Syntázy oxidu dusnatého (NO synthases - NOSs) Oxid dusnatý (NO) Zlozenie: atóm kyslíku a dusíku viazané dvojnou väzbou Atom kyslíku nesie 2 páry (nevazebnýchJ elektónov Atom dusíku má 1 pár nevazebných elektrónov a jeden nepárový elektrón y Fab*rt F Fureh^aíi Laufe J k-.hr>o Pnnú Murnd 1915 1ŮÍI ISM D«pi. oř Pharmacology. Cwp4. flf McifcaJjf nnc [(«gl. d InlĽ^tf-YP ĹHwJcgy SUNY Ktalih &s*ní* turner Mf^fBl ftun™«fcigy Phjmiscíiaay 3"0 MůwYMk «LA 5ttKd of Ptlyiiukljr U«»!! Llmmily d1 Taxík Mrdca L«.Ang«tt 5CÄWH. HWjSHHl MU, Oj H]N H I N -OH y*1 o, P.SHAÖPH 1, řH,H' 'COO L-Arglnin H;0 K.hr(l;.If.L.Í-n,M,r NH: N«0 HifiT "GW L-Cl|™iin Cm«! duinaty • V savčí ch buňkách je NO tvořen oxidaci terminálního guanidino dusíku L-angininu molekula mim kysl i ke m; kromě NO vzniká L-citrulin. * Celo u ko mplex n í rea kci katalýz uje jed i ný e nzym, NQ syntáza. která existuje 3 isoformách. L-arg + 02 —► NO + L-cit £ 1 ■ I 400 42S *BŮ Ignarrova spektrálni analýza Ignarro pomoci spedrálni analýzy prokázal. ±e EDRF j& totoJný s NO. Hemoglobin fílutý) exponovaný endďďiálním bitkám amdtikuiicím EDRF fkcnverzg OKyhemogtofcoriLi na malhenncglofcin). Hemoglobin t,lfutý) exponovaný primo imO. Pqs .ju u BOSOrbínl kí vr.e je icenlický (EDRF ; NO; A relaxuj factor from ih: erukiihe-liuni - EDRF - wa* discüVcfcJ NA - Noradrenaline Furchgott Civ sandwich FuTchgoR prgkézal. ie relgjww cév indukovprid acetylcuolinsm je závislá na cnuolďu. Použil dva kousky aoriy. u ledrntio odstranil eprlelium NO I GTP Phosphat- Phosphate cGMP ŕ/ Myosin (relaxation) Guany ly l c yclase MÄDH pMinrimpma« FMNH2 FADHa FMNH NADPH NO + Citrulline 02 + Arginine Electrons (e) are donated by NADPH ta (he reductase domain of the enzyme and proceed via FAD and FMN nidox carriers to Ihe oxygenase domain. There they interact with trie heme iron and EH, ai tne active site lo catalyse the reaction of oxygen whh L-Arginine. generating citrulline and NO as products. Electron Flow through! the reductase domain r&quires Ihe presence of bound Ca:'.fCaM. Name Present in Stimuli Description Neuronal NOS InNOS or NOS1 Central and peripheral neurons, platelets, pancreatic ß cells. epithelial Cells MMOA. insulin, thrombin Produces NO in neuronal tissue in both Ihe central and peripheral nervous system. Neuronal HOS also performs a role in ceil communication and is associated with plasma membranes. nNOS action can be inhibited by NPA (N-propyl-L-argmmc;. Inducible NOS (iNOS or NOS2) Endothelial NOS (eNOS or NOS J or constitutive/ cNOS) Macrophages, endothelial cells. chondrocytes., hepatocytes, smooth muscle calls Endothelial cells, neurons, cardiac myocytes Endotoxin, IFN-y. IH.TNF-0 Acetylcholine, ADP, Ihrombin. shear stress. VEGF Can be found in the immune system but is also found m (he cardiovascular system. It uses the oxidative slrass or 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 tells and Uie membranes of Golgi bodies wilhm ceils. Za fyziologických podmienok sa oxid dusnatý tvoři v organizme v nízkych koncentráciách (pM). Je rozpustný vo vode a v lipidoch, a preto veľmi rýchlo a ochotne difundují* cez cyioplazmatické aj plazmatické membrány, V takomto pripade prevláda jeho regulačné pôsobenie: cGMP-dependentné účinky -NO akiivuje enzým guanylát eyklázu. čim sa zvyšuje koncentrácia cyklického guanozín-3,5-monofosfátu {cGMPj v cieľových bunkách. cGMP polom priamo reguluje mnohé bunkové funkcie. Riadi niektoré bunkové kanály, znižuje intraceluíárnu koncentráciu Ca"' iónov a inhibuje konlrakdlný aparát v hladkom svalstve. Okrem toho reguluje vázodilatáciu ciev, moduluje srdcovú kontraktililu a znižuje zrážanhvosť krvi. Nemenej dôležitý je jeho funkčný podiel na neurotľansmisii a Ivorbe pamäťovej stopy. cGMF-indeoedentné účinky - V tomto pfipade sa oxid dusnalý uplatňuje pri inhibídí syntézy ONA a aj celkového energetického metabolizmu bunky. Reguluje metabolizmus řeleza. Pri zápalových procesoch sa jeho koncentrácia v organizme mnohonásobne zvyšuje (pM). Vtedy sa NO a aj jeho reaktívne metabolily účastnia na protizápalových, antibakieriálnych, antiviraIných a antiqxidaCných procesoch. Cytotoxické a cytostatické účinky oxidu dusnatého sprostredkovávajú bunky imunitného systému, zúčasiňujúce sa zápalových procesov. 5ú to nsutrofily. monocyty a makrofägy. NADPH NADP+ NOS FAD HEME FMN HB, lí CaM GTP cGMP + PP< Generator ce Target cel ■ -../ Neuronal Nitric Oxide Synthase (nNOS. Type II Glutamat« Homodimers with 2 subunits (130-160 kDa) nNOS have binding sites for NADPH. FAD, and FMN near the csrboxyl terminus (the reductase domain), and binding sites for tetrahydrobiopterin (BHj and heme near the amino terminus (the oxygenase domain). The reductase and oxygenase domains are linked by a calmodulin (CaM) binding site Occupation of this site facilitates electron transfer from the cefaclors in the reductase domain to heme during nitric oxide production. nNOS is associated with the post-synaptic density protein (PSD-95) in the neuronal membrane. In response to increased intracellular Ca2"\ nNOS interacts with CaM The Ca2*-CaM complex, in combination with BH4, binds to nNOS and induces its translocation from the plasma membrane to the cytoplasm. The dephosphorylatton of nNOS by calcineurin initiates the production NO, NO activates g u any I yl cyclase (GC) and activates the various cGMF-reoulated signaling pathways. nNOS is inactivated by phosphorylation by protein kinase A (PKA) or protein kinase C ŕPKCV Neuronal nitric oxide synthase (nNOS) has been implicated in a wide variety of physiological and pathological processes. These include neurotransmission, neurotoxicity, skeletal muscle contraction, sexual function, body fluid homeostasis and atherosclerosis, among others. L-Cit L-Arg NO I cGMP re gula lion Long-Term potentiation Neurotransmission NMDA receptor (HMDAR) is an itinotrrj;iii receptor for grulamate N M DA tN-methyl d-asoartate) is a name of its selective specific agonisl Activation of NMDA receptors results in the opening of an ton channel that is nonselective to cations. This allows flow of Ma" and smaN amounls of Caž* ions into the cell and K' oul of the cell. Calcium flux through NMDARs is Ihought to play a critical role in synaptic plasticity, a ceälular mechanism for learning and memory. The MMDA receptor is dislinct in ihat it is both linand-aated and voltaae-decendent. Activation of NMDA receptors requires b'nding of glutamate or aspartate (aspartate does not stimulate the receptors as strongly). In addition, NMDARs also require the binding of the co-agonist glycine for the efficient opening of the ion channel, which is a part of this receptor. Endothelial nitric oxide synthase (eNOS or NOS3 or constitutive/ cNOS) • localised to caveolae • eNOS is a lipid raft/caveolar protein apparently regulated by caveolin • Agonist stimulation induces calcium dependent association of protein cofactors and kinases ultimately resulting in generation of nitric oxide from Arginine Regulation of Endothelial Nitric Oxide Synthase Classical regulation bv calcium • All NO-svnthases 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 nMOS, 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. VEGF, insulin receptors Caveolae :fc- Bradykinin receptor .•••' .y #■' ľ% Eilraccllulnr Intracellular Inactive eNOS **o* lei u m pump L-Arg Akt PAK MAPK AMP-PK > NO Calcium-independent regulation Activity of eNOS is acutely dependent on intracellular localization and also dependent on phosphorylation at specific aminoacids, Intracellular localization * 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 MO synthetic activity in the intact cells. Membrane disiribution is probably needed by the presence in the same localization of other proteins important for eNOS activity: the cat ion i c amino acid transporter C AT-1 (involved in the uptake of L-arginine. substrate for NO synthesis), calcium pump and the bradykinin 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 by 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-translationai modifications: * Mvristovlation distinguish eNOS from oNQS and iNOS, that are predominantly cytosolic proteins * Palmitoylation is also required for a proper localizaiion of eNOS in the membrane Phosphorylation: Tyr-Phosphorylation. 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-anginine 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 Covers and Rabefink Am J Physiol 2001. 280.F193] 'EGF, insulin receptors Caveolae EmnctHluliir Bradykinin receptor %&> LArQ Ca 2+ L-Arg Akt PAK MAPK AMP-PK Intracellular ľ% Inactive eNOS Calcium pump '**. > NO Regulation of e NO 3 » factors that regulate the transcription of eNOS gene (shear stress, eslnogen and hypoxia) * factors that modulate the stability of It» mRNA ((umor necrosis factor alfa or TNF-alfa. lipopolysacharide or LPS, and vascular endothelial group factor or VEGF) * permanent changes of the eNOS protein e.g. mvristoylatioru palrmtoylation, myristoylalion seems a critical factor lo 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 synlhetizes NO or in some cases superoxide ign (this later circunslance can take place when the substrate, Ľarginine, or tetrahydrohiopterin nre deficient and has pathophysiological consequences). Then, all these non-oermaneni modifications of eNOS revert and eNOS is deactivated A cycle of aciivation-deactivation occurs in parallel with a cycle of association and dissociation from the caveoale al the plasma membrane- [Based on Covers' and Rabelink, Am J Physiol 2001, 230:F193) AAAAAA/ eNOS gene + shear stress transcriDtionV5^6" . hypoxia caveolae Degradated mRNA + TNF-cr, LPS VEGF eNOS mRNA Protein protein interaction eNOS + other proteins phosphorylation translation eNOS protein mvristovlatiorť cavcolac 1 myrlstoyl-eNOS palmitoyl-eNOS palmitovlation caveolae ACTIVE phosphorylated-eNOS Caveolae * small invaginations fvesiclesl of Die plasma membrane with a welt-defined size (50-100 nrn) and a particular lipid content ■ localised in plasma membrán? (lipid rafts-rich in glycgsphingolipids and cholesterol) * Involved in transcytosis, lipid trafficking and more recently signal transduction * very dynamic organelles lhal can pinch off the plasma membrane in a process thai requires the hydrolysis of GTP ■ mediate trans-epithelial transport of small molecules across the cell by fusing together to form (fans-cellular channels ■ mediate the uotake of particular molecules and ions Prom the exterior and then redistribute these compounds in intracellular compartment through a process called potocytosis ■ cvcle between the plasma membrane and the ER for delivery of molecules inside Ihe cell * many receptors and cytosolic signaling proteins that do not require lipid modifications to associate with membranes, such as PKCa, are reportedly found in caveolae - number of viruses, parasites and bacteria utilize caveolae {or caveolae-like domains) as an alternative route to enter cells. Proteins called caveolins • represent major components of the caveolar coat * important for the structure of caveolae, than ks to their abil ity to oligomenze and bind cholesterol • caveolin-1 and 2 have a similar tissue distribution, being mainly expressed in endothelial, epithelial and muscle cells * caveolin-3 expression is limited to muscle cells caveolin-1 adaptor molecule or scaffolding protein in signal transduction Caveolin-1 functions as a tumor suppressor in human colon carcinoma cells hnhil si .B« |3^1«.CjdmIm and —wnUi Hi» n—rt—j» dcnmnj n.j*fc 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 giycosphirifloii&ids, and c ho I a sterol than do non-raft parts of Ihe membrána. Rafts are also distinguished by a different assortment of proteins. Certain types of proteins cluster together in rafts, while others remain mostly outside of rafts. ■ Although Ihe existence of lipid rafts in cellular membranes remains controversial, many scientists believe ihev 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 link lipid rafts with a variety of diseases, including AIDS, Alzheimer's, anthrax, and atherosclerosis. _U caveolae w_ 3>^ phospholipid Tyrosine pnospnoryistion Sítí produkcia prozápalových mediátorov (IL-1, IL-2,TNF-a, IMO.„) IL-»(*lbwlF»-Tŕ STATI) IL.A {ti hiflh IFW.Y ■' STAI1) hMO*^*AMwiHHff»4n -a ISRE ka Regulation of iNOS induction at the molecular level. Transcription factors compelling induction of tha iNOS gens Activated STAT1 induces transcription of Hie IRF-1 and iNOS genes, an effect which is competed by scijvaicd Statí. iRF-1 interacts physically wih nF'kB. binds to me distal xS- binding site of trie iNOS promoter region, and stimulates Iransciption Only when NF-*B is absem IRF-2 can bind to in« iSfiE sit« and block Iranscfipbun Stimulátory pathways are indioalad by green arrows (—>, and inhibitory pathways are drawn in red fEltermann-Eriksen Virology Journal 20Q6). Regulation of iNOS induction at the cellular level Cytokines controlling the iNOS induction in maencťriaíJSS during eorly HSV inr*airjn IFN-y. p*CKJue*J ms*ikr by NK «Us. stimulate* 'NÜ5 production. This IFN-y-mduced production of iNOS tan be inhfcited by IL-J. Upon HSV (herpss simlex virus} infection of macrophages thay produce TNF which synergizas with the iFN-v-induied pathways and inhibits tha inhibitory signals of IH. Thus, tha wus overrules the rest.rir.1ive signals end opens up for an otherwise closed pathway. Stimulatory pathways are indicated by green arrows í —. ), and inhibilory pathways at« drawn m red (Elermann-Entsen Virology Journal 2005). Toll-like receptory • tra osmerribrá nově proteiny fmonocvtv . makrofäQv. nftutrnfilv. denririfekŔ bunkv. intestina I ne epitel iálna a endoteliálne bunky • 13 členov (niektoré su lokalizované intra- iné extraceluláme) • Rozpoznávajú molekuly významné pre infekciu a zápal (LPS, Lipoproteiny, Fragmenty bakterálnych bičíkov. Bakteriálnu DNA, Dvojvláknovú (virálnu) DNA) • Akt i váda: * Zahájenie rýchlych obranných reakcií • Dve sig ná I ne dráhy (N F-kB t ra nsk ripčn é faktory, Mitogén-aktivovaně protein kinázy (MAPK)) • Konečné produkty TLR-4 - cytokiny (IL-1b, -6, -12. TNF) a RKM LPS • Súčasť bakteriálnej steny Gnarn-neg. baktérií • crniüUjxi'i • vyvoláva zápaíové odpovede • imeraguje s LPS-viaiúcinn proteínom (I.PS-BP) v sáre a vytvára kjwnlax ktorý sa v prítomnosti CDU viaže na TLR-4 • Polr-ebna prítomnosť sekreíného proteinu MD-2 IFN-Y.IL-1, TNF receptory • IFN-Y indukuje aktiváciu ilMOS prostredníctvom Jak-STAT signálnej dráhy TGF-p.TNF-a. IL- 4, IL-10, IL-11, IL- 13, LPS, IFN-oLp lFN-r.TNF-g, JL-1, IL-Í, IL-10, MIF, LPS, IFN-u.ľi L-Arg LPS OOCOOCO^OOOOOOOOÓOOOOOOOOOf^ IL-4, IL-10, IL-13, TGF- P, LPS OOOOOOOOOOOCOOOOOOOCOXXXXXXXXX) Ar-; ■■ ■-;:, succinate AS NO / Antimicrobial activity Tumoricldsl activity Signaling and immunoregulalioni C yto!ox ici ty.'t iss u e destruction FAD 1 ',•.'', Arg in a se | NADPH + Ornithin» + citrullincľ ODC _J L OAT + + i * * i 4-ir-JOS ■^Proinflammatory cytokines Proline NO pathways Regulation and function of inducible ntlnt oxide synthase, arg=nase and related pathways in mouse macrophages. The activity of iNOS u reguislefl fcy eyioKme* and microbial produds (judi a* IPS), which affect (he uptake of L-arginina (L-Arg) by cationic amino acid transporters (CAT), the synthesis of cefaclors, the expression of iNOS mRNA and prolem. the enzymatic recycling of citrglline lo nrgmine and the depletion of árgmine by arginase. Polyanwias- products of lhe 3rginase-ODC paihway, act as immunosuppressants aid can further downiegulate 1lie produclion at NO. A high argirsaie activity in lhe aUsence cl NQS can also be associated wilfi (issue fibrosis tesuUinů item the increased syMNesis ůi prdine tit m« argíňasí-OAT palh*ay, which is required for collagen synlhests (tar example, by Fibroblasts). AL, argminosuccinalie řvase, AS. any nmcBucciriale synthelase: MIF. macrophage migralwn inhibitory facwr. ODC. wniwine gecaroonylase: OAT gmi1h.ne aminotransfarasg. Regulácia iNOS sprostredkovaná MAP kinázami • ERK (extracelulámymi signálmi regulované kinázy). • c38 MAPK • JNK (c-Jun amino-terminálne kinázy) • Sprostredkúvajú fc-sforyláciu tfaliich proteínov (protein kinázy, fosfalipázy, transkričné faktory a proteiny cytoskeletu) • V bunkách majú rôzne Funkcie * ERK regulu! u iú bunkovú proliferáeiu a diferenciáciu * p38 MAPK a JNK sprostredkovávajú apoptôzu * d38 MAPK a ERK su zapojene aj do regulácie expresie niektorých prozápglgivých génov {iNOS, 11-6} Regulácia iHQS sprostredkovaná NF-kB • štrukturálne a evolučné konzervovaná rodina proteínov pozostáva z piatich členov: NF-KB1 (p105/p50)t NF-KB2 (p100/p52), RelA (p65), RelB a c-Rel • Transkripŕné faktory NF-kB sa v nestimulovaných bunkácn nachádzajú v inaktivnei forme (diměry) a nevyznačujú sa žiadnym účinkom na transkripciu príslušných génov • Ich aktivácia je kontrolouavanä inľiibičnou podjednotkou zo skupiny inhibitorov kappa B (IkB) • K aktivácii NF-kB dochádza pod vplyvom různých faktorov; • Nešpecifický • Specificky pomocou lioandav TNF-o. IL-1. CD-40L a LPS -> aktivujú transkripčné faktory NF-kB prostredníctvom špecifických receptorov • aktivácia IkB kináz (IKK) (fosforylujú IkB zložku inaklivneho komlexu NFkB-IkB) • Uvoľnením inhibitoru sa NF-kB stávajú aktívnymi a sú translokované do jadra • Vazba na svoj responzívny element a spustenie expresie cierových génov • N F-k B reguluje expresi u génovL ktoré hrajú veľmi dôležitú úlohu v nešpecifickej imunite: cytokíny (IL-1, IL-2, IL-6. IL-12, TNF-c, Lta, Ltp a GM-CSF), adhezívne molekuly (ICAfvl, 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 aktivně proteiny IMF-kB. • Aktivácia NF-kB ie nevyhnutná ore LPS indukovanú exorasiu iNOS. a používaním NF-kB inhibitorov dochádza k blokovaniu iNQS expresie a produkcie oxidu dusnatého v makrofáqoch. proinflammatory cytokines (IL-1 p. THF-a) INF- IhHt SEK1IMKK4 ; KAFKs.'JNK MKKI.ft MEK 1,1 1 pM ERK1Í Cytokines TNF-n. IL-1P. IL-6. GM-CSF (IL-4, IL-5) Chfimnkirfis 1 Š MIP '.i MCP-3. frataxin Enzyme iNOS COX-2 tPLAÍ J-LO Adhesion molecule* ICAM-1 VCAM-1 E-sulf.-ul:n R«csi5tofs IL-2R a) hi NOS inhibitor Inhibitors of NOS have been described which interact with the NOS enzymes in a variety of ways: • different sites (l-arginine, biopterin site, haem-binding inhibitors, flavoprotein and CaM inhibitors) • differing time-dependence • substrate-dependence • mechanism of inhibition • widely used inhibitors - l-NMMA, l-NNA and its methyl ester prodrug (WG-nitro-l-arginine methyl ester, l-NAME) - effect on nNOS dimerization but did inhibit both NOS and NADPH oxidase activity of nNOS, eNOS and iNOS in a time-dependent manner Inhibitors of NOS • widely used in experimental research • still in under investigation for clinical application • Treatment with NOS inhibitors (chronic inflammatory diseases, eg. 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 iNOS should be advantageous in septic shock and in chronic inflammatory diseases SildenafiM (Viagra) • inhibitor of the phosphodiesterase subtype 5, which is selective for cGMP • removes cGMP and thus terminates the action of NO (if protein nitrosylation is neglected) • Sildenafil was not developed with its now-famous application in mind; instead, the idea was to come up with another vasodilatator, Its enhancing effect on penile erection gave rise to the discovery that NO actually is the transmitter that triggers this process • Thus, without NO. no one of us would even be here today! h,N— argji K,N----- N^-methy I j i r i 11 i ne N^-nitiojigJniiie iikIIk IlSFCI K^A s—CA ^n hjN—c—coo« S-clhyl-lhkúbourca S-im.lhyl-uio[hi(wilrLilLiiK> ARL 17477 (nNOS-Mlcctive) 6-cyc lohcxyl- 2-i m i nopi pcrid i nc (iNOS-selective) http://watcut.uwaterloo.ca/webnotes/Pharm acology/noNoslnhibitors_ws.png Detekce NO NO Detection by Gas Phase Chemiluminescence Priame stanovenie NO: • Gas-phase chemiluminescence assay • Electron paramagnetic resonance (EPR) ■ Electrochemical detection • cell-permeabilni fluorescenční indikátory (4,5-dtaminofluoregcein diacelale (DAF-2 DA) Nepriame stanovenie NO: »celková koncentrace nitrátů/nítritíi (Griessova metoda) • aplikace NO donorti compounds, NO scavengers a guanylyl-cyklásy • NOS aktivita v buněčných homogenatech měřením enzymatické konverze argininu na cítrulín během tvorby NO • inhibitory NOS (L-NAME) • aplikace protilátek k isoformám NOS (imunocytochemie, imunoblotting) • exprese genu pro iNQS Detection Principle: 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 N02* -----* N02 + ft 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? Chemiluminescence reaction 1 -<— Ozone Duo* 18 Atol* rerordj úala directlyJrom NOMKI1, EVOMX, IS02, pH electrodes and Ion Selective Etectroúes\ Micromanipulator M3301 t •H C t 'S, 5BQ S 4» | 200 0 "^ J 300 600 900 1200 /WV^ Concentration / njň f Jt 8nM 3 U t 4bM 2nM Time / S Reaction chamber Sample capillary Vacuum Photon counter Bioimaging of Nitric Oxide Using Diaminofluorcsceine-2 (DAF-2) Sensing WPI Element Membrane __ 100 nm non-fluorescent H ¥t non-U uorescent nqn,T ľľ Vr Ex4S5 nm, Em 515 nm ce 11 -permeable ^^ -^ Advantages; Sensitivity for NO {S nhl in vitro} with high temporal and spatial resolution. No cross-reactivity to NO^/NO,- and ON ÜO Koprna etnl.. Btol.Fhjrm. Bulí. (1997) Assay limitations: Possible Interference by reducing agents and divalent cations, requires standardized illumination condition« A home-made miniature saturated silver/silver chloride reference eleclrode B porphyrinic rmicrosensor working electrode C platinum win? cgun|gr electrode ISO-NO Mark II NO meter (World Precision Instruments (WPI)) HyuUHHB DUO 18 two-channel data-system (WPI) Thermostat RAW 264.7 (1x106)(100x) porphyrinic microsensor (7 urn in diameter, typical length 0.5-1 mm) RESULTS Measurement of NO release from RAW 264.7 cell line using microsensor ■ RAW264.7without stimulation ■RAW 264.7 stimulated by LPS 5 10 15 20 Time / h .AMT 2 4 6 8 10 12 14 16 18 20 Time (h) ,MEM 25- —•— RAW 264.7 -AMT / +LPS ,r- 20- --#-- RAW 264.7-AMT/-LPS / _l —m— RAW 264.7 +AMT / +LPS / O 15- E i ľ~ 10- O z . 5- +-.-r'~...$!£r*.+.................... --I iii 25- ^-20- !_i Ö 15- O 2 5- 10 Time (h) 15 20 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 2O.0 Time (h) 200' ~150- ^100- c < *-» '« 100- 0) Q 50- 023456 16 20 C Time (h) I without AMT I with AMT JDU 0 4 6 Time (h) 7 8 9 10 11 12 13 14 Time (h) 1.6-1 1.5- 1.4- 1.3- BAEC - bovine aortic endothelial cells Cal 1 ug/ml Cal 2,5 ug/ml Cal 0,5 ug/ml Cal 5|ig/ml 1.2- 50 100 150 Time (min) 200 250 BAEC + L-NAME 0.7n 0.6- 0.5- Cal 1,0ng/ml M199 Cal 2,5 ng/ml L-arginine (50 nM) Cal 5 ng/ml 0.4- 0.0 0.5 1.0 1.5 Time (h) 2.0 2.5 Upld Peroxidation -^^ ^^^ <>HA Braafcs Membrane Damage t NP-SH Oridation L-Arfl ^ EC Proiilerfllion/Higrfltion Anglogenesia cGMP fc VtaBodlrfltfon —fc- Blood Flow r ^ c^r.iP-Gflted cGMP- PKG bn Chjnrul PDE Activation Parkinson's Disease % *« »I Programmed Cell Death \/ NO« Antimicrobial Agent Cardiovascular Disease Impotence (Viagra®) TľiľltrWHitaľT irrunrtTKijr-ílTn