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 enzyme nitric oxide synthase (NOS) which has three isoforms: endothelial nitric oxide synthase, neuronal nitric oxide synthase, macrophagal nitric oxide synthase. Due to mechanisms 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 induction by bacterial endotoxins or cytokines, do not depend on Ca2+/calmodulin concentration and are being considered as pathological isoforms. It is thought that nitric oxide synthase catalyses transport of electrons for reactions between molecular oxygen and L-arginine. This consideration based on fact that flavine co-enzymes and hem are found as structural 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 differences of activation pathways as well as specificity of inhibitors. The characteristic features of nitric oxide and functional differences among the nitric oxide synthase isoforms determine its different role in regulation of many physiological and pathophysiological processes.

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/4πDr)erƒc(r/2√Dt), 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 µm (approximate diameter of nerve ending), the concen- The physical features of nitric oxide tration at the surface will be 1 µM (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- erƒc(r/2√Dt)=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 µM (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 µm. 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 µm, 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 µm, but in 170 µm 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 activates 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 production, 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). isoforms of NO synthase are identified, it became Inducible forms (mNOS) appear in cells after in- evident that the role of NO in the organism determined duction by bacterial endotoxins and cytokines not only the unique qualities of NO itself, but also the (interleukin-1, interleukin-2, interferon, tumor necro- particularity of localization of various NO synthases sis factor) (1, 2, 14). The activity of the isoform does isoforms and regulation of its activity. There are known not depend on Ca2+ concentration, therefore called three different isoforms of NOS, which vary in a se- Ca2+/calmodulin independent isoenzyme (5). It is con- quence of amino acids in their protein part and by sidered that almost all cells of the organism in patho- mechanisms of activity regulation: logical conditions are able to express NOS and this 1. Endothelial NO synthase (eNOS) found in throm- may be of great importance in immunological reac- bocytes, and endothelial cells (1, 2). tions. The activity of inducible NOS appears 6-8 hours 2. Neuronal NO synthase (nNOS) found in thromb- after induction (time for gene activation and synthesis ocytes, β-cells of pancreas (16, 17), muscle (2), lung of enzyme). Synthesized iNOS begins to generate large (3, 18), stomach, uteri epithelial cells and in endot- amounts of NO (1000 times greater than constitutive helial cells of afferent and efferent arterioles (2). forms) (2, 14). Also, the time of generating of NO is 3. Macrophagal NO synthase (mNOS) found in mac- markedly longer. Distinctly from cNOS, iNOS is con- rophages, neutrophils, Kupffer cells, macro- and sidered to be pathological isoform. It appears only in microglia (2, 19). pathological conditions and generates big amounts of All the isoforms are products of different genes. NO toxically for the cells (2, 14). It is considered, that The gene of nNOS is localized in 7th chromosome, the mainly iNOS plays an important role in suppression of gene of mNOS – in 17th, and the gene of eNOS – in activity of bacterial and tumors cells, in pathogenesis 12th chromosome (1, 2). of arterial hypertension and in the appearance of lip- Although all isoforms catalyze the same reaction, ids peroxidation disorders. each of them do has its own structural and functional specificity, that’s why there are constitutive (cNOS) The structure of nitric oxide synthase and inducible (iNOS) forms of NOS distinguished. Complex studies of NOS have demonstrated that Constitutive forms (eNOS and nNOS) are Ca2+/ NOS was one of the most complicated enzymes both in calmodulin dependent enzymes. Their activity depends its structure and regulation having a lot of cofactors. directly on concentration of Ca2+ and is maximal at There are six very important cofactors participating in concentration 1 mM of Ca2+ (1, 2, 19). These enzymes NO synthesis: nicotinamide adenine dinucleotide phos- constantly generate small amounts of NO, create so phate (NADPH), flavin adenine dinucleotide (FAD),

MEDICINA (2003) Vol. 39, No.6 - http://medicina.kmu.lt 538 Vagan Arzumanian, Edgaras Stankevičius, Alė Laukevičienė, Egidijus Kėvelaitis flavin mononucleotide (FMN), tetrahydrobiopterin Nitric oxide synthase activation pathways 2+ (BH4), calcium (Ca ) and calmodulin (19, 20). Different localization of isoforms (eNOS on cell Mechanism is still unclear but studies showed that membrane, mNOS and nNOS in cytosol) deter- even without L-arginine NOS might produce reduced mines the different pathway of activation (1, 2) oxygen and peroxide (H2O2) (1). Continuous NADPH (Fig.3.). oxidation without L-arginine shows that flavin cofac- One pathway - the binding of mediators to ionotropic tors may participate in electron transportation in the receptors when ion channels in membrane are opened process of occurrence of reduced oxygen (1, 4, 19, and Ca2+ entering to cytosol from extracellular fluid is 20). This type of reaction is similar to oxidation reac- increased. Extracellular Ca2+ gets into cell and induces tion in mitochondria (20). Ca2+ mobilization from endoplasmatic reticulum. It cre- NOS exist in cells as dimer and are active in such ates a complex of Ca2+/calmodulin, which activates mode (14, 20) (Fig. 2.). Every subunit consists of re- NOS. ductase, calmodulin-binding and oxygenase domains The other pathway – the action through (2, 5, 20). Reductase domain has flavin cofactors FAD metabotropic receptors: activated metabotropic re- and FMN. FAD is a primary electron acceptor from ceptor separates G protein (αβγ) from guanosine ADPH; FMN transports electrons from FAD to heme diphosphate (GDP) (in cytosol αβγ makes compound of oxygenate domain. Oxygenase domain has heme with GDP). When GDP is separated from G protein, L-arginine and tetrahydrobiopterin binding sequence guanosine triphosphate (GTP) comes in, because the (19, 20). It is thought that Ca2+/calmodulin induces NOS concentration of GTP in cells is higher that GDP (be- to conformative state, which is needed for electron cause of active enzyme in cell, phosphorylating GDP transportation from NADPH to heme of oxygenase to GTP). GTP-αβγ quickly degrades to monomer domain where electrons are used in the L-arginine and GTP-α and heterodimer βγ. GTP-α and βγ (mes-

O2 reaction. The strength of calmodulin and NOS bind- sengers) bind to membrane by lipidation mode and ing determines catalytic qualities of each NOS isoform. diffuse through membrane to enzymes-targets: mem- The activity of nNOS and eNOS strongly depends on branous NOS forms, adenylyl cyclase, phospholi- 2+ Ca when calmodulin and mNOS are bound so tightly pases A and Cβ (FLA2 and FLCβ), and cGMP phos- that doesn’t need Ca2+ (5, 19). The activity of NO phodiesterase. During one cycle metabotropic recep- isoforms may determine the role of NO as physiologi- tor separates a lot of, sometimes up to 1000, G pro- cal regulator or as toxic agent. teins from GDP (21).

Fig. 2. The structure of nitric oxide synthase (21) Domains of subunit: 1 - reductase domain has cofactors nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN); 2 - calmodulin-binding domain has calmodulin (CAM) binding locus; 3 - oxygenase domain has heme (Fe), L-arginine (ARG) and tetrahydrobiopterin (BH4) binding locus. FAD is a primary electron acceptor from ADPH; FMN transports electrons from FAD to heme of oxygenate domain, participating of tetrahydrobiopterin. Electrons are used in the L-arginine and O2 reaction, in which NO molecule is synthesized. MEDICINA (2003) Vol. 39, No.6 - http://medicina.kmu.lt Mechanisms of nitric oxide synthesis and action in cells 539

calmodulin

Fig. 3. The pathways of NO synthase activation IR - ionotropic receptor, ER - endoplasmatic reticulum, cNOS- cytosolic NOS form, MR - metabotropic receptor, GDPαβγ - guanosine diphosphate and G protein complex, GDF – guanosine diphosphate, GTP- guanosine triphosphate, βγ and GTP-α - signal transductors, FLCβ-phospholipase Cβ, PIP2 – inositol phosphatidyl diphosphate, IP3 – inositol triphosphate, DAG - diacylglycerol, mNOS - membranous NOS form, L-arg - L-arginine, NO – nitric oxide.

FLCβ substrate is inositol phosphatidyl diphos- Nitric oxide synthesis inhibition pathways phate (PIP2), which is on inner layer of membrane. NO synthesis regulated by activators and inhibi-

FLCβ degrades PIP2 to inositol triphosphate (IP3) tors during different physiological (pathophysiological) and diacylglycerol (DAG) (second messenger) (21, states. There are several important ways of NOS in-

22). Negatively charged IP3 is water-soluble and hibition: carries signal in cytosol. It binds to a specific re- 1.Concurrent inhibition by L-arginine derivates. This ceptor on membrane of endoplasmatic reticulum group is represented by N-monomethyl-L-argin- and induces the release of Ca2+ from endoplasmatic ine (L-NMMA), NN-dimethyl-L-arginine (L- reticulum to cytosol (Ca2+ mobilization) (21, 22). ADMA) and N-nitro-L-arginine methyl ester (L- Ca2+ binds to calmodulin, cytosolic NOS is acti- NAME), which non-selectively competes with L- vated. So, the mobilization of Ca2+ from arginine and equally inhibits all NOS (1, 24). Also endoplasmatic reticulum to cytosol may be induced there are known selective iNOS (L-canavanine, not only by Ca2+ current from extracellular space N-amino-L-arginine (L-NAA), amidin-N- but also by second messengers appeared due to ac- iminoethyl-L-ornitine (L-NIO), amino guanidine) tivation of metabotropic receptor. and eNOS (N-omega-cyclosporin-L-arginine (L- Inducible NOS appear only after specific cell re- OCA)) inhibitors (1, 2, 24). ceptors (tyrosine kinase) are excited by bacterial en- 2.NADPH using flavin enzyme inhibition. One rep- dotoxins or peptide of cytokine group (interleukins, tu- resentative of this group is diphenyleneiodonium, mor necrosis factor), which regulate cell division, dif- which inhibits irreversibly all enzymes, which use ferentiation and apoptosis (1, 2, 14). Activated tyrosine NADPH (1). kinase phosphorylases amino acid tyrosine. Most of 3.Competitive inhibition of calmodulin receptors. signal proteins bind tyrosine phosphate (that is recog- Calmodulin antagonists (trifluoperazine, chlorpro- nize the signal). Among signal proteins there are sig- mazine, calmidazolium) inhibit all NOS dependent nal transductors activating transcription (STAT), of Ca2+/calmodulin. Inhibition is specific because the which diffuse to nucleus of cell and by phosphorylat- receptors of NOS isoforms are specific (1). ing, activates transcription factors, which induce iNOS 4.Tetrahydrobiopterin (BH4) (19, 25) synthesis inhi- gene expression, and 2 hours later, the synthesis of bition. Representative 2,4-diamino-6- iNOS begins (21, 23). hydroxypyrimidine (DAHD) inhibits GTP

MEDICINA (2003) Vol. 39, No.6 - http://medicina.kmu.lt 540 Vagan Arzumanian, Edgaras Stankevičius, Alė Laukevičienė, Egidijus Kėvelaitis

cyclohydrolase-I, which initiates synthesis of BH4. Summary Methotrexate inhibits dihydropteridine reductase The research of nitric oxide qualities, features and N-acethyl-5-hydroxytryptamine inhibits and mechanisms of action, using theoretical mod- sepiapterin reductase, which participates in con- els, laboratory findings and interpretation of new

version of sepiapterin to BH4 (1). results gives us a view of mechanisms of nitric ox- 5.Glucocorticoids - inhibit iNOS gene expression in ide synthesis, role of different NO synthases cells (2, 22). isoforms in nitric oxide synthesis, the mechanisms 6.NO itself inhibits NOS by negative feed-back of reaction with substrates-targets and the role of mechanism. This inhibition is caused by NO reac- nitric oxide in different physiological and pathologi- tion with NOS heme group. cal processes.

Azoto oksido sintezė ir veikimo mechanizmai ląstelėse

Vagan Arzumanian, Edgaras Stankevičius, Alė Laukevičienė, Egidijus Kėvelaitis Kauno medicinos universiteto Fiziologijos katedra

Raktažodžiai: azoto oksidas, azoto oksido sintazė, hemas.

Santrauka. Azoto oksidas – tai dujinio pavidalo laisvasis radikalas, kuris in vivo susidaro iš molekulinio deguonies ir L-arginino. Didelis azoto oksido molekulės judrumas lemia greitą šio junginio erdvinį pasiskirstymą aplink jį pagaminusias ląsteles, o nesuporintas elektronas paverčia jį labai reaktyviu junginiu, sąveikaujančiu su esančiais metalais fermentų sudėtyje, hemu, superoksido anijonais, deguonimi ir kt. Azoto oksido sintezę ląstelėse katalizuoja fermentas azoto oksido sintazė, turinti tris izoformas: endotelinę, neuroninę bei makrofaginę. Dėl skirtingų sužadinimo ir aktyvumo reguliavimo mechanizmų azoto oksido sintazė dar skirstoma į konstitucinę ir indukuojamąją formas. Konstitucinių formų skaičius išlieka beveik vienodas viso ląstelės ciklo metu ir jų aktyvumas priklauso nuo Ca2+/kalmodulino koncentracijos. Jos lokalizuojasi citozolyje arba membranoje ir dalyvauja reguliuojant fiziologinius procesus. Indukuojamosios formos sintezuojamos tik patologijos sąlygomis, paveikus ląsteles bakteriniais endotoksinais arba citokinais. Jos laikomos patologinėmis formomis ir jų aktyvumas nepriklauso nuo Ca2+/kalmodulino koncentracijos. Azoto oksido sintazė katalizuoja elektronų pernešimą molekulinio deguonies ir L-arginino reakcijai. Tai vyksta azoto oksido sintazės struktūroje esančių flavininių kofermentų ir hemo dėka. Nors visos izoformos sužadina tą pačią reakciją, kiekviena iš jų turi savo unikalią struktūrą ir lokalizaciją. Tai nulemia aktyvinimo bei slopinimo būdų skirtumus. Azoto oksido savybės bei azoto oksido sintazių izoformų skirtumai lemia skirtingą azoto oksido vaidmenį fiziologinių ir patologinių procesų metu.

Adresas susirašinėjimui: V. Arzumanian, KMU Fiziologijos katedra, A. Mickevičiaus 9, 3000 Kaunas El. paštas: [email protected]

References 1.Knoweles RG, Moncada S. Nitric oxide synthases in mam- donor, on an experimental model of tumor cells line HELA.) mals. Biochem J 1994;298:249-58. 2002. Available from: URL: http://1.cellimm.bio.msu.ru/ 2.Malkoch AV, Majdannik VG, Kurbanova EG. Fiziologiches- groups/kvf/egor.html kaya rol’ oksida azota v organizme. (The physiological role of 6.Romanova T, Krasnov T, Avramov P. Elektronnaja struktura nitric oxide in the organism.) 2001. Available from: URL: http:/ kompleksa gema gemoglobina s oksidom azota i dinamika /www.invencardio-m.ru/rus/references/phis_role_ of_NO.htm. atomnogo ostova pri fiziologicheskoj temperature. (Electronic 3.Roderberg AD, Chaet SM, Bass CR, Arkovitz SM, Garsia FV. structure of hem complex of hemoglobine with nitride oxide Nitric oxide: An overview. Am J Surgery 1995;170:292-9. and dynamics of atomic base under physiological tempera- 4.Garthwaite J, Boulton CL. Nitric oxide signaling in the central ture.) Voprosy medicinskoj khimii 2001. Available from: URL: nervous system. Annu Rev Physiol 1995;57:683-706. http://medi.ru/doc/8810304.htm 5.Plotnikov E. Izuchenie citotoksicheskoj aktivnosti donora 7.Lorkovic IM, Wedeking K, Zanella AW, Works CF, Massick oksida azota 3-nitro-4-fenilfuroksana na eksperemental’noj SM, Ford PC. Reactions of nitrogen oxides with heme models. modeli kletok opukholevoj linii HELA. (Investigation of Characterization of NO and NO(2) dissociation from Fe(TPP) citotoxic activity of 3-nitro-4-phenylfuroxane, a nitric oxyde (NO(2))(NO) by flash photolysis and rapid dilution tech-

MEDICINA (2003) Vol. 39, No.6 - http://medicina.kmu.lt 541

niques: Fe(TPP)(NO(2)) as an unstable intermediate. J Ameri- dm.htm can Chemical Society 2002;36:565-8. 17.Schmidt HH, Warner TD, Ishii K, Scheng H, Murad F. Insulin 8.Tsoukias NM, Popel AS. Erythrocyte consumption of nitric secretion from β-pancreatic cells caused by L-arginine- oxide in presence and absence of plasma-based hemoglobin. derived nitrogen oxides. Science 1992;258:1376-8. Amer J Physiol 2002;282:2265-77. 18.Atz AM, Lefler AK, Fairbrother DL, Uber WE, Bradley SM. 9.Stasch JP, Schmidt P, Alonso-Alija C, Apeler H, Dembowsky Sildenafil augments the effect of inhaled nitric oxide for K, Haerter M, et al. NO-and haem-independent activation postoperative pulmonary hypertensive crises. J Thorac Car- of soluble guanylyl cyclase: molecular basis and cardio- diovasc Surg 2002;124:628-9. vascular implications of a new pharmacological principle. 19.Michael A Marletta. Nitric oxide syntase structure and Brit J Pharmacol 2002;136:773-83. mechanism. J Biol Chemistry 1993;17:12231-4. 10.Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, et al. S- 20.Stuehr DJ. Structure-function aspects in the nitric oxide nitrosylation of matrix metalloproteinases: signaling pathway syntases. Annu Rev Pharmacol Toxicol 1997;37:339-59. to neuronal cell death. Science 2002;297:1186-90. 21.Gutmanas A, Kėvelaitis E. Ląstelių dirglumas. Signalų per- 11.Lesauskaitė V, Ivanovienė L. Programuota ląstelių mirtis: davimas ląstelėse ir tarp jų. (Cells excitibility. Signal trans- molekuliniai mechanizmai ir jų nustatymo metodai. (Prog- ductions in the cells and between them.) In: Kėvelaitis E, rammed cell death: molecular mechanisms and detection.) Illert M, Hultborn H, sudarytojai (editors). Žmogaus fizio- Medicina (Kaunas) 2002;38:869-75. logija. (Human Physiology). Kaunas: Kauno medicinos uni- 12.Brown GC. Nitric oxide, mitichondria and cell death. IUBMB versiteto leidykla (Kaunas University of Medicine Press); Life 2001;3-5:189-95. 1999. p. 25-43. 13.Lamy M, Nys M, Deby-Dupont. Reactive nitrogen and oxigen 22.Stankevičius E, Kėvelaitis E, Vainorius E, Simonsen U. Fizio- species: Role and evidence of their production in humans. loginis azoto oksido ir kitų endotelio išskiriamų faktorių vaid- Tree Radic Biol Med 2001;13:284-300. muo. (Physiological roles of nitric oxide and other endothe- 14.Sharpe MA, Ollosson R, Stewart VC, Clark JB. Oxidation lium-derived factors.) Medicina (Kaunas) 2003;39:333-341. of nitric oxide by oxomanganese-salen complexes: a new 23.Monteiro H. Signal transduction by protein tyrosine nitration: mechanism for cellular protection by superoxide dismutase/ competition or cooperation with tyrosine phosphorylation- catalase mimetics. Biochem J 2002;366:97-107. dependent signaling. Free Radical Biology 2002;15:765. 15.Sosunov A. Oksid azota kak mezhkletochnyj posrednik. (Ni- 24.Stankevicius E, Martinez AC, Mulvany MJ, Simonsen U. tric oxide as intracellular messenger.) Biologija 2000. Available Blunted acetylcholine relaxation and nitric oxide release in from: URL: http://www.pereplet.ru/obrazovanie/stsoros/ arteries from renal hypertensive rats. J Hypertension 2002; 1128.html 20:1571-9. 16.Nikolaev A, Osipov E, Ksoeva S. Biokhimija insulinza- 25.Tiefenbacher CP, Am J. Tetrabiopterin: a critical cofactor for visimogo diabeta. (Biochemistry of insulin dependent dia- eNOS and a strategy in the treatment of endotelial disfunc- bet.). Available from: URL: http://biochemistry.vov.ru/dm/ tion? Heart Circ Physiol 2001;6:H2484-8.

Received 13 November 2002, accepted 13 December 2002