Nitric oxide

Question 1

Where do we get NO from?

How do we get NO from NO3-? [3]

Is oxygen needed for this pathway? [1]

Answer 1

Diet derived

1. Salivary glands and oral bacteria: nitrate (NO3-) to nitrite (NO2)
2. Nitrite is reduced to NO in acidic and hypoxic tissues

O2 independent pathway

Question 2

Name 3 sources of endogenous NO [4]

Answer 2

Endogenous NO is derived from:
- Endothelium
- Nerve fibres
- Skeletal Muscle
- Tunica intima

Question 2

Name 3 sources of endogenous NO [4]

Answer 2

Endogenous NO is derived from:
- Endothelium
- Nerve fibres
- Skeletal Muscle
- Tunica intima

Question 3

What are the 2 endogenous forms of NOS need to know? [2]

Where are each found? [2]

Are they Ca2+ dependent or independent? [2]

Answer 3

Nitric oxide synthase enzyme (NOS)

NOS type I: neuronal NOS, nNOS
- Found in central & peripheral neuronal cells and muscle
- Calcium dependent

NOS type III: endothelial NOS, eNOS
- Vascular endothelial cells
- Calcium dependent

Question 4

Decsribe the structure of NOSs xx

Answer 4

NOS: oxidoreductase homodimer enzymes:

• Oxygenase domain: binding for NADPH, FMN & FAD
• Reductase domain: binding for BH4, heme and L’arginine (substrate
• Calmodulin binding site: in between reductase and oxidase domains

Question 5

What happens to NOS if there is / isn’t BH4 present?

Answer 5

BH4 prescene: causes dimerisation and proper catalytic activity for NO formation

BH4 absence: causes monomer, becomes a superoxide

Question 6

For the formation of NO using NOS, what substrates are essential? [3]

Which co-factors are essential? [5]

Answer 6

For the formation of NO using NOS, what substrates are essential? [3]
- L-arginine
- O2
- NADPH

Which co-factors are essential? [5]
- FAD
- FMN
- BH4
- haem
- calmodulin.

OXYGEN is essential for the synthesis. Because of the oxygen requirement, NO synthesis is inhibited in hypoxic tissue

Question 7

Explain MoA of endothelial NO synthase creating NP

Answer 7

• Shear stress promotes wall stretching, which promotes the dissociation of eNOS from caveolae (which are invaginations of plasma membrane)
• This allows eNOS release into the cytoplasm and its activation (through binding of a Ca2+/ calmodulin (CaM) complex)
• M2-muscarinic acetylcholine receptor activation or other stimulation initiates an influx of Ca2+ that binds to calmodulin.
• eNOS dissociates from Cav1 and then combines to Ca/CaM.
• eNOS is activated leading to synthesis of NO.

Question 8

[] shear stress favors the activation of eNOS by releasing endothelium-dependent agonists.

What are they? [4]

Answer 8

Increased shear stress favors the activation of eNOS by releasing endothelium-dependent agonists.

Agonists stimulated NO formation:
* Acetylcholine (M3muscarinic)
* Bradykinin
* Substance-P
* Adenosine
*

Question 9

Neuronal NOS synthase pls

Question 10

Explain MoA of NO evoking vasodilation

Answer 10

• NO activates guanylate cyclase (GC) in vascular smooth muscle
• This converts guanosine triphosphate to cyclic guanosine monophosphate cGMP
• cGMP acts through cGMP-dependent protein kinases (PKGs) to inactivate myosin.
• The net effect of raised cGMP is to inhibit contraction.

Question 11

NO in endothelial cells serves to maintain []?

What is tonic vasodilation? [1]

Answer 11

NO in endothelial cells serves to maintain basal vascular tone

Continuous release of NO from the endothelium tonically reduces basal microvascular tone in vivo—an effect termedtonic vasodilatation.

Question 12

Explain how NO release, during exercise counteracts sympathetic NS xx

Answer 12

During exercise:

• Sympathetic nervous system produces a general vasoconstriction of arterioles in muscle during exercise (mediated by alpha-1 receptors). This tends to reduce local muscle blood flow

BUT

• During exercise the blood flow in active muscles increases over TEN FOLD.
• Due to release of NO and adenosine and Ca2+ influx (linked to NO formation

Together:

• Vasodilation in active & vasoconstriction in inactive muscles effectively redistributes blood to the active muscles

Question 13

Why does muscular dystrophy occur?

Answer 13

In dystrophic muscle:

• nNO is reduce, less NO is generated during muscle contraction, resulting in unrestrained sympathetic vasoconstriction and transient functional muscle ischemia

Question 14

Why does muscular dystrophy occur?

Answer 14

In dystrophic muscle:

• nNO is reduce, less NO is generated during muscle contraction, resulting in unrestrained sympathetic vasoconstriction and transient functional muscle ischemia

Question 15

During physical exercise, oxygen is consumed and lactic acid is produced, these inhibit NOS activity which is oxygen dependent.

So how does blood flow in muscles increase during exercise?

Answer 15

• In hypoxic conditions during robust physical exercise, Xanthine oxidoreductase using the nitrate-nitrite-NO reductive pathways in skeletal muscle, generates nitrite and NO from nitrate reservoir in muscle

Xanthine oxidoreductase activity increased by exercise-induced lactic acid accumulation and possibly by hypoxia.

Major sources of nitrate in skeletal muscle are believed to be from dietary sources delivered via transport by the blood

Question 16

Explain MoA of nitroglycerine treating angina / MI

Answer 16

GTN is converted to nitrite by mitochondrial enzymes including mitochondrial aldehyde dehydrogenase.

The nitrite is then converted to nitric oxide and dilates the coronary vessels to improve oxygen supply to the heart.

Nitric oxide is particularly useful for treating angina and AMI as it does not just dilate but also opens collateral arteriolar vessels in the heart. This enables blood to get to hypoxic tissue downstream from a blocked or partially blocked coronary artery.

Question 17

Whats the craic why NO & fetal lungs when they take their first breath?

Answer 17

Baby takes his or her first breath: the increase in oxygen in the lungs stimulates the synthesis of nitric oxide in the pulmonary endothelium. Causes pulmonary vasodilation

Increased oxygen tension after birth upregulates the expression of eNOS and PKG, which act in concert with the maturational increase in sGC and PKG activities, to contribute to a low postnatal PVR

Question 18

What causes persistent pulmonary hypertension of the newborn? [1]

Answer 18

• Too little NOS (or inhaled meconium prevents inhaled oxygen reaching the NOS): severe hypoxia
• causes raised pulmonary vascuclar resistance

Question 19

How does NO contribute to penile erection?

Answer 19

• NO promotes penile vasodilation and blood flow by diffusing across the smooth muscle membrane causing relaxation of cavernosal smooth muscle leading penile erection

Question 20

How does erectile dysfunction occur? listen to lecture

Answer 20

Endothelial dysfunction: smooth muscle cavernosal contraction

Question 21

Explain the relationshop between NO and intravascular coagulation

Answer 21

NO exerts an antithrombotic action by inhibiting platelet adhesion and aggregation

Question 21

Explain the relationshop between NO and intravascular coagulation

Answer 21

NO exerts an antithrombotic action by inhibiting platelet adhesion and aggregation

Question 22

What is endothelial dysfunction?

Answer 22

Pathological state of the endothelium characterized by a reduction in the bioavailability of vasodilators, essentially nitric oxide.

Endothelium becomes prothrombotic and proinflammatory

Question 23

What can endothelial dysfunction be caused by? [4]

Answer 23

Consumptive processes that transform bioavailable NO into other species:

• Reactive oxygen species such as -O2 reacts readily with NO forming peroxynitrite (ONOO−)
• Uncoupling of NOS: BH4 insufficiency results in uncoupled NOS, which produces superoxide anions instead of NO

Deficiencies in production of NO in the endothelium:
- Reduced bioavailability ofl-arginine o
- Presence of its inhibitor, asymmetric dimethyl-l-arginine (ADMA)

Question 24

Endothelial dysfunction leads to vascular and metabolic disorders such as WHAT? [6]

Answer 24

Hypertension, hyperlipidemia, atherosclerosis, insulin resistance, and diabetes mellitus