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Mechanisms of Nitric Oxide Synthesis and Action in Cells

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Mechanisms of Nitric Oxide Synthesis and Action in Cells MEDICINA (2003) Vol. 39, No.6 - http://medicina.kmu.lt 535 Mechanisms of nitric oxide synthesis and action in cells Vagan Arzumanian, Edgaras Stankevičius, Alė Laukevičienė, Egidijus Kėvelaitis Department of Physiology, Kaunas University of Medicine, Lithuania Key words: nitric oxide, nitric oxide synthase, heme. Summary. Nitric oxide (NO) is a free radical gas, which is a product of reaction between molecular oxygen and L-arginine. Great diffusibility of nitric oxide determines its quick three-dimensional distribution around the cell which is a source of nitric oxide. Single electron makes it into very reactive radical, interacting with metals incorporated in enzyme structure, heme, superoxids, oxygen, etc. The synthesis of nitric oxide is catalyzed by en- zyme nitric oxide synthase (NOS) which has three isoforms: endothelial nitric oxide syn- thase, neuronal nitric oxide synthase, macrophagal nitric oxide synthase. Due to mecha- nisms of action NOS enzymes are also classified as constitutive nitric oxide synthase and inducible nitric oxide synthase. Constitutive forms are found in cytosol and membranes, they are dependent on Ca2+/calmodulin concentration and are extremely important in the regulation of physiological processes. Inducible forms are synthesized in cells after induc- tion by bacterial endotoxins or cytokines, do not depend on Ca2+/calmodulin concentra- tion and are being considered as pathological isoforms. It is thought that nitric oxide syn- thase catalyses transport of electrons for reactions between molecular oxygen and L-argi- nine. This consideration based on fact that flavine co-enzymes and hem are found as struc- tural units in nitric oxide synthase. Although all isoforms catalyze the same reactions, every one of them has its own unique structure and localization. These features determine differ- ences of activation pathways as well as specificity of inhibitors. The characteristic features of nitric oxide and functional differences among the nitric oxide synthase isoforms deter- mine its different role in regulation of many physiological and pathophysiological pro- cesses. Introduction sight can be gained by examining the theoretical cal- Nitric oxide (NO) is inorganic, colorless, relatively culations based on established diffusion equations. NO stable gas, dissolvable in water and lipids (1). The single concentration (C) in the infinite medium at any dis- electron turns the NO to a very active radical, which tance (r) from a point source after the time (t) can be penetrates through the biological membranes and eas- described with the following equation (4): ily reacts with other substances (2). Half-life period of this agent is 5-6 seconds. In vivo NO is quickly neutral- C=(S/4pDr)er¦c(r/2ÖDt), ized by hemoglobin, and superoxide anions (3). Because of its high diffusibility NO gets not only to the every S - the strength of source, D - the coefficient part of the cell but also creates a field of action around of diffusion, which was measured in experiment and the cells. This feature enables NO to act on targets not equal 3.3×10-5 cm2/sec. If S= 21×10-18 mol/sec in only in cytosol but also beyond the cell’s membrane (4). the center of spherical structure of diameter of 1 mm (approximate diameter of nerve ending), the concen- The physical features of nitric oxide tration at the surface will be 1 mM (measured di- NO has a feature to diffuse easily in lipids and water rectly from the surface of a stimulated endothelial (3, 4). Extreme diffusibility determines the rapid three- cell). At steady state when t in the equation is large, dimensional spread of agent and easy access to neigh- er¦c(r/2ÖDt)=1. Then the concentration of NO is bor cells (4). It is important to know how far NO trav- inversely proportional to the distance from the source. els before its concentration becomes too low to have Accepting that the biologically active concentration act. In absence of experimental evidence, some in- of NO is 0.001 mM (based on relaxing effect of NO Correspondence to V. Arzumanian, Department of Physiology, Kaunas University of Medicine, A. Mickevičiaus 9, 3000 Kaunas, Lithuania. E-mail: [email protected] 536 Vagan Arzumanian, Edgaras Stankevičius, Alė Laukevičienė, Egidijus Kėvelaitis on aortic smooth muscle) the volume of tissue in which nism NO regulates local blood circulation in different NO may exert physiological effects become equal to organs, increases preload, induces bronchodilatation and a sphere of diameter 1000 mm. Such volume will have adaptive relaxation of stomach, intestines and bladder. about one billion synapses. According to the fact that The increase of cGMP concentration in thrombocytes the half-life time of NO is about 5 seconds, physi- inhibits their adhesion and aggregation, in neurons – ologically important NO concentration will be in a changes their electrogenesis. There are more and more sphere of diameter 340 mm, because marked de- data about the role of NO as a neuromodulator in the crease of NO concentration may be expected not in central nervous system. Profound investigations are 500 mm, but in 170 mm distance. It is still a very large made about NO ability to release the glutamate. quantity comparing with dimension of synapse (4). Neuromodulator glutamate activates N-methyl-D-as- The field of action of NO determines not only half- partate (NMDA) receptors of postsynaptic membrane, life period but also a very high reactivity (2-5). NO which are responsible for activation of Ca2+ channels. 2+ reacts actively with O2 and superoxide radicals, thi- After Ca entries the postsynaptic neuron the NO syn- ols, metals of enzymes and is oxidized to inactive ni- thase is activated. Newly synthesized NO moves back trate (NO-2) and nitrite (NO-3) ions (2, 3, 5). to synaptic ending and as a retrograde messenger acti- vates soluble guanylyl cyclase. The increase of cGMP NO reactions with heme induced by guanylyl cyclase activates the release of One of the main targets of NO is heme-containing glutamate facilitating release of nerve impulse in the enzymes (cytochrome 450, cytochrome C oxidases, synapses (the positive feed-back). It is thought that this catalases, peroxidases, NO synthases themselves and mechanism participates in a long-term synaptic poten- others) (6). NO changes activity of these enzymes by tiation, which is a basis of learning and memory (2). precipitating on heme. Pathological amounts of NO (thousands pM) cause Semiempirically, using quantum-chemical mode, nitrosylation of metals containing enzymes (10), block atomic and electronic comparative analysis of heme-ni- electron transportation in mitochondria, by bounding tric oxide and of heme-oxygen derivates was performed. to cytochrome heme group (6, 7), suppress activity of It was proved that internuclear distance markedly changes antioxidation enzymes (catalase, prostaglandin H per- in molecule when oxygen binds to heme. The distance oxidase (PGH), superoxide dismutase) and DNA ri- between oxygen atoms increases 0.046 Å, when distance bonucleotide reductase, damage DNA by between nitrogen and oxygen increases by 0.064 Å, what deaminization, inactivate glyceraldehyde-I- shows stronger link between hem and NO. It was found phosphodehydrogenase by blocking synthesis of gly- 2+ that NO binds to atom of Fe covalently when O2 and colytic ATP (2, 5). DNA and mitochondria damage heme is linked in ion-dipole mode. induced by NO is one of the factors causing apoptosis In the complex of O2 and heme the participation of (genome controlled cell death) (11, 12). ligand electron completing Fe2+ orbitales is small, while Indirect NO effect on cells is the action of active single electron of NO enters Fe2+ d-orbital forming d7 NO forms appearing after NO reaction with peroxide orbital (as a consequence NO gets positive charge) anions or O2 (13). Such active forms as peroxynitrite (6). Research also revealed that physiological changes (ONOO-) induce nitrosylation stress (increased gen- of temperature, markedly changes the participation of eration of nitrosamines, S-nitrosothiols) or oxidation NO electron in forming Fe2+ d-orbital, greatly influ- stress, when the amounts of oxidative substances (hy- encing the reactivity of the complex (7). NO and he- droxyl, nitryl and chlorine radicals) increase markedly moglobin compound (methemoglobin) may be consid- (5, 13) (Fig. 1.). One of the possible explanations why ered as transportable form of NO (8). cells generating big amounts of NO are not damaged is great activity of superoxide dismutase, which cata- Biological action of nitric oxide lyzes the degradation of toxic radicals (14). NO acts in cells directly and indirectly. One of the most important substrates of NO is cytosolic guanylyl Nitric oxide synthase cyclase (9). Guanylyl cyclase activated by physiologi- NO is formed during the complex oxidation reac- cal NO concentration (any pM) increases the produc- tion, from L-arginine and oxygen (O2). NO is produced tion of cyclic guanosine monophosphate (cGMP) which with participation of enzyme NO synthase, which was leads to disconnection of actin-myosin links. Already first found in endothelial cells of blood vessels. En- after 10 seconds from the beginning of NO action the zyme is called synthase, not synthetase, because there relaxation of smooth muscles is observed. By this mecha- is no need of adenosine triphosphate (ATP) for its MEDICINA (2003) Vol. 39, No.6 - http://medicina.kmu.lt Mechanisms of nitric oxide synthesis and action in cells 537 NADPH oxydase L - arginine – – – – – Fig. 1. Generation of free radicals with participation of nitric oxide (14) – – O2 – superoxide anion, NO – nitric oxide, O2 – oxygen, Cl – chlorine anion, H2O2 – hydrogen peroxide, – – ONOO – peroxynitrite, NO2 – nitrite anion, HOCl – hypochlous acid, NO2Cl – nitrylchloride, § § § OH – hydroxyl radical, NO2 – nitryl radical, Cl – chlorine radical. action (15). Almost all cells of human and animal or- called basic level of NO and by this way participate in ganisms are able to synthesize NO. As different regulation of physiological processes (1, 2, 14).
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