Nitric oxide (NO) is inorganic, colorless, relatively stable gas, dissolvable in water and lipidis. The arrangement of one atom of nitrogen and one of oxygen leaves an unpaired electron, this single electron turn the NO to a very active radical, which penetrates through the biological membranes and easily react with other substances. Half-life period of this agent is 5-6 seconds. Because of its high diffusibility NO gets not only to the every part of the cell but also creates a field of action around the cells.
NITRIC OXIDE SYNTHASE
NO is synthesised by a complex family of enzymes called NO synthases (NOS). These are some of the most complex enzymes known and require many factors and co-factors for activity.
NOS enzymes synthesize NO and L-citrulline from L-arginine and molecular oxygen in a reaction that is dependent on NADPH, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), tetrahydrobiopterin(BH4), and calmodulin (CaM).
may we assume that NOS1 and NOS3 belong to non proliferating cells and NOS2 to proliferating cells?
There are three NOS isoforms, each produced by a distinct gene and named in order of discovery:
• NOS1 was the first to be purified and cloned from neural tissue and it is therefore also called nNOS. This is expressed constitutively in the brain, central nervous system and peripheral nerves, but has also been found in skeletal muscle and epithelial cells.
• NOS3 is the isoform first found in endothelial cells, also called eNOS. Expression of eNOS is also constitutive and is found in endothelial cells, cardiac myocytes and thrombocytes.
NOS1 and NOS3 may themselves be induced under certain conditions (such as pregnancy).
• NOS2 is an inducible, calcium-independent isoform, also called iNOS or macrophage NOS. Expression of iNOS is induced by cytokines and lipopolysaccharides (LPS) in many cell types.
NOS2 PRODUCTION is found in various cells including:
• VASCULAR CELLS
• MACROPHAGES and NATURAL KILLER CELLS in the immune system;
• It is constantly expressed by large airway epithelium;
• In the basal keratinocyte layer of normal skin;
• In NORMAL SALIVARY DUCTS.
• SKELETAL MUSCLE
In chronic inflammatory conditions, NOS2 expression is therefore dependent on the overall cytokine balance.
All the isoform are products of different genes. The gene of nNOS is localized in 7th chromosome, the gene of iNOS in 17th, and the gene of eNOS in 12th chromosome.
Although all isoform catalyzed the same reaction, each of them do has its own structural and functional specificity, that's why there are constitutive (nNOS and eNOS) and inducible (iNOS or mNOS) forms of NOS distinguished.
• Constitutive forms are Ca2+/calmodulin dependent enzymes. Their activity depends directly on concentration of Ca2+ and is maximal at concentration 1mM of Ca2+.
These enzimes constantly generate small amounts of NO, create so called basic level of NO and this way partecipate in regulation of physiological processes.
• Inducible forms appear in cells after induction by bacterial endotoxins and cytokines.
iNOS is INDUCED by:
- BACTERIAL LIPOPOLYSACCHARIDE;
- INTERFERON-GAMMA (IFN-γ);
- CYTOKINES like:
- TUMOR NECROSIS FACTOR-ALFA (TNF-α);
- INTERLEUKINS: IL-1, IL-10 and IL-12 ;
- PLATELET ACTIVATING FACTOR (PAF);
- NUCLEAR FACTOR-kB (NF-kB).
iNOS IS INHIBITED by:
- NO itself;
- TRANSFORMING GROWTH FACTOR-BETA (TGF-β);
- IL-4, IL-6, IL-8 and IL-10 (although paradoxically IL-10 may also induce NOS2),
- Certain IMMUNOSUPRESSIVE DRUGS, such as glucocorticoids, cyclosporin and tacrolimus.
An important regulator of NOS2 is the tumour suppressor gene p53, which senses raised cellular NO and inhibits NOS2 by a negative feedback loop.
It is considered that almost all cells of the organism in pathological conditions are able to express NOS and this is important in immunological reactions.
The activity of iNOS appears 6-8 hours after induction. Once induced the enzyme continues to produce much higher NO concentrations than the other two NOS isoforms, for many hours or even days until the protein is eventually inactivated. So, distinctly from eNOS and nNOS, iNOS is considered to be PATHOLOGICAL ISOFORM.
THE BIOLOGICAL ACTION OF NO ARE DIRECTLY AND INDIRECTLY.
• One of the main ways in which NO exerts its effect indirectly is by binding to the heme group of the soluble guanylyl cyclase (sGC), thus activating synthesis of the cyclic nucleotide cGMP. cGMP can act at a number of different cellular targets including cGMP-gated ion channels, cGMP-dependent phosphodiesterases (PDE) and cGMP-dependent protein kinases (PKG). Effects following NO-induced guanylate cyclase activation include the regulation of vascular tone, and a complex role as a neuromodulator in the central nervous system.
• Another mechanism by which NO can modulate target protein activity is by direct nitrosylation of thiol groups (for instance, glutathione, proteins) or nitration of amines (for instance, nucleotides): these NO effects are cGMP-independent. It has immunocytotoxicity against both pathogens and tumours. DNA and mitochondria damage induced by NO is one of the factor causing apoptosis (genome controlled cell death). However, the NO concentrations found in human cancers are unlikely to be sufficient to produce tumour cell death or apoptosis, and instead are thought to be responsible for enhancing angiogenesis tumour dissemination.
• Indirect NO effect on cells is the action of active NO forms appearing after NO reaction with superoxide, oxygen, thiol groups (-SH). Such active form as respectively peroxynitrite (ONOO-), nitrite (NO2-) and nitrosothiols (R- SNO). Peroxynitrite, formed when both NO and superoxide radicals are present, is very toxic, particularly to DNA and to enzymes involved in DNA repair. All of these so-called reactive nitrogen oxide species (termed RNS) react with transition metals (such as zinc and iron in haem-containing proteins) and thiol groups in various enzymes and proteins. The resulting effects can lead to inhibition of enzymes such as cytochrome c oxidase, resulting in decreased ATP production and cell death.
THE PATHOLOGICAL AMOUNT OF NO (thousand pM) PRODUCED BY NOS2 IN INFLAMMATION causes:
• NO directly inhibited the assembly process of NADPH oxidase;
• NO directly reacts with the binuclear metal centre of cytochrome c oxidase and so diminished the production of ATP;
• NO directly binds the four iron-four sulphur domain of mammalian cytoplasmic and mitochondrial enzymes (ex:aconitases) and heme group;
• N2O3 can leads to DNA damage by nitrosation of primary amines on DNA bases which leads to deamination or by deamination of bases (ex: guanine);
• NO directly causes nitrosylation of thiol groups containing enzymes (ex: glutathione)
• Suppress activity of antioxidant enzymes (catalase, prostaglandin H peroxidase, superoxide dismutase);
• ONOO- suppress activity of DNA ribonucleotide reductase;
• Reactive Nitrogen Oxide Species have oxidative action and lead to:
- lipid peroxidation causing membrane damage,
- protein oxidation;
- DNA lesions: reactions of the nuclear and mitochondrial DNA with Timine produce the break of the single filament of DNA.
Virus and Bacterial cell damage through NOS2 induction:
- Epstein-Barr virus
- Human immunodeficiency virus type 1 Tat protein stimulates inducible nitric oxide synthase expression and nitric oxide production
- All Gram negative bacteria can be involved in damaging cells thanks to their LPS which induces NOS2. Among them we can find:
Nitric oxide synthase
Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme.1990
Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication 1989