Syntázy oxidu dusnatého (NO synthases - NOSs) Oxid dusnatý (NO) • • •• Zloženie: atóm kyslíku a dusíku viazané dvojnou väzbou Atom kyslíku nesie 2 páry (nevazebných) elektónov Atom dusíku má 1 pár nevazebných elektrónov a jeden nepárový elektron HaN- H;N H N-OH NH, NH o2 -V? 1.0 NADPH \ / NH H30 *HjN' L-Arginin *H3N o2 0.5 NADPH \ H,0 coo / NH N=0 N -Hydroxy *L*Arginín *H3N^ ^COO L-Citrulín Oxid dusnatý • V savčích buňkách je NO tvořen oxidací terminálnfho auanidino dusíku L-aroininu molekulárním kyslíkem; kromě NO vzniká L-citrulin. • Celou komplexní reakci katalyzuje jediný enzym, NO syntáza, která existuje 3 isoformách. L-arg + 02 —> NO + L-cit Furchgottův sandwich Furchgott prokázal, že relaxace cév indukovaná acetylcholinem je závislá na endoteliu. Použil dva kousky aorty, u jednoho odstranil epitelium Krc&sure gauges' Eiuioihir litím removed -acetylcholine rcsull. in contraction Aecty Ich oline (AC« Contraction tiiŕv e Endothelium prewnt -;ic«lylclioline results in ,,|.iWio:i Robert F Furchgott 1916 Dept. of Pharmacology, SUNY Health Science Center New York A «laxirtg. factor from the cmk>thelium - EPK F -was dii<:ovctc<3 NA = NoratJrc neline Ignarrova spektrální analýza Ignarro pomocí spektrální analýzy prokázal, že EDRF je totožný s NO. Hemogíobin (žlutý) exponovaný endoteliálním buňkám produkujícím EDRF (konverze oxyhernoglobinu na methemoglobin). Hemoglobin (žlutý) exponovaný přímo NO. Posun v absorbční křivce je identický (EDRF = NO). Louis J Ignarro, 1941 Dept. oř Molecular and Medical Pharmacology UCLA School of Medicine Lo& Angeles Muradova enzymatická aktivace Murad věděl, že nitroglycerin působí relaxaci hladké svaloviny. Enzym guanylát cyklasa byla aktivována a indukovala zvýšení cGMP s následnou ralaxací svalu. Působí nitroglycerin cestou uvolňování NO ??? Probublával NO přes tkáň obsahující enzym - cGMP se zvyšoval. cGMP Ferid Murad 1936 Dept. of Integrative Biology Pharmacology and Physiology University of Texas Medical School, Houston Myosin (relaxation) Guanylyl cyclase (GC) activated GC Něco málo chemie o NO NO ie radikál (lichý počet valenčních elektronů, konkrétně 11 - o 1 víc než N2, o 1 míň než 02) to, zeje to radikál, se někdy zdůrazňuje tečkou (NO), to ale není nutné, "radikálovost" je implicitní v označení NO z N2 a 02 se tvoří jen za specifických podmínek při vysokých teplotách, např. při blesku: taky vzniká ve spalovacích motorech a tepelných elektrárnách samovolně se nerozkládá, jen za vyššího tlaku - při něm pozvolna vzniká 2-3 % toxického N02 za měsíc (pozor na skladování v bombách!) poměrně málo rozpustný ve vodě (-1.7 mmol/l při 25°C), t.j. řádově podobně jako 02 či N2 • v přítomnosti kyslíku podléhá autooxidaci za vzniku N02: 2 NO + 02 —► 2 N02 autooxidace je asi 200x rychlejší v roztoku než v plynné fázi autooxidace je rychlá (několik sec), je-li NO i 02 hodně, ale celkem pomalá, je-li NO málo - jako je tomu většinou v tkáních, kde je NO méně než 10 |jM (poločas NO tam může být až 500 sec) ve vodném roztoku jsou produktem autooxidace nitritv (NOA pouze v přítomnosti hemoproteinů proběhne oxidace až na nitráty (NOO v přítomnosti superoxidu vzniká extrémně rychle peroxynitrit: NO + 02~ -► "OgNO OONO není radikál, aleje velmi reaktivní a cytotoxický NO je velmi rychle inaktivovan oxidací s železem oxyhemoglobinu za vzniku N03" FYZIOLOGIE Neurotrans ptiíJŕý i násle k silnější odpovědi postsynaptického Zabíjí viryrbaktérie, pai Kortokostéftoidy) Inhibuje mitochondria I n ¥äzod Nataše jrksuce hTuauska (.Ortcentrace NO ve iQtyjiou r^^š^^j odf listáli^^c^^ectcf je koncentrace NO pod detekčním limitem vysoce citlivé chem j^iniscenůrif metody {Chest 110:9^.-938: 1996^ a ns Nejvíc MS J je končentr. chiernilj(iminiscenrn[nrietodv(Cjjesf 110:9^.-938: 1996^ ^ m PravdeDodobne důlezi Za fyziologických podmienok sa oxid dusnalý tvoři v organizme v nízkych koncentráciách (pM). Je rozpustný vo vode a v lipidoch. a preto vermi rýchlo a ochotne difunduje cez cytoplazmalické aj plazmatické membrány. V takomto prípade prevláda jeho regulačné pôsobenie: cGMP-dependentné účinky - NO aktivuje enzým guanylát cyklázu, čim sa zvyšuje koncentrácia cyklického guanozin-3.5-monofosfátu (cGMP) v cieľových bunkách. cGMP potom priamo reguluje mnohé bunkové funkcie. Riadi niektoré bunkové kanály, znižuje intracelulárnu koncentráciu Ca-" iónov a inhibuje kontraktilný aparát v hladkom svalstve. Okrem toho reguluje väzodilatáciu ciev. moduluje srdcovú konlraktilitu a znižuje zrážanlivost krvi. Nemenej dôležitý je jeho funkčný podiel na neurotransmisii a tvorbe pamäťovej stopy. cGMP-indepedentné účinky - V tomto prípade sa oxid dusnatý uplatňuje pri inhibícii syntézy ONA a aj celkového energetického metabolizmu bunky. Reguluje metabolizmus železa. Pri zápalových procesoch sa jeho koncentrácia v organizme mnohonásobne zvyšuje (uM). Vtedy sa NO a aj jeho reaktívne metabolity účastnia na protízápalových, antibakteriálnych. antivirálnych a antioxidačných procesoch. Cytotoxické a cytostatické účinky oxidu dusnatého sprostredkovávajú bunky imunitného systému, zúčastňujúce sa zápalových procesov. Sú to neutrofily. monocyty a makrofágy. NADPH NADP* NOS FAD HEME FMN HB4 n CaM GTP Generator cell Target cel cGMP + PPi ^.7 Syntásy oxidu dusnatého • neuronální syntása oxidu dusnatého (NOS1 = nNOS) • inducibilní syntása oxidu dusnatého (NOS2 = iNOS) • endothelials syntása oxidu dusnatého (NOS3 = eNOS) Každá z těchto syntáz: • má rozdílnou tkáňovou distribuci • lokalizovaná na různých chromozomech Name Neuronal NOS [nNOS or NOS1 Present in Central and peripheral neurons, platelets, pancreatic ß cells, epithelial cells Stimuli NMDA, insulin, thrombin Description Produces NO in neuronal tissue in both the central and peripheral nervous system. Neuronal NOS also performs a role in cell communication and is associated with plasma membranes. nNOS action can be inhibited by NPA (N-propyl-L-arginine). Inducible NOS (iNOS or NOS2) Endothelial NOS (eNOS or NOS3 or constitutive/ cNOS) Macrophages, endothelial cells. chondrocytes, hepatocytes. smooth muscle cells Endothelial cells, neurons, cardiac myocytes Endotoxin, IFN-y, IL-1,TNF-a Acetylcholine, ADP. thrombin, shear stress, VEGF Can be found in the immune system but is also found in the cardiovascular system. It uses the oxidative stress of NO (a free radical) to be used by macrophages in immune defence against pathogens. Generates NO in blood vessels and is involved with regulating vascular function. A constitutive Ca2+ dependent NOS provides a basal release of NO. eNOS is associated with plasma membranes surrounding cells and the membranes of Golgi bodies within cells. Všetky 3 isoformy NO syntázy • Sú aktívne jako h o m od i m éry • Obsahujú v aktívnom centre hem • Sú ste reo specific ké (D-arginín nie je substrátom) • Jako kofaktory vyžadujú: •NADPH • 6(R)-5,6,7,8-tetrahydrobiopterin (BH4) • FAD FMN • kalmodulin (ten se k NOS typu I a III váže po navázání Ca na kalmodulin, NOS II váže kalmodulin trvale) FADH O2 + Arginine ■NO + Citrulline NADP+ Electrons (e') are donated by NADPH lo the reductase domain of the enzyme and proceed via FAD and FMN redox carriers to the oxygenase domain. There they interact with the heme iron and BHaat the active site to catalyse the reaction of oxygen with L-Aroinine, generating citrulline and NO as products. Electron flow throught the reductase domain requires the presence of bound Ca'VCaM. Flavin adenine dinucleotide f faď> is it* pckkw nviiwvfc mediate the uptake of particular molecules and ions from the exterior and then redislribute these compounds in intracellular compartment through a process called potocytosis * cvcle between the plasma membrane and Ihe ER for delivery of molecules inside the cell * many receptors and cvtosolic signaling proteins that do not require lie-id modifications to associate with membranes, such as PKCa. are reportedly found in caveolae ■ number of viruses, parasites and bacteria utilize caveolae {or caveolae-iike domains) as an alternative route to enter cells. Proteins called caveolins • represent major components of the caveolar coat • important for the structure of caveolae. Ihanks to their ability to oligomerize and bind cholesterol • caveolin-1 and 2 have a similar tissue distribution, being mainly expressed in endothelial, epithelial and muscle cells • eaveolin-3 expression is limited to muscle cells caveolin-1 adaptor molecule or scaffolding protein in signal transduction Caveolin-1 functions as a tumar suppressor in human colon carcinoma cells hndkr «i ■!. Am Rh SI |M02h >1*IM. CivmHh jUimdIii Hiramm tmu naUk t^xntg »"H »•*—» OľWmm» <* dowutHin town H* m« lunurf fvepwtt' eft»* (+.«Ji-1 Lipid rafts ■ In artificial membranes, different lipids separate from each other based on their physical properties, forming small islands called lipid rafts. These rafts have a higher concentration of certain specialized lipids, called glycoschingolipids. and cholesterol ihan do non-raft parts of Ihe membrane. Rafls are also dislinguished by 3 different assortment of proteins. Certain types of proteins cluster together in rafts, while others remain mostly outside of rafts. ■ Although the exislence of lipid rafts in cellular membranes remains controversial, many scientists believe ihey serve as communication hubs by recruiting proteins that need to come together in order to transmit a signal, Thev are important signal transduction centers in the Plasma membrane, coordinating and integrating incoming signals, especially in tyrosine kinase signalling. Researchers are beginning to linií lipid rafts with a variety of diseases, including AIDS, Alzheimer's, anthrax, and atherosclerosis. Caveolae iho teste ro I Slycospiingolipd Tyrosine phospnoryiseon Site (arc) sre family kinase Caveolln-1 pliCu http://www.scielo.cl/fbpe/img/bres/v35n2/img06-01.gif Transmembrane proteins GPI-tethered proteoglycans Glycol Sphingolipids ^__________ V Lipid raft Cholesterol _____J http://www.steve.gb.com/images/science/lipid_raft.png Regulation of Endothelial Nitric Oxide Synthase Classical regulation by calcium All NO-synthases required for its activation to be bound to a calcium regulatory protein: calmodulin. iNOS tightly binds calmodulin even at resting calcium concentrations, and then it is active once it is synthetized. Constitutive enzymes, eNOS and nNOS, only bind calmodulin when the intracellular calcium concentration increase up to a certain value. Agents that increase intracellular calcium concentration, either by allowing calcium entrance from the outside or by stimulating calcium mobilization from intracellular stores, can activate these constitutive enzymes. In endothelial cells various substances increase intracellular calcium and in consequence NO synthesis: bradykinin, histamine, serotonin. • Calcium-independent regulation Activity of eNOS is acutely dependent on intracellular localization and also dependent on phosphorylation at specific aminoacids. Intracellular localizatipn • eNOS is predominantly localized in caveolae (specialized invaginations of the plasma membrane), where it is closely regulated by interaction with caveolin-1. Modifications preventing membrane localization of eNOS also result in the absence of NO synthetic activity in the intact cells. Membrane distribution is probably needed by the presence in the same localization of other proteins important for eNOS activity: the cationic amino acid transporter CAT-1 (involved in the uptake of L-arginme. substrate for NO synthesis), calcium pump and the bradvkinin receptor are also present in caveolae. • Although membrane distribution is an essential requirement for eNOS activity, at plasma membrane the enzyme activity is closely regulated by caveolin-1. This intrinsic protein strongly reduces eNOS activity bv interfering with calmodulin binding. Intracellular calcium increase or shear stress displace caveolin-1 and allow eNOS activation. • Membrane localization of eNOS is modulated by certain post-translational modifications: • Mvristovlation distinguish eNOS from nNOS and iNOS, that are predominantly cytosolic proteins • Palmitoylation is also required for a proper localization of eNOS in the membrane Phosphorylation: Tvr-Phosphorvlation. Ser/Thr-Phosphorylation Oxygen free radicals • In addition to direct regulation of NO-synthases, NO availability is also dependent on the quantity of oxygen free radicals generated by cells surrounding NO-producer cell. In fact, eNOS may generate superoxide instead of NO in certain conditions (e.g. low L-arginine levels). Whatever the origin of superoxide (eNOS, xanthine oxidase,...) this compound rapidly reacts with NO to form peroxynitrite. In certain pathological circunstances an increase in superoxide formation can be determinant in reducing NO availability. [Based on GovofS and Rabetink. Am J Physiol 2001. 280:F193J , insulin receptors ►♦•• Caveolae Extracellular Bradykinin receptor •••• ____J 3 Intracellular. Inactive eNOS ^„fr L'Ar9 Ca 2+1 Caveolin Calcium pump L-Arg Akt PAK MAPK AMP-PK ► NO Regulation of eNOS factors that regulate the transcription of eNOS gene (shear stress, estrogen and hypoxia) factors that modulate the stability of its mRNA (tumor necrosis factor alfa or TNF-alfa, lipopolysacharide or LPS, and vascular endothelial group factor or VEGF) permanent changes of the eNOS protein e.g. myristoylation. palmitovlation myristoylation seems a critical factor to allow the final location of the enzyme at certain specific domains of the membrane. non-permanent changes of eNOS protein e.g. phosphorylation and specific interactions with another proteins. After those modifications the eNOS protein is active and synthetizes NO oÄ some cases superoxide ion (this later circunstance can take place when the substrate, L-arginine, or tetrahydrobiopterin are deficient and has pathophysiological consequences). Then, all these non-permanent modifications of eNOS revert and eNOS is deactivated. A cycle of activation-deactivation occurs in parallel with a cycle of association and dissociation from the caveoale at the plasma membrane. [Based on Govers and Rabelink, Am J Physiol 2001, 280:F193] f\r\r\pj\f\r cNOS gene + shear stress tr9n?prip,ipnV^o"x|a - . . . +TNF-O.LPS Degradated mRNA -VEGF eNOS mRNA Protein protein interaction caveolae eNOS + other proteins phosphorylation o translation caveolae palmitoyl-eNOS mvristovlation^y / palmitovlation ; O myristoyl-eNOS ACTIVE phosphorylated eNOS Inducible nitric oxide synthase (iNOS, NOS II) generates NO independently of intracellular calcium concentrations induced by immunostimulatory cytokines, bacterial products or infection in a number of cells e.g. endothelium, hepatocytes. monocytes, mast cells. macrophages and smooth muscle cells (function in host defense against microbial and viral pathogens) responsible for formation of NO radicals or S-nitrosothiols or ONOO- in the host cell or in the microbe itself participate in the pathology of inflammatory diseases including atherosclerosis, rheumatoid arthritis, diabetes, septic shock, transplant rejection, and multiple sclerosis, leading to cell death (f. Aktan, Life sciences 75 (2004) 639-653) • Indukcia a regulácia iNOS expresie Indukcia iNOS: nešpecifická (oxidatívny stres. UV-žiarenie) Pomocou špecifických receptorov (liaandvTNF-g. IL-1. CD-40Ľ LPS) Regulácia iNOS sa uskutočňuje na BUNEČNEJ a MOLEKULÁRNEJ úrovni Kľúčovým faktor pre syntézu NO je zvýšenie expresie génu pre iNOS a regulácia transkripcie tohoto génu (väzobné miesta pre transkripcne faktory, ktoré sa nachádzajú na iNOS promotore) -^Miesta určené pre špecifické transkripcne faktory: jadrové faktory-kappaB (NF-kB), aktivačný proteín-1 (AP-1), CCAAT/enhancer-binding protein C/EBP a cyklický-AMP-responzívny element viažuci protein (CREB) Makrofágy (tkanivová forma monocytov) - veľmi dôležitú úlohu v zápalových procesoch (LPS, IFN-y, TNF...). Odpoveď n« stimuláciu —► produkcia prozapalovych mediatorov (IL-1, IL-2,TNF-a, NO...) IL-4 (31 tow IFN-7' STATI) IL-4 (at high iFN-v i STATI) L NO ''"*■ Anti-viral eTfett kB ISRE Regulation of iNOS induction at the molecular level. Transcription faclors controlling induction of the iNOS gene. Activated STAT1 induces transcription of [he IRF-1 and iNOS genes, an effect which is compeled by activated 5TAT6. IRF-1 interacts physically wilh NF-kB, binds to the distal k.B-binding sile of the iNOS promoter region, and slfrnulales transcription. Only when NF-kB is absent. IRF-2 can bind to íha IS RE site and block transcription. Stimulstory paihways are indicated by green arrows (—>}, and inhibitory pathways are drawn in red (Ellermann-Eriksen Virology Journal £005). HSV IFN-y NO Regulation of iNOS induction at the cellular level Cytokines controlling the iNOS induction in macrophages during early HSV infection. IFN-y. produced mainly by NK cells, stimulates iNOS production. This IFN-y-induced production of iNOS can be inhibited by IL-4. Upon HSV (herpes Sim lex virus) infection of macrophages they produce TMF which synergisms- with the IFN-y-induced pathways and inhibits the inhibitory signals of IL-4. Thus, the virus overrules the restrictive signals and opens up for an otherwise closed pathway. Stimulatory pathways are indicated by green arrows (—). and inhibitory pathways are drawn in red {Ellermann-En'ksen Virology Journal 2O05). Toll-like receptory . transmembránové pre bunky, intestinálne ep . 13 členov (niektoré si IFN-Y, IL-1, TNF receptory x Rozpoznávajú molekí Fragments DNA) --"^ Regulácia iNOS sprostredkovaná NF-kB štrukturálne a evolučné konzervovaná rodina proteínov pozostáva z piatich členov: NF-kB1 (p105/p50), NF-KB2 (p100/p52), RelA (p65), RelB a c-Rel Transkripčné faktory NF-kB sa v nestimulovaných bunkách nachádzajú v inaktívnej forme (diméry) a nevyznačujú sa žiadnym účinkom na transkripciu príslušných génov Ich aktivácia je kontrolovavaná inhibičnou podjednotkou zo skupiny inhibítorov kappaB (IkB) K aktivácii NF-kB dochádza pod vplyvom rôznych faktorov: Nešpecifický • Špecificky pomocou liqandov TNF-g, IL-1, CD-40L a LPS —> aktivujú transkripčné faktory NF-kB prostredníctvom špecifických receptorov aktivácia IkB kináz (IKK) (fosforylujú IkB zložku inaktívneho komlexu NFkB-IkB) Uvoľnením inhibitoru sa NF-kB stávajú aktívnymi a sú translokované do jadra Vazba na svoj responzívny element a spustenie expresie cieľových génov NF-kB reguluje expresiu génov, ktoré hrajú veľmi dôležitú úlohu v nešpecifickej imunite: cytokíny (IL-1, IL-2, IL-6, IL-12, TNF-a, Lta, Ltß a GM-CSF), adhezívne molekuly (ICAM, VCAM)#proteiny akútnej fázy (SAA) a inducibilné enzýmy (iNOS a COX-2). Väzbou NF-kB na DNA dochádza zároveň k spätnej indukcii transkripcie IkB. Inhibitor sa znova viaže na aktívne proteiny NF-kB. Aktivácia NF-kB je nevyhnutná pre LPS indukovanú expresiu iNOS. a používaním NF-kB inhibítorov dochádza k blokovaniu iNOS expresie a produkcie oxidu dusnatého v makrofágoch. pto-nflammatory cytokines (IL-1ß, TNF-Q) TNF Cytokines Chemokines Enzymes Adhesion Receplofs TNF-Q. II- iL-8. MIP-ta. (NOS molecules IL-2R 1ß. IL-6 MCP-3, COX-2 ICAM-1 GM-CSF eotaxin CPLA2 VCAM-1 (IL-4. IL-5> 5-LO 6-soleclin Regulácia ilMOS sprostredkovaná MAP kinázami ERK (extracelulárnymi signálmi regulované kinázy), p38 MAPK JNK (c-Jun amino-terminálne kinázy) Sprostredkúvajú fosforyláciu ďalších proteínov (protein kinázy, fosfolipázy, transkričné faktory a proteiny cytoskeletu) V bunkách majú rôzne funkcie ERK reaululujú bunkovú proliferáciu a diferenciáciu p38 MAPK a JNK sprostredkovávajú apoptózu p38 MAPK a ERK sú zapojené aj do regulácie expresie niektorých prozápalových génov (iNOS, IL-6) • pto-nflammatory cytokines (IL-1ß, TNF-Q) TNF Cytokines Chemokines Enzymes Adhesion Receplofs TNF-Q. II- iL-8. MIP-ta. (NOS molecules IL-2R 1ß. IL-6 MCP-3, COX-2 ICAM-1 GM-CSF eotaxin CPLA2 VCAM-1 (IL-4. IL-5> 5-LO 6-soleclin Inhibitors of NOS widely used in experimental research still in under investigation for clinical application Treatment with NOS inhibitors (chronic inflammatory diseases, e.g. rheumatoid arthritis) Some such drugs are derivatives of arginine alkyl derivatives of isothiourea are very potent inhibitors of NOS Some experimental inhibitors that indeed do show some preference for iNOS and nNOS selective inhibition of iN^5 should be advantageous in septic shock and in chronic inflammatory Wseases a) H,N— *V—E CCH, -------l^V-M urginme N^-methylaiginine M^-nitroainirLine methyLesier b) ¥—°A J ifi—';—yAi-y.- ^■. .1:" ■ ' I i. - -. ii - .; S- mclliy] - i S4.JL h i n ľ i 1 ru 11 i iif ARL 17477 (nNOS-selective) 6-cyclohcxyl- 2-i m i nopiperidine (iNOS-*dccti\e) http://watcut.uwaterloo.ca/webnotes/Pharm acology/noNoslnhibitors_ws.png Detekce NO Priame stanovenie NO: Gas-phase chemiluminescence assay Electron paramagnetic resonance (EPR) Electrochemical detection cell-permeabilní fluorescenční indikátory (4,5-diaminofluorescein diacetate (DAF-2 DA) Gas-phase chemiluminescence assay NO Detection by Gas Phase Chemiluminescence Detection Princiole: NO is purged from an aqueous solution using an innert gas such as Ar or He and transferred to a mixing chamber where it reacts with 03 under reduced pressure. NO + O3 —► N02* + 02 k —► N02 + h u The light emitted by excited N02 upon returning to the ground state is measured by photon counting (fmol-pmol). Not very useful when attempting to quantify NO in physiological fluids such as serum, plasma or urine. Why? Ozone C hem i I u mi nescence reaction Reaction chamber Sample capillary Vacuum Photon counter cell-permeabilní fluorescenční indikátory (4 diaminofluorescein diacetate (DAF-2 DA) Bioimaging of Nitric Oxide Using Diaminofluoresceine-2 (DAF-2) AF-2 DA DAF-? DAF-2 T coo coo non-fluorescent, cell-permeable non-fluorescent fluorescent, Ex 495 nm, Em 515 nm Advantages: Sensitivity for NO (5 nM in vitro) with high temporal and spatial resolution. No cross-reactivity to N027N03- and ON 00' Kojima era/., BloLPharm. Bull. (1997) Assay limitations: Possible interference by reducing agents and divalent cations, requires standardized illumination conditions Electrochemical detection Duo* 18 Now records data directly from NQMKIt, EVOMX, IS02, pH electrodes and Ion Selective Electrodes! Micromanipulator M3301 itaWcimt Included} Magnetic Holding Device MID 1 g 0 300 600 900 T200 Concentration / nM Time / S Ag/AgCl Sensing WPI Element Membrane 100 nm Nepriame stanovenie NO: celková koncentrace nitrátů/nitritů (Griessova metóda) aplikace NO donorů compounds, NO scavengerů, a guanylyl-cyklásy NOS aktivita v buněčných homogenatech měřením enzymatické konverze argininu na citrulin během tvorby NO inhibitory NOS (L-NAME) aplikace protilátek k isoformám NOS (imunocytochemie, *nunoblotting) exprese genu pro iNOS Lipid Peroxidation -+-*^ ^^+- OMA Breaks XXXXX + Mc-m branc Damage t HP-SH Qicidation 4- ON00 L-Aifl ■^ EC Proiilerfllign/Higrfltwn A^igiocjenisis cGMP fr Vasodilation —^ Blood Flow r*^ cUMP-Gated cGMP- PKG k?n Ch-annel PDE Activation Parkinson's Disease M' % 4 Programmed Cell Death Cardiovascular \/ NO« /\ Antimicrobial Agent Impotence (Viagra®) r riPiiiivx-^T^i^F imm-ii4rtitn«n