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21 Endothelium-derived NO acts locally on underlying vascular

smooth muscle or on adherent monocytes or platelets. The potential for action at a distance is neatly demonstrated by

Rhodnius prolixus, a blood-sucking insect that produces a salivary vasodilator/platelet inhibitor with the properties of a nitrovasodilator. This consists of a mixture of nitrosylated haemoproteins, which bind NO in the salivary glands of the insect but release it in the tissues of its prey.

The consequent vasodilatation and inhibition of platelet activation presumably facilitates extraction of the bug's meal in liquid form.

A strong, but still controversial, case has been made that NO can also act at a distance in the mammalian circulation via reversible interactions with haemoglobin

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• Haem has an affinity for NO > 10 000 times greater than for oxygen. In the absence of oxygen, NO bound to haem is relatively stable, but in the presence of oxygen NO is converted to nitrate and the haem iron oxidised to methaemoglobin.

• Distinct from this inactivation reaction, a specific cysteine residue in globin combines reversibly with NO under physiological conditions.

• The resulting S-nitrosylated haemoglobin is believed to be involved in various NO-related activities, including the control of vascular resistance, blood pressure and respiration.

23• Key features of the proposed mechanism include the following. • Nitrosylation of haemoglobin is reversible. • It depends on the state of oxygenation of the haemoglobin,

which consequently takes up NO in the lungs and releases it in tissues, in concert with release of oxygen.

• Haemoglobin acts as an O2 sensor and could regulate vascular tone (and hence tissue perfusion) in response to the local partial pressure of O2 by releasing NO in this way. This mechanism is impaired in sickle cell disease (a common inherited disorder caused by a molecular variant of haemoglobin).

• NO is not released into the cytoplasm of erythrocytes (where it would promptly be inactivated by haem), but is transported out of the red cells via cysteine residues in the haemoglobin-binding cytoplasmic domain of an anion exchanger called AE1.

• S-nitrosylated albumin also constitutes a source of circulating NO bioactivity. An alternative view is that nitrite anion, rather than nitrosylated protein, is the main intravascular NO storage molecule

24 Nitric Oxide in the Immune System

NOS II catalyzes synthesis of NO used in host defense reactions Activation of NOS II is independent of Ca+2 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 gums This kills bacteria in the mouth that may be harmful to

the body

25 Nitric Oxide 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 releasing neurons

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Nitric Oxide MetabolismNO may also be involved in the regulation of protein activity through S-nitrosylation. In the extracellular milieu, NO reacts with oxygen and water to form nitrates and nitrites.

NO toxicity is linked to its ability to combine with superoxide anions (O2–) to form peroxynitrite (ONOO–), an oxidizing free radical that can cause DNA fragmentation and lipid oxidation.

In the mitochondria, ONOO– acts on the respiratory chain (I-IV) complex and manganese superoxide dismutase (MnSOD), to generate superoxide anions and hydrogen peroxide (H2O2), respectively.

27 By analogy with cytochrome P450, it is believed that the

flavins accept electrons from NADPH and transfer them to the haem iron, which binds oxygen and catalyses the stepwise oxidation of L-arginine, via a hydroxyl-arginine intermediate, to NO and citrulline.

In pathological states, the enzyme can undergo structural change leading to electron transfer between substrates, enzyme cofactors and products becoming 'uncoupled', so that electrons are transferred to molecular oxygen, leading to the synthesis of superoxide anion rather than NO.

This is important, as superoxide anion is a reactive oxygen species and reacts with NO to form a toxic product (peroxynitrite anion)

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Nitric Oxide MetabolismSIGMA-ALDRICH

29 New research ideas involving Nitric Oxide

The role NO might play in neuronal development

The mechanism of NO inhibiting the different forms of NOS

Diazeniumdiolates as NO releasing drugs Excessive NO release as the cause of most

brain damage after stroke

30Silent Killer !!!

31 Carbon Monoxide

Carbon monoxide (CO) is a colorless, odorless, and tasteless gas that is slightly less dense than air. It is toxic to humans when encountered in concentrations above about 35 ppm

Carbon monoxide consists of one carbon atom and one oxygen atom, connected by a triple bond that consists of two covalent bonds as well as one dative covalent bond. It is the simplest oxocarbon

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In biology, carbon monoxide is naturally produced by the action of heme oxygenase 1 and 2 on the heme from hemoglobin breakdown.

This process produces a certain amount of carboxyhemoglobin in normal persons, even if they do not breathe any carbon monoxide. Following the first report that carbon monoxide is a normal neurotransmitter in 1993, as well as one of three gases that naturally modulate inflammatory responses in the body

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Carbon monoxide is produced naturally by the human body as a signaling molecule. Thus, carbon monoxide have a physiological role in the body, such as a neurotransmitter or a blood vessel relaxant. 

Because of carbon monoxide's role in the body, abnormalities in its metabolism have been linked to a variety of diseases, including neurodegenerations, hypertension, heart failure, and inflammation.

34The most common symptoms of carbon monoxide poisoning may resemble other types of poisonings and infections, including symptoms such as headache, nausea, vomiting, dizziness, fatigue, and a feeling of weakness.

Affected families often believe they are victims of food poisoning. Infants may be irritable and feed poorly. Neurological signs include confusion, disorientation, visual disturbance, syncope and seizures.

Some descriptions of carbon monoxide poisoning include retinal hemorrhages, and an abnormal cherry-red blood hue

35Sources of Carbon Monoxide carbon monoxide has received a great deal of clinical

attention as a biological regulator. In many tissues, all three gases are known to act as anti-inflammatories, vasodilators, and encouragers of neovascular growth.

However, the issues are complex, as neovascular growth is not always beneficial, since it plays a role in tumor growth, and also the damage from wet macular degeneration, a disease for which smoking (a major source of carbon monoxide in the blood, several times more than natural production) increases the risk from 4 to 6 times.

Endogenous Exogenous Methylene chloride

36Sources of Carbon Monoxide Endogenous:

› Normal heme catabolism: Only biochemical

reaction in the body known to produce CO.

› Levels increased in: Hemolytic anemia. Sepsis

37Sources of Carbon Monoxide Exogenous:

› House fires.› Gas–powered

electrical generators.› Automobile exhaust.› Propane-powered

vehicles.› Heaters.› Camp stoves.› Boat exhaust.› Cigarette smoke.

38Sources of Carbon Monoxide

Methylene chloride:› Paint and adhesive

remover.› Converted to CO in

the liver after inhalation.

39What’s the problem with CO?

Kills 5,000 people in US each year

Survivors of CO poisoning can suffer from brain damage, loss of sight or hearing, or heart problems

40What are the signs of CO

poisoning? Headache Nausea Vomiting Dizziness Confusion Tiredness

Weakness Sleepiness Tightness in

chest Trouble

breathing All of these

are flu- like symptoms

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42 Mechanism of action of CO CO combines reversibly with the oxygen binding

sites of hemoglobin and has an affinity Hemoglobin about 220times that of oxygen. The product formed carboxyhemoglobin, cannot transport oxygen.

43 Oxyhemoglobin

oxygen

Thus brain and heart are most affected

The free carboxy group present combines with hemoglobin, thus forming carboxy hemoglobin. Therefore Reduces the oxygen supply to the tissues.

441. CO binds to platelet hemoproteins

and increases NO efflux.2. Platelet-derived NO reacts with

neutrophil-derived superoxide which activates platelets and causes platelet-neutrophil aggregates.

3. Reactive products and adhesion molecules promote firm aggregation and stimulate degranulation of neutrophils.

4. Endothelial cells acitaved by myeloperoxidase facilitating firm neutrophil adhesion and further degranulation.

5. Reactive oxygen species (ROS) initiate lipid peroxidation and adducts interact with brain myelin basic protein. The altered myelin basic protein triggers an adaptive immunologic response that causes neurologic dysfunction.

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CO regulates blood flow and blood fluidity

Vascular tone SMC proliferation

Platelet aggregation

By inhibiting

46 Cross talk between CO AND NO CO AND NO are two endogenously produced gases that can act as second

messenger molecules

Heme oxygenase and nitric oxide synthase are the enzyme systems responsible for generating CO and NO share similar properties, such as ability to activate soluble guanylate cyclase to increase cyclic GMP.

it is becoming increasingly clear that these 2 gases do not always work independently, but rather can modulate each others activity.

Although much is known about the heme oxygenase/CO and nitric oxide synthase/nitric oxide pathways, how these two imp systems interact is Less well understood.

The current known relationship between CO and NO it relates to their production and physiological function.

47 REFERENCES

• RANG and DALE’S Pharmacology. Sixth edition (2007). 265-274.

• The sources on the world wide web.

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Thank you