Nitric Oxide Flashcards
What is the role of endothelium in modulation of vascular smooth muscle tone
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
Examples of vasoactive mediators from vascular endothelium
Endothelium/(angiotensins) (peptides)
EDRF/ Nitroc oxide (free radical)
Prostaglandins (PGE2/PGI2)
EDHFs (isoprostanes?)
EDCFs (isoprostanes?)
Isoprostanes are formed by…
Direct oxidation of membrane phospholipids
What is a free radical
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
Nitrate-containing compounds for treatment of …… since ….
Angina
1840s
(Nitroglycerin)
What is EDRF
Endothelium derived relaxant factor
Discovery of EDRF
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
Endothelium-dependent response requires… (mechanism)
Physical/electrical coupling between endothelium and vascular smooth muscle
Release of paracrine mediator/s from endothelium to act on vascular smooth muscle
Ach in HIGH concentrations acts…..
Directly on smooth muscle (modest contraction)
Ach in LOW concentrations acts on…
Endothelium to cause an endothelium-dependent relaxation of underlying smooth muscle
Describe the characterisation of EDRF
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)
Vasoactive stimuli that release vaso relaxant paracrine mediators from endothelium (chemical, pharmacological, physical)
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
Describe the biosynthesis of NO
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
NOS exists in what forms
Constitutive enzyme (eNOS, nNOS)
Inducible Enzyme (iNOS)
Constitutive enzyme of NOS
ENOS nNOS
Shorter, intermittent release of pmol amounts of NO
Activity regulated by Ca2+ - causes cal modules to bind tightly with NOS
Describe inducible enzyme of NOS
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
Localisation of constitutive NOS
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
NOS-independent sources of NO?
Especially under Pathophysiological conditions eg myocardial ischemia
Enzymatic sources - nitrite reductase, xanthine oxidoreductase
Describe the metabolism of NO
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
HbO2 + NO ->
MetHB + NO3
Biological selectivity of NO (‘receptor’-independent action) depends on:
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
What are the 3 mechanisms of action of NO
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
O2- also ……. For interaction with ……. Explains why……
O2- also reduces NO bioavailability for interaction with GC, explains why antioxidant superoxidase dismutase potentials NO vasorelaxation
Effects of NO in vasculature (physiological effects)
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
Effects fo NO in platelets
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?
NO actions outside the vasculature
• 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)
‘Janus’ Effect of NO (beneficial effects of eNOS activation)
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
‘Janus’ effect of NO (aberrant iNOS activation in wrong place/wrong time)
=detrimental effects.. exacerbates
Respiratory inflammation
Neurodegenrative disorders
Psychiatric disease
Epilepsy and Brain injury
Inflammatory bowel disease
Inflammatory skin disorders
Heart failure
Apoptosis
What are self protection from deleterious effects of NO
• 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
Diseases linked to NO as a pathogenic component
Excessive NO production
Enhanced iNOS activity/benefit of iNOS inhibitors
Neurological, bronchopulmonary diseases, tumourgenesis, ischaemia-reperfusion, sepsis, arthritis, inflammatory bowel
Diseases linked to NO deficit/impaired bioavailability
• 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?)
describe NO and atherosclerosis
• 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
What compounds are frequently used in NO research
-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
NO synthase inhibition
• 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)
Therapeutic use of NO-related drugs in NO excess
• 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?
Therapeutic of NO-related rug in NO deficiency
• 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?
NO augmentation of drugs (hybrids)
Pro-drugs requiring actions of …… to release NO
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
