Nitric Oxide

Question 1

What is the role of endothelium in modulation of vascular smooth muscle tone

Answer 1

Physical barrier preventing hormones reaching muscle layer
Extraction/metabolism of mediators - 5-HT, catecholamines, kinins
Conversion of precursors to vasoactive products - angiotensins (ACE enzyme)
Secretion of excitatory/inhibitory paracrine mediators in response to vasoactive stimuli

Question 2

Examples of vasoactive mediators from vascular endothelium

Answer 2

Endothelium/(angiotensins) (peptides)
EDRF/ Nitroc oxide (free radical)
Prostaglandins (PGE2/PGI2)
EDHFs (isoprostanes?)
EDCFs (isoprostanes?)

Question 3

Isoprostanes are formed by…

Answer 3

Direct oxidation of membrane phospholipids

Question 4

What is a free radical

Answer 4

A species of independent existence containing 1 or more unpaired electrons occupying an atomic or molecular orbital by themselves thus imparting chemical reactivity
Eg Nitric Oxide

Question 5

Nitrate-containing compounds for treatment of …… since ….

Answer 5

Angina
1840s
(Nitroglycerin)

Question 6

What is EDRF

Answer 6

Endothelium derived relaxant factor

Question 7

Discovery of EDRF

Answer 7

Ach (vasoconstrictor) exerted vasodilator effect on rabbit aorta in vitro if vessel wall carefully preserved (1980)
Deliberate localised damage to endothelial layer (mechanical treatment/collagenase) abolished vasodilation to Ach

Question 8

Endothelium-dependent response requires… (mechanism)

Answer 8

Physical/electrical coupling between endothelium and vascular smooth muscle
Release of paracrine mediator/s from endothelium to act on vascular smooth muscle

Question 9

Ach in HIGH concentrations acts…..

Answer 9

Directly on smooth muscle (modest contraction)

Question 10

Ach in LOW concentrations acts on…

Answer 10

Endothelium to cause an endothelium-dependent relaxation of underlying smooth muscle

Question 11

Describe the characterisation of EDRF

Answer 11

Bioassay of intact rabbit thoracic aorta in series with pre-contracted rabbit coronary artery lacking endothelium
EDRF release continually in basal state and in elevated state in response to Ach
Compared biological activity of EDRF and NO by bioassay (87)
Quantified NO as chemi-luminescent product of reaction with ozone
Quantitatively similar effects
(Equally unstable, activity inhibited by Hb, activity enhanced by superoxide dismutase)

Question 12

Vasoactive stimuli that release vaso relaxant paracrine mediators from endothelium (chemical, pharmacological, physical)

Answer 12

Chemical:
Ach, ATP/ADP, vasopressin, substance P, thrombin, histamine, Bradykinin, Endothelin
Pharmacological:
A23187 (calcium ionophore), Poly-L-Lysine (polycation)
Physical :
Mechanical (sheer stress, pulsatile flow), Hypoxia

Question 13

Describe the biosynthesis of NO

Answer 13

From L-arginine, molecular O2 by NO synthase (NOS)
NOS cleaves N from guanidine terminus of L-arginine in a 2 step reaction to produce L-citrulline and NO
Requirement for co-factors (NADPH, FAD, FMN, Haem, Mg, Ca, tetrahydrobiopterin)
Inhibited by L-NMMA (L-arginine analogue) - competes with L arginine for substrate binding sites

Question 14

NOS exists in what forms

Answer 14

Constitutive enzyme (eNOS, nNOS)
Inducible Enzyme (iNOS)

Question 15

Constitutive enzyme of NOS

Answer 15

ENOS nNOS
Shorter, intermittent release of pmol amounts of NO
Activity regulated by Ca2+ - causes cal modules to bind tightly with NOS

Question 16

Describe inducible enzyme of NOS

Answer 16

iNOS
Sustained release of nmol amounts of NO
Activity not regulated by Ca2+ - cal modules already tightly bound at physiological Ca2+
Expression incr by bacterial LPS, cytokines, TNF-a, decreased by glucocorticoids, TGF-beta

Question 17

Localisation of constitutive NOS

Answer 17

ENOS acetylation by Myristate, palmitate
Caveolin proteins anchor eNOS near membrane, decr activity
-CaM displaces inhibitory interaction, increased activity (promotes electron flux)
nNOS anchored near NMDA receptor in synaptic membrane
- postsynaptic density proteins link receptors and nNOS

Question 18

NOS-independent sources of NO?

Answer 18

Especially under Pathophysiological conditions eg myocardial ischemia
Enzymatic sources - nitrite reductase, xanthine oxidoreductase

Question 19

Describe the metabolism of NO

Answer 19

Unpaired outer e- renders NO chemically reactive radical
T1/2 sec-min dependent on concentrations or chemical environment
Reversible reaction of NO with thiol (-SH) groups
-serving as reservoir or/carrier for paracrine/endocrine NO action? Eg glutathione, albumin
Haemoglobin acts as intravascular scavenge
Main metabolic mathway for NO, limits reaction of physiological amounts of NO with cellular components, oxidation in plasma to nitrites and (in presence of oxyhaemoglobin) to nitrates
Explains why activity of EDRF attenuated by haemoglobin

Question 20

HbO2 + NO ->

Answer 20

MetHB + NO3

Question 21

Biological selectivity of NO (‘receptor’-independent action) depends on:

Answer 21

Concentration of NOS (NOS isoform involvement)
- compartmentalisation of NOS, temporal, spatial gradients of NO, 1-30nM physiological regulation/survival, >300nM cytotoxicity/apoptosis
Reactivity of other molecules (especially radicals)
Proximity of target cells
Machinery/way target cells programmed to respond - speed, duration; proteins, 2nd messengers etc available

Question 22

What are the 3 mechanisms of action of NO

Answer 22

Binding to metal centres (haem groups) of proteins
-Guanylate cyclase (cyclic GMP production incr x 200)
-Cytochrome c oxidase (decr ATP production, o2 consumption)
S-nitrosylation reactions (interacts first with o2)
-Reversible interaction with thiol groups (Cys amino acids)
-Implications for conformation and function of many enzymes, gene transcription factors, SERCA etc
Nitration (interacts first with superoxide, o2-)
-Of lipids, nucleotides, Tyr amino acids (3-nitrotyrosine)
-Implications for function, turnover, other interactions

Question 23

O2- also ……. For interaction with ……. Explains why……

Answer 23

O2- also reduces NO bioavailability for interaction with GC, explains why antioxidant superoxidase dismutase potentials NO vasorelaxation

Question 24

Effects of NO in vasculature (physiological effects)

Answer 24

Regulation of peripheral vascular resistance
Homeostasis/microcirculatory flow adjustment (in hypoxia)
General increased vasodilation in pregnancy
Decr leukocyte adhesion, smooth muscle proliferation and endothelial cell senescence
Protects against atherosclerosis, plaque initiation/rupture
Promotes angiogenesis

Question 25

Effects fo NO in platelets

Answer 25

Conflicting evidence for/against role of NO
Decr platelet adhesion/aggregation/thrombus formation in vitro
- Limits thrombus formation in Vivo?
Hb prevents endothelial-derived NO from affecting platelet function under physiological conditions
NO biosynthesis within platelets as autocrine agent?
Increased nitroprusside or GTN required to alter platelet function?
Addition of GTN to standard therapy does not improve mortality in patients with acute coronary thrombosis?

Question 26

NO actions outside the vasculature

Answer 26

• non-specific host defence and cytotoxicity against numerous pathogens and tumour cells
– excess ONOO- or NO can damage host tissues by nitration and nitrosylation
• neuromodulator (and neurotransmitter) in peripheral nervous system
• physiological role in central nervous system (memory, pain, sleep, appetite)

Question 27

‘Janus’ Effect of NO (beneficial effects of eNOS activation)

Answer 27

Protects from excessive bronchoconstriction
Neurotransmission
Memory and learning
Cerebral blood flow
Gastric mucosal blood flow
Cutaneous blood flow
Peristalsis, intestinal secretion
Non-specific host defence
Skin barrier function
Cardioprotection
Cell survival

Question 28

‘Janus’ effect of NO (aberrant iNOS activation in wrong place/wrong time)

Answer 28

=detrimental effects.. exacerbates
Respiratory inflammation
Neurodegenrative disorders
Psychiatric disease
Epilepsy and Brain injury
Inflammatory bowel disease
Inflammatory skin disorders
Heart failure
Apoptosis

Question 29

What are self protection from deleterious effects of NO

Answer 29

• cells producing NO from cNOS – ↑ Ca2+ also inhibits GC – cells producing NO have limited capacity to respond?
• cells producing NO from iNOS – ↑↑ levels of GSH than cells not producing NO
• ↑Antioxidant enzyme activity – ↑expression of SOD (↓ superoxide, ↓ peroxynitrite), catalase and glutathione peroxidase
• expression of NOS-associated protein NAP110 – inhibits catalytic activity of iNOS in macrophages

Question 30

Diseases linked to NO as a pathogenic component

Answer 30

Excessive NO production
Enhanced iNOS activity/benefit of iNOS inhibitors
Neurological, bronchopulmonary diseases, tumourgenesis, ischaemia-reperfusion, sepsis, arthritis, inflammatory bowel

Question 31

Diseases linked to NO deficit/impaired bioavailability

Answer 31

• endothelial NO biosynthesis impaired in disorders predisposing to atheroma (hypercholesteraemia, diabetes) – cause or effect?
– impaired L-arginine transport into endothelium?
– ↓ BH4/dysfunctional uncoupled eNOS produces O2- rather than NO?
• NO deficiency contributes to hypertension, PAH, local vasospasm in variant angina, sub-arachnoid haemorrhage, pre-eclampsia
– requirement for strategies to preserve/maintain NO signalling (eNOS gene transfer, L-arginine supplements?)

Question 32

describe NO and atherosclerosis

Answer 32

• Anti-atherogenic: excess NO terminates lipid peroxidation by interaction with LipidOO- to form non- radical stable end product LipidOONO
• Pro-atherogenic: excess superoxide binds NO to form toxic peroxynitrite ONOO-
– Oxidised LDL stimulates NADPH oxidase generating superoxide in blood vessel wall
– ONOO- can damage BH4 and ‘uncouple’ eNOS
antioxidants reduce O2- (and ONOO-) and spare NO

Question 33

What compounds are frequently used in NO research

Answer 33

-elevation of NO levels
Inhaled NO, precursors of synthesis (L-arginine), NH4 supplementation to decouple eNOS, NO donors
-reduction of NO levels
NO scavengers (Hb) NOS inhibitors (L-NNA, L-NAME, L-NMMA)
Problem lack of selectivity for individual NOS isoforms

Question 34

NO synthase inhibition

Answer 34

• nitric oxide (EDRF) - paracrine mediator
– ↓vascular tone, cardiac contraction, platelet aggregation
• impaired production of NO in man
– ↑ hypertension, atherosclerosis
• L-NAME (NOS inhibitor) given in drinking water
– incr BP, incr LVH, myocardial ischemia and oxidative stress
– incr cardiac, renal function
• non-surgical, technically easy to perform, low mortality
• relevance of experimental model? (insufficient data)

Question 35

Therapeutic use of NO-related drugs in NO excess

Answer 35

• Inhibitors of NO biosynthesis (L-NMMA) beneficial (depending on dose) in patients with hypotension from multiple organ failure secondary to sepsis
• iNOS-specific inhibitors or gene therapies tospare constitutive NOS activity/physiological effects of NO?

Question 36

Therapeutic of NO-related rug in NO deficiency

Answer 36

• NO donors (nitroprusside, organic vasodilators) in angina (and severe hypertension, acute heart failure)
– spontaneous or enzyme-associated release of NO (mitochondrial aldehyde dehydrogenase, mtADH?)
• Inhaled NO in adult respiratory distress syndrome, acute right heart failure, neonatal PAH?

Question 37

NO augmentation of drugs (hybrids)
Pro-drugs requiring actions of …… to release NO

Answer 37

Esterases
• NO-NSAID derivatives
– Protect against NSAID-induced GI toxicity
– Maintain mucus, bicarbonate barrier, promote gastric capillary blood flow, enhance ulcer healing
– Retain anti-inflammatory, anti-pyretic, analgesic actions
• NO donating drugs (NODDs)
– NO-paracetamol, NO-prednisolone, NO-mesalamine
– Nicorandil (NO donor and K+ATP channel opener, angina)
– Nitrosylated analogs of a-adrenoceptor antagonists
• Treatment of erectile dysfunction
• Target similar signalling pathway to Viagra
• Additive benefit derived from antagonist + NO
– Nipradilol (NO donor and b-adrenoceptor antagonist)
– NO-statins currently in development