Application of a nitric oxide sensor in biomedicine

Biosenso s 2014, 4, 1-17; doi:10.3390/bios4010001 biosenso s ISSN 2079-6374 www.mdpi.com/jou nal/biosenso s/ Re iew Applica ion o a Ni ic Oxide Senso in Biomedicine Ca lo a Saldanha 1,*, José Ped o Lopes de Almeida 1,2 and Ana San os Sil a-He dade 1 1 Ins i u o de Bioquímica, Ins i u o de Medicina Molecula , Faculdade de Medicina de Lisboa, Edi ício Egas Moniz, A . P o . Egas Moniz, 1649-028 Lisboa, Po ugal; E-Mails: [email p o ec ed] (J.P.L.A.); [email p o ec ed] (A.S.S.-H.) 2 Se ico de Imunoale gologia, Cen o Hospi ala Lisboa No e-Hospi al de San a Ma ia, 1649-028 Lisboa, Po ugal * Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed]; Tel.:+351-91-8985450; Fax: +351-21-7999477. Recei ed: 19 Decembe 2013; in e ised o m: 21 Janua y 2014 / Accep ed: 23 Janua y 2014 / Published: 4 Feb ua y 2014 Abs ac : In he p esen s udy, we desc ibe he biochemical p ope ies and e ec s o ni ic oxide (NO) in in ac and dys unc ional a e ial and enous endo helium. Applica ion o he NO elec ochemical senso in i o and in i o in e y h ocy es o heal hy subjec s and pa ien s wi h ascula disease a e e iewed. The elec ochemical NO senso de ice applied o human umbilical enous endo helial cells (HUVECs) and he desc ip ion o o he s NO ypes o senso s a e also men ioned. Keywo ds: endo helium e y h ocy e ni ic oxide; mic oelec ode 1. In oduc ion Ni ic oxide (NO) is a soluble gas syn hesized om L-a ginine by he enzyme ni ic oxide syn hase (NOS) [1]. Th ee iso o ms o NOS ha e been desc ibed, namely he neu onal (nNOS o NOSI), he inducible (NOSII o iNOS), and he cons i u i e endo helial o m (eNOS o NOSIII), which was he i s o be disco e ed [1–3]. Bo h neu onal and endo helial enzymes a e ac i a ed by he calcium and calmodulin complex, and he inducible o m binds o calmodulin bu is independen o in acellula calcium concen a ion [4]. These isoenzymes a e la op o eins ha ac on L-a ginine, in he p esence o oxygen and NADPH, and equi e e ahyd obiop e in (BH4). In he absence o BH4, a supe oxide OPEN ACCESS Biosenso s 2014, 4 2 anion is o med ins ead o NO [5]. The iso o m eNOS is also exp essed in pla ele s and in ca diac myocy es [6,7]. NO p oduced in he endo helial cell di uses o he lumen whe e i is cap u ed by ed blood cells (RBC) and ans e ed in o muscle cells whe e i induces elaxa ion, elici ing asodila ion. In his mechanism, guanosine 3′,5′-cyclic monophospha e (cGMP), o med om guanosine 5′- iphospha e (GTP) by he ac ion o guanyla e cyclase (GC), is ac i a ed by NO [3]. cGMP modula es he myosin ligh chain (MLC) phospha ase posi i ely and MLC kinase nega i ely, esul ing in he dephospho yla ion o MLC wi h subsequen muscle elaxa ion [3]. NO-induced asodila ion is dependen on hema oc i , blood low and he hemoglobin ee concen a ion in he ci cula ion [8]. The p esence o ee hemoglobin educes asodila ion in pig co ona y a e ies, induced p e iously by se o onin o by shea s ess [8]. The inc ease in ascula pe meabili y, inhibi ion o pla ele agg ega ion, pla ele adhesion, p oli e a ion, and mig a ion o smoo h muscle cells a e e ec s media ed by NO-dependen cGMP [9]. The in ac ascula endo helium es ablishes a dynamic in e ace be ween blood and issues, allowing gas and me aboli es exchanges and pa icipa es in hemos asis, in h ombosis, and in in lamma o y and an i-in lamma o y mechanisms [10,11]. The pheno ype o endo helial cells is dependen on he loca ion in he ascula ield and p esen s speci ic esponses o a ious s imuli [11]. Endo helial cells o pos capilla y enules espond o in lamma o y signals and hose in he a e ial ascula ne wo k elease asoac i e subs ances in o he blood [11]. Among he endogenous asoac i e compounds, ace ylcholine (ACh) ac s like an au oc ine o pa ac ine signal in endo helial cells, s imula ing eNOS, wi h he o ma ion o NO [1,3]. The apid ime cou se o hese e en s and he sho hal -li e o NO includes se e al me hods, based on elec ochemical, chemiluminiscen , and spec opho ome ic p inciples, which ha e been de eloped in o de o measu e NO o i s de i a i e molecules. The me hod mus be sensi i e o in si u measu emen s. NO mic osenso s ha e been de eloped o eal ime assessmen in i o [12–14]. The eal ime measu emen o NO, in esponse o s imuli and d ugs, will con ibu e o he apeu ic ad ances in endo helial dys unc ion. 2. Ni ic Oxide Senso s The u iliza ion o NO senso s allows he quan i ica ion o NO concen a ion anging om subnamola o mic omola alues [15]. Usually, he biosenso consis s o a bio ecogni ion elemen , a signal ansduce and a de ec o . The elec ochemical senso s a e, o now, he mo e eliable ool o NO de ec ion in eal ime. They ope a e ia he applica ion o a po en ial a he elec ode su ace posi i e o nega i e o elec ochemically oxidize o educe NO. The esul ing ans e o elec ons is measu ed as a cu en p opo ional o he NO concen a ion. Ou s udies o NO measu emen in human e y h ocy es suspensions we e pe o med wi h he amiNO-IV senso (Inno a i e Ins umen s Inc., Tampa, FL, USA) [16]. The igu e o he elec ode can be seen in he webpage: h p://www.2in.com. The senso has a sha p me allic ip comple ely co e ed wi h a se ies o memb ane, including a gas pe meable memb ane. The ―amiNO‖ se ies o ni ic oxide senso s, wi h ip diame e anges o 7 µm o 600 µm, do no equi e an ex e nal e e ence elec ode has high sensi i i y abolishing he e o s due o baseline d i associa ed wi h empe a u e changes and hey a e shielded om elec ical noise. They Biosenso s 2014, 4 3 we e designed o in i o and la ge su aces (cul u ed cells), wo ks wi h he inNO-T me e wi h easy calib a ion p ocedu es. The inNO-Tcombine bo h a NO con igu ed po en ios a and a so wa e con olled da a acquisi ion sys em included in one ba e y powe ed uni . The ―amiNO‖ se ies senso is co e ed wi h a iplecoa gas pe meable memb ane o gua an ee selec i i y and as esponse ime. The NO di uses h ough he memb ane and is hen oxidized a he wo king pla inum elec ode, esul ing in an elec ic cu en . The edox cu en is p opo ional o he NO concen a ion ou side he memb ane and is con inuously moni o ized wi h an inNO-TM so wa e ( e sion 1.9 supplied by Inno a i e Ins umen s Inc.) ins alled on a PC compu e . The calib a ion cu e and i s ep esen a i e appea in Figu e 1 o ou published p e ious wo k [16]. B ie ly, he senso is calib a ed by a simple, economical, and a eliable chemical eac ion o NO p oduc ion. This eac ion is based on he con e sion o ni i e o ni ic oxide in acidic solu ion in he p esence o iodide ion. The eac ion has a a io o one o one, meaning ha he amoun o NO p oduced in his eac ion equal o he amoun o ni i e added. The amino-IV senso wi h i s NO-pe meable memb ane iplecoa a oid a b oad ange o in e e ing molecules o ming du ing he elec ochemical eac ion o NO on me al su aces a posi i e elec ode po en ials ia elec on oxida ion mechanism: Alloys o pla inum [17], ca bon ibbe [18], and glassy ca bon [19] a e ma e ials addi ionally de eloped o co e he su ace o he elec odes ha show a iable sensi i i y, selec i i y, and signal s abili y [20–23]. The mos common modes o elec ode ope a e a e by elec o educ ion o NO, di ec elec ooxida ion o NO and ca aly ic elec ooxida ion o NO [20–23]. The elec o educ ion has he ad an age o elimina e he in e e ing molecules bu , a a iance, has low sensi i i y and pH and elec ode su ace cha ac e is ics dependence [24–26]. In hese ypes o elec ode oxygen molecules in e e es and is a p oblem in biological applica ions due o i s sca enge p ope ies as men ioned in he p e ious sec ions. In he elec ode ope a e by elec o educ ion o NO he in oduc ion o a ansi ion me al o me allop o eins such as haemoglobin ha e p o en o be use ul o imp o e sensi i i y and measu emen s o NO a low ange o sub-mic omola concen a ions [24,25]. In he elec ode ope a e by he di ec elec ooxida ion o NO he e a e b oad ypes o senso s wi h di e en elec ode ma e ial composi ion o NO-selec i e memb anes, and di e se diame e s o igina ing a la ge a ie y o alues o limi o de ec ion anging om 0.083 nM o 75 nM [27,28]. Thus, hey a e dependen on he pe mselec i e memb ane. In he ype o elec ode ha ope a es by ca aly ic elec ooxida ion o NO a edox media o o example a me allopo phy ins immobilized on he elec ode su ace o inco po a ed in a polyme is u ilized [18]. The unc ion o he media o is o ac s as ca alys s o he oxida ion o NO. Howe e , non-po phy in complexes ha e showed also simila esul s [29]. The senso s desc ied in he li e a u e o measu e NO in solu ion a e classi ied as belonging o h ee classes as ollows: Shibuki-s yle; solid pe mselec i e; and solid ca aly ic and hei cha ac e is ics and composi ion a e summa ized in he Table 1 [20,30]. The i s one s yle o senso de e mine NO by elec ooxida ion and he o he s wo by elec ooxida ion o elec o educ ion [20,30]. All he h ee ypes o senso s in eg a e a e e ence elec ode ha is wi hin he elec oly e illing solu ion in he Shibuki-s yle. The ca aly ic s yle comp ise a media o (me allopo phy ins o me al ph alocyanines) o ca alyze he oxida ion o educ ion o NO. While senso s applied o in i o, NO measu emen s in blood has con ined in humans [31] o he s o de e mina ions o NO eleased in biological issues like hea , b ain, o lung a e used only in animal Biosenso s 2014, 4 4 expe imen al models [32–34]. Fo example an elec ochemical mic osenso has been inse ed in o a human hand ein o de ec NO in blood essels o heal hy pe sons [31]. I is con i med in i o, in human beings, ha he endo helium de i ed elaxing ac o is he NO om he s imula ion wi h ACh [31]. Table 1. Cha ac e is ics and composi ion o ni ic oxide senso s. Senso class In e nal illing solu ion Composi ion Sensi i i y Mini u iza ion Shibuki-s yle Elec oly e Pla inum and sil e Va iable o e ime and be ween senso s No possible Solid pe mselec i e Elimina ed Ca bon Mul iple memb anes disc imina e in e e ence molecules Possible Solid ca aly ic Elimina ed Media o inco po a ed in elec ode su ace o in pe mselec i e memb ane Minimize in e e ence molecules Possible 3. Ni ic Oxide in A e ial Endo helium The asoac i e unc ion o ACh could be comp omised by he e y h ocy e agg ega ion endency ha is inc eased in a ew ascula diso de s including hype choles e olemia, a e ial hype ension, acu e myoca dial in a c ion, and diabe es [35–38]. The ascula endo helium is dys unc ional when i is no able o egula e i s one o main ain s uc u al o ganiza ion con ibu ing o he ins alla ion and p og ession o hype ension and a he oscle osis [11,39]. These a e ial diseases a e conside ed ca dio ascula isk ac o s and a e associa ed wi h s imula ion o NAPH oxidase and gene a ion o eac i e oxygen species [40,41]. Dismu a ion o he supe oxide anion hyd ogen pe oxide is o med, s imula ing he exp ession o eNOS ha was e i ied o be associa ed wi h hese wo ca dio ascula isk ac o s [42,43]. In his case, NO p oduc ion is insu icien o o e come consump ion by supe oxide anion wi h he gene a ion o pe oxyni i e ha de egula es (uncouples) eNOS, swi ching o he p oduc ion o supe oxide anion ins ead o NO [44]. Vasodila ion o he essels ails o appea by lowe concen a ions o NO, which suppo pla ele and leukocy e adhesion s imula ing he in lamma o y esponse in he pa hogenesis and p og ession o a he oscle o ic disease [45]. I s e iology is ecognized as complica ed and mul i- ac o ial [45]. Thus, any manipula ions o he pa hway o eNOS appea o be p omising ea men s [46,47]. In he composi ion o he a he oma ous plaque he e a e muscle cells, mac ophages ha a e in he apop o ic s a e and a e inges ed by phagocy es, which dec ease he in lamma o y esponse and plaque eg ession [48]. P o-apop o ic unc ions and an i-a he ogenic p ope ies o NO a e impo an , along wi h des abiliza ion o he plaque, which could occu due o enhancemen o muscle cell apop osis induced by NO [48]. The mos e ec i e way o inc ease NO syn hesis is by adap a ion o hypoxia ha is obse ed in hype ensi e s age 1 pa ien s cha ac e ized by dec eased concen a ions o NO [49,50]. Pa ien s wi h g ade 1 hype ension submi ed o in e mi en condi ions o no moba ic and hypoxia no malize blood p essu e and NO syn hesis [50]. The cons an p esence o hypoxia can lead o dec eased syn hesis o NO and in u n o he onse o pulmona y a e ial hype ension. Inhaled e hyl ni i e has shown p omising esul s in neona es wi h Biosenso s 2014, 4 5 pulmona y hype ension [51]. Inhibi o s o he enzyme phosphodies e ase ha ca alyze he decomposi ion o cGMP may be an al e na i e he apy o he inhala ion o NO applied in pulmona y hype ension [52,53]. Ni oglyce in (GTN) ac s by inducing asodila ion independen o he endo helium and is used as a he apeu ic agen in co ona y a e y disease wi h some condi ions [54]. Ch onic use o NTG eg ess NO le els and main ains endo helial dys unc ion [54]. The e a e many exogenous compounds leading o ni ic oxide a ailabili y including ni i e, S-ni oso hiols, N-ni oso-p o eins, and i on ni osyl complexes [55]. Nons e oidal an i-in lamma o y compounds (NSAID) ela ed o de i a i es o NO molecules ha e been syn hesized o e i y i s e ec i eness as NO dono s and as ca diop o ec i e agen s [56]. Among he NSAIDs, aspi in de i a ized wi h NO dono s dec eases in es inal oxici y and s imula es he eNOS enzyme in a pulsa ile manne [56,57]. The s a ins, he ype 1 ecep o blocke s o angio ensin II and es ogen, inc ease he syn hesis o e ahyd obiop e in. S a ins also inhibi NADPH oxidase and p o ec he co ac o om oxida ion induced by eNOS [58]. Polyphenols p esen in ed wine, by inhibi ing he o e exp ession o NAPH oxidase educe oxida i e s ess and p o ec endo helial cells by main enance o he basal le el o NO, educing he endency o pe oxini e o ma ion [59]. Pa ien s wi h dep ession ha e diminished le els o he na u al subs a e o NO, L-a ginine; i is es ima ed ha dep ession can lead o he onse o co ona y a e y disease (CAD) [60]. Howe e , whe he supplemen a ion o L-a ginine p o ides imp o emen o CAD is unknown [60]. I is known ha a die wi h high le els o black beans, endi e, and spinach can be a sou ce o ni a e ha gi es ni i es in he sali a y-en e ic ci cula ion, ollowing a sequence o ca alysis eac ion in ol ing acidic oxides o ni ogen ha lead o NO in he blood and issues [61]. The e is e idence ha a die ich in ege ables and supplemen s o ni a es dec eases blood p essu e and he isk o b ain ischemic occu ences [62]. The isk o ca dio ascula e en s in CAD is associa ed in e sely wi h he ca aly ic ac i i y o e y h ocy e glu a hione pe oxidase [63]. In addi ion, published s udies demons a ed he exis ence o NOS3 polymo phisms associa ed wi h some g oups o pa ien s wi h CAD and wi h g oups o insulin-dependen diabe ics who will de elop CAD [64,65]. The implica ion o polymo phisms o NOS isoenzymes in he appea ance o CAD equi es u he s udy. Recen ly, measu emen s o NO in i o and in humans di ec ly using an elec ochemical elec ode senso ha e been de eloped and applied in he co ona y ci cula ion [14]. The s udy was pe o med in pa ien s wi h dila ed ca diomyopha y and in heal hy con ols and NO was measu ed in he p oximal g ea ca diac ein wi h a ca he e - ype senso [14]. The au ho s gi e a con ibu e o he clinical quan i ica ion o endo helial dys( unc ion) by measu ing in i o endo helium a ailabili y o NO [14]. The de e mina ion o NO associa ed wi h he e alua ion o co ona y diame e and co ona y blood low could gi e use ul in o ma ion o he applica ion o adjus ed he apeu ics. 4. Ni ic Oxide in he Venous Endo helium Human umbilical ein endo helial cells (HUVECs) in he p esence o ACh, he na u al subs a e o ace ylcholines e ase (AChE), libe a e highe alues o NO han basal alues [66]. Va iance when elnac ine (AChE inhibi o ) was added o he HUVECS lowe s alues o NO han hose obse ed o ACh [66]. These esul s we e ob ained using he elec ochemical senso amino-IV NO [66]. Fu he s udies a e needed o cla i y he signal ansduc ion mechanisms in endo helial cells unde he Biosenso s 2014, 4 6 in luence o ac i a o s and inhibi o s o ace ylchonis e ase. O he s biosenso s o NO e alua ion a e a ailable as desc ibed abo e. The appea ance o s uc u al changes in connec i e issue, smoo h muscle, and o unc ional changes in enous endo helium, de ec s in he mic oci cula o y ne wo k, and de icien supplies o nu ien s in he enous sec o a e induce s o a icosi y appea ance [67,68]. In enous disease, he up igh posi ion o igina es a dec ease o oxygen pa ial p essu e in issues, capilla y s asis, and hypoxia ha ac i a es endo helial cells wi h inc eased cy oplasmic calcium. This is c ucial o he elease o p oin lamma o y ac o s (such as PAF, leuko iene B4, p os aglandins E2 and D2) [69–72]. The elease o his amine and se o onin s imula e he mig a ion o leukocy es o he endo helium and exp ession o adhesion molecules in bo h cells [73]. Mo eo e , endo helial cells p oduce cy okines (IL-1be a, IL-6, TNF alpha) and p o h ombo ic ac o s ( on Willeb and ac o ) ha elici monocy es and ac i a ed T lymphocy es wi h he in lamma o y esponse [73]. In lamma ion does no occu only in he pos -capilla y enules, bu also in he la ge eins which con ibu es o a be e unde s anding o he e iology o enous h ombosis and pulmona y embolism [74]. Flow-media ed asodila ion is impai ed in pa ien s wi h spon aneous enous h omboembolism, which is an indica o o endo helial dys unc ion [75]. In he enous endo helium o heal hy indi iduals, as happens on he a e ial side, he e is elease o ni ic oxide which egula es and main ains enous one [76]. The NO sec e ion by ascula endo helial cells is depending on shea s ess [77]. In ch onic enous insu iciency, he diame e o he ein is inc eased, dec easing shea s ess and he p oduc ion o NO by endo helial cells du ing he ini ial phase. A e he exp ession o inducible ni ic oxide syn hase, highe amoun s o NO a e eleased ha in e ac wi h he supe oxide anion (p oduced by leukocy es and mac ophages) p oducing pe oxyni i e ha causes issue oxida ion o ch onic enous ulce s. The es o a ion o basal le els o NO is a he apeu ic a ge o he healing o ulce s [76]. The success o wound healing in pa ien s ecei ing monoc oma ic in a ed ene gy and submi ed o s e ching and esis ance exe cise may be associa ed wi h inc eases o NO in he blood [78]. I hea ailu e is accompanied wi h enous ascula dys unc ion, i bene i s om he apeu ics ha ac s on es o ing enous ascula unc ion dependen on NO [76]. Applica ion o an eNOS inhibi o on he enous ne wo k no malizes enous ascula esis ance and blood p essu e alues in he lowe limbs, al hough hese esul s a e s ill p elimina y [79]. Hype emia obse ed in he elde ly wi h passi e mo emen o he legs is absen in pa ien s wi h pe iphe al a e ial disease. This suppo s he hypo hesis ha NO is p esen in enous endo helial cells [80]. The esul s ob ained wi h inhibi o s o eNOS con i m his hypo hesis [81]. The au ho s conside ha he passi e mo emen o he legs may become a nonin asi e ins umen o analyzing he unc ion o he endo helial NO in enous issue [80]. Endo helial unc ion can be e alua ed using non-in asi e echniques including high- esolu ion ul asonog aphy ha measu es asodila ion in he adial, emo al, and b achial a e ies [81]. The asodila ion esul ing om he ac ion o asodila o s is used as a benchma k. Ni oglyce in is used as a diagnos ic es o po al hype ension (PHT) [82]. The ex en o PHT is quan i ied in clinical p ac ice by measu ing he hepa ic po al ein p essu e g adien [83]. In PHT, he insu icien elease o NO om endo helial cells in luences he inc ease o ascula esis ance a he le el o he in a-hepa ic mic oci cula ion [84,85]. The e is excessi e p oduc ion o NO in he splanchnic ci cula ion ha mus be accoun ed o when NO dono s a e used o educe po al p essu e [86]. Many o he s udies Biosenso s 2014, 4 7 ega ding he in luence o NO dono s on he enous ascula sec o a e pe o med in expe imen al animal models, sugges ing he bes ea men s a egies dependen on NO can unc ion in he esolu ion o human enous disease. 5. Ni ic Oxide in E y h ocy es In physiological condi ions, he e y h ocy e senses oxygen pa ial p essu e (PaO2) in he ascula u e sca enging bo h oxygen and ni ic oxide in highe PaO2 o deli e ing hem in lowe PaO2 (NO) [87]. The cap u e and dona ion o bo h gases is dependen on he haemoglobin (Hb) con o ma ion s a es being i s elaxed s a e is associa e wi h sca enging and i s ense s a e wi h bo h gases dona ion [88,89]. E y h ocy es media e he a ailabili y o NO and collec ion o and om he endo helium [87]. This ec ui ing cycle, ese a ion and dona ion o NO by e y h ocy e applies o bo h NO syn hesized in he lung o inhaled [87]. P e ious s udies ha e demons a ed he p esence o NO inside he e y h ocy e using luo escence mic oscopy [90].The in lux o NO o ed blood cells occu s h ough he band 3 p o ein also known by he chlo ide/bica bona e anionic channel [91]. The in lux depends on he endency o he band 3 p o ein o adop he s uc u e o he dime o he e ame and on he deg ee o dena u a ion o hemoglobin, i.e., he p esence o Heinz bodies and me hemoglobin [92]. The p esence o e ame s o me hemoglobin and Heinz bodies a e un a ou able and blocks he en y o NO in o e y h ocy es [12]. The a e o in lux in he e y h ocy es is highe in he hemoglobin deoxygena ed s a e han in he oxygena ed s a e [93]. Condi ions o inc eased hema oc i a o he in lux in oxygena ed RBCs [93]. The e y h ocy e ac s as a ca ie and as a ni ic oxide dono . The ou pu o NO om he e y h ocy es occu s h ough he band 3 p o ein dependen on i s deg ee o phospho yla ion [94]. The molecula o ms o NO, namely he ni osilhemoglobin (NO bound o heme i on o hemoglobin) he ni osohemoglobin (NO bind o he hiol g oup o cys eine 93 o he be a chain o hemoglobin) and ni osoglu a hione egula e he a ailabili y o NO by e y h ocy e [95]. Glu a hione is an abundan molecule inside e y h ocy es which has a hiol g oup ha can eac wi h ni ic oxide, o ming ni oso hiols such as S-ni osoglu a hione (GSNO) [96]. The NO ese oi a ibu ed o glu a hione could be in luenced by he inac i a ion o glu a hione educ ase induced by oxida i e s ess [97]. The hiol/disul ide eagen s such as educed o oxidized glu ha hione (which a e p esen a high le els inside RBCs), ha e a sui able edox po en ial ha is use ul o p o ein egene a ion. Fo ins ance, di hio h ei ol (DTT) is a hiol- educing agen capable o egene a ing disul ide-con aining p o eins and also able o es ablish in e changeable hiol-disul ide eac ions wi h glu a hione [98]. The p esence o DTT induces e y h ocy e changes in enzyma ic ac i i y s a es o example in p o ein y osine phospha ase (PTP) and p o ein y osine kinase (PTK) [99,100]. DTT signi ican ly mobilizes e y h ocy e NO o gene a e ni i es/ni a es and SNOHb, he eby dec easing NO e lux [100]. Using he same expe imen al model, DTT was p o en o enhance he le els o GSNO, ni i e/ni a e concen a ions [101]. When au o-oxida ion o hemoglobin occu s, i p oduces pe oxide anion which gene a es pe oxyni i e a e eac ing wi h NO [102]. The decomposi ion o pe oxyni i e molecules yields ni i e and ni a e [103] and he eac ion be ween pe oxyni i e and hemoglobin gene a es SNOHb, which could decompose o ni oso hiol and ni a e [104]. Howe e , NO may educe oxyhemoglobin o Biosenso s 2014, 4 8 me hemoglobin along wi h he o ma ion o ni a e wi hou any a ia ion in he me hemoglobin concen a ion [104,105]. This could be associa ed wi h he p esence o hemoglobin educ ase coupled wi h NADH ha is p oduced in he glycoly ic pa hway [106]. In i o, he NO dono , Spe mine NONOa e, induces an inc ease in he me hemoglobin concen a ion and dec eases he P50 alues, which means ha hemoglobin oxygen a ini y inc eases [105]. An elec ochemical me hod was desc ibed o quan i ica ion o he e lux o NO om e y h ocy es using an NO elec ode senso in e y h ocy e suspensions con aining ace ylcholine [16]. Fu he s udies documen ed a signal ansduc ion mechanism in e y h ocy es in ol ing he AChE-ace ylcholine (ac i e enzyme—subs a e complex), he Gi p o ein, he band 3 p o ein—dependen o he deg ee o phospho yla ion [107,108]. In he p esence o elnac ine, an inac i e enzyme complex is o med and lowe le els o e y h ocy e e lux we e obse ed han hose ob ained in he p esence o ace ylcholine [107,108]. Timolol is an inhibi o o e y h ocy e AChE [109] and does no change e y h ocy e NO bioa ailabili y in e y h ocy e suspensions [110]. This can be conside ed an ad an age i he ascula lumen is unde high le els o eac i e oxygen species, as he o ma ion o pe oxini i e will no be a o ed. The an ioxidan p ope ies o imolol ha e been epo ed in i o and in i o [111–113]. The e y h ocy e sca enging p ope y o NO is p ese ed by ib inogen binding o he RBC memb ane [114]. The same was e i ied mimicking hipe ib inogenemia condi ions in he absence o p esence o ace ylcholine [115,116]. Howe e , a e e se si ua ion is ob ained when highe ib inogen concen a ions a e simul aneously p esen wi h phospho yla ion o band 3 p o ein [115]. This esul could be conside ed a use ul he apeu ic ool in blood s o age o u he ans usion. Fib inogen binds o he e y h ocy e memb ane CD47 and when he agonis pep ide o CD47 is added o e y h ocy es, he same NO sca enging p ope y was e i ied [117]. High NO elease om RBC samples was obse ed in i o om pa ien s wi h hypoxia and in lamma o y s a es, namely sickle cell disease, hype choles e olemic, and hype ensi e pa ien s; also, impai men in e y h ocy e de o mabili y was documen ed [118]. Vasocons ic ion and ischemia may occu when pa ien s a e submi ed o blood ans usions o igina ing om blood-bank-s o ed blood, which ha e a lowe abili y o elease bo h oxygen and NO [119]. NO consump ion by e y h ocy es is egula ed unde hypoxic condi ions by deoxygena ed Hb ha binds o i on heme (NO occupies he acan si e le by oxygen). The NO-heme hemoglobin adduc (HbFe (II) NO) has been de ec ed du ing NO inhala ion he apy used o pulmona y hype ension ea men , bu i also occu s when deoxygena ed blood en e s a ascula bed in which NO is p oduced, such as he pulmona y ci cula ion [120,121]. In si ua ions o hype emia, he ni osyla ed hemoglobin ecen ly measu ed in i o by a modi ied sub ac ion me hod using elec on pa amagne ic esonance co ela ed wi h he endo helium unc ion measu ed by onome y [122]. Unde hypoxic condi ions es ablished in i o in segmen s o mesen e y a e ies o Wis a a s pe used wi h e y h ocy e suspensions asodila ion occu s due o he libe a ion o NO om e y h ocy e in dependence o he shea s ess [123]. Inducible NOS exp ession in he endo helium inc eases in ischemia/hypoxia condi ions o ischemia/ epe usion in which blood low dec eases, he eby a o ing HbFe (II) NO o ma ion [124]. F om all hese esul s, he use o NO dono s mus be accompanied by NO in si u moni o ing wi h a mic oelec ode senso . Biosenso s 2014, 4 9 6. Conclusions Ni ic oxide is a signaling molecule in luen ial in se e al ascula diseases. By NOS uncoupling, NO lowe s i s le els and endo helial dys unc ion is ins alled. The endo helial dys unc ion gene a es eac i e oxygen species ha impai he NO concen a ion by i s combina ion wi h supe oxide anion. The endency o apply NO dono s seems o be he choice o moni o ing he he apeu ic op ion. Howe e , ca e mus be aken wi h NO measu emen s in si u esul ing in he exp ession o he inducible NOS. The a ailabili y o e y h ocy e o sca enge o elease NO, measu ed using a mic oelec ode senso , is a e lex also o he endo helium and ecommended o moni o ca dio ascula disease. The signal ansduc ion mechanisms e idenced o he ACh–AChE ac i e complex can be a ou ed o he apeu ic con ol o NO bioa ailabili y o he e y h ocy e. The elec ochemical senso s a e well es ablished class o in i o senso s, which o e almos eal- ime NO de e mina ions Acknowledgmen s This s udy was suppo ed by g an s om he FCT—Fundação pa a a Ciência e a Tecnologia (p ojec e e ence EXCL/Ma -NAN/0114/2012). 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Sci. 2002, 47, 528–534. © 2014 by he au ho s; licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion license (h p://c ea i ecommons.o g/licenses/by/3.0/).