Nitric Oxide Flashcards
Why are the diverse biological functions of NO surprising?
NO is also an atmospheric pollutant and a free radical.
How is NO found naturally?
NO is formed in the soil by denitrifying bacteria, and is a major component of cigarette smoke.
NO is part of the series of oxides of nitrogen, including nitrous oxide (N2O), nitrogen dioxide (NO2), nitrite (NO2-) and nitrate (NO3-).
How is NO broken down in the body non-catalytically?
Nitric oxide is converted to nitrogen dioxide by reaction with molecular oxygen (O2). Within the body this is a very slow reaction due to the stoichiometry.
The requirement for two NO molecules to simultaneously interact with dioxygen means that solubilised NO is stable at concentrations below 1M.
What kind of half life does NO have in the body and why?
In the body, the lifespan of NO is very low due to its high-affinity association for haem-compounds, particularly haemoglobin.
How was NO’s biological relevance discovered?
NO was first known by its function before chemical characterisation, initially being designated endothelium dependent relaxing factor (EDRF).
Isolated EDRF was identified as being NO by the unique spectrum of the nitrosylhaemoglobin it formed as well as the distinctive chemiluminescence produced by its reaction with ozone.
How is NO biosynthesised?
This is done by Nitric Oxide Synthase (NOS) using L-arginine as a substrate. This amino acid is in high concentration within cells in the form of its intermediate N-hydroxyarginine. The conversion of L-arginine to L-citrulline allows nitration of O2.
This reaction forms part of a cycle of deamination and re-amination of L-arginine, which involves the Krebs cycle and is linked to the ornithine cycle.
What factors are required for NOS?
Several cofactors are required for this, including NADPH (two of which are oxidised to NADP+), calmodulin/Ca2+, tetrahydrobiopterin, Haem, FMN and FAD.
Describe the NOS family.
There are three NOS isoforms, designated I, II and III, which serve different biological roles.
All have significant homology with Cytochrome P450 Reductase enzyme, but this is unable to make NO as it lacks the specific reductase domain required.
All three genes are present on different chromosomes.
Describe the features of NOS I
Neuronal NOS shares most with NOS III, but differs in its acquisition of a PDZ domain which targets the protein to neuronal synapses and its lack of plasma membrane anchoring sites.
What is the role of NOS I?
NOS I produces NO as a neurotransmitter at low levels constitutively (though is calcium-dependent), and is involved in many responses, including in the GI tract, penile erection, sphincter relaxation, and blood flow.
It is also known to be involved with synaptic plasticity and modulation of cellular response to glutamate.
What is the role of NOS II?
Inducible NOS is activated as part of the non-specific immune response to micro-organisms and the inflammatory response.
It is induced to great levels of activity by various pathogenic stimuli, including bacterial lipopolysaccharides, reaching its maximum activity levels after 24hrs – after which is it not further activates by increased calcium.
Like NOS I, it lacks plasma membrane anchoring sites.
What are the features and roles of NOS III?
Endothelial NOS, like NOS I, produces low levels of NO constitutively though calcium-dependently, and is activated in response to a variety of stimuli. Uniquely, eNOS is anchored to the plasma membrane by myristoylation and palmitoylation.
NOS III is involved in blood flow and pressure, as well as inhibiting platelet activation. Thus it is the one important in the context of CVD.
How is the baseline NO level maintained and what effect does it have?
The gene expression and enzymatic activity both being constitutive, eNOS produces low levels of NO constantly within the endothelium, where it diffuses both into the lumen and into the vessel wall, leading to inhibition of platelet activation and smooth muscle cell relaxation in those places respectively.
NOS III is directly activated primarily by two pathways, each of which are sensitive to a number of stimuli.
What pathways directly regulate eNOS?
Activation of the PI3K/Akt pathway by oestrogens and pulsatile shear stress leads Akt phosphorylating a serine in the calcium-binding domain of eNOS, activating it.
NOS III is also activated by increased calcium concentration, which results from increased levels of ATP and acetylcholine, amongst others.
How is eNOS indirectly upregulated?
NOS III can also be upregulated indirectly by the availability of L-arginine and its cofactors. The cofactor availability can also affect the reaction performed by eNOS – it is also prone to production of superoxide anions and hydrogen peroxide.
What is the effect of the hydrogen peroxide by product?
The production of H2O2 may be part of the biological function; it is believed to be one of the endothelium-dependent hyperpolarising factors that relax the resistance vessels that are insensitive to NO.
Vessel relaxation is in fact controlled by a wide variety of regulators, including prostaglandin, endothelin and thromboxane (see endothelial dysfunction lecture).
What inhibits NOS?
Experimentally, L- N methyl arginine is used as a competitive inhibitor.
There are however two natural inhibitors, produced from L-arginine, that are found in blood and tissues: asymmetric and symmetric dimethyl arginine (ADMA and SDMA).
What controls the production of natural NOS inhibitors?
ADMA and SDMA are competitive inhibitors produced from L-arginine by protein-arginine methyltransferases (PRMTs), and are themselves converted to citrulline by dimethylarginine dimethylaminohydrolase (DDAH).
