Modulatory Effect of 2-(4-Hydroxyphenyl)amino-1,4-naphthoquinone on Endothelial Vasodilation in Rat Aorta

Resea ch A icle Modula o y E ec o 2-(4-Hyd oxyphenyl)amino-1,4-naph hoquinone on Endo helial Vasodila ion in Ra Ao a Ja ie Palacios,1F edi Ci uen es,2Jaime A. Valde ama,1Julio Beni es,1 Da id Ríos,1Cons anza González,1Ma io Chiong,3Benjamín Ca es-Saa ed a,3 Ca los La ou cade,4U sula Wyneken,4Pamela González,5Ga e h I. Owen,5 Fabián Pa do,6Luis Sob e ia,6,7,8 and Ped o Buc Calde on1,9 1Depa amen o de Ciencias Qu´ ımicas y Fa mac´ eu icas, Facul ad de Ciencias de la Salud, Uni e sidad A u o P a , Iquique, Chile 2Expe imen al Physiology Labo a o y (EPhyL), Ins i u o An o agas a, Uni e sidad de An o agas a, An o agas a, Chile 3Ad anced Cen e o Ch onic Diseases, Facul y o Chemical and Pha maceu ical Sciences, Uni e si y o Chile, San iago, Chile 4Labo a o io de Neu ociencias, Cen o de In es igaciones Biom´ edicas, Uni e sidad de los Andes, Monse˜ no Al a o del Po illo 12.455, 7550000 San iago, Chile 5Facul ad de Ciencias Biol´ ogicas y Cen o UC In es igaci´ on en Oncolog´ ıa (CITO), Pon i icia Uni e sidad Ca ´ olica de Chile, Chile 6Cellula and Molecula Physiology Labo a o y (CMPL), Di ision o Obs e ics and Gynecology, School o Medicine, Facul y o Medicine, Pon i icia Uni e sidad Ca ´ olica de Chile, 8330024 San iago, Chile 7Depa men o Physiology, Facul y o Pha macy, Uni e sidad de Se illa, 41012 Se ille, Spain 8Uni e si y o Queensland Cen e o Clinical Resea ch (UQCCR), Facul y o Medicine and Biomedical Sciences, Uni e si y o Queensland, He s on, QLD 4029, Aus alia 9Toxicology and Cance Biology Resea ch G oup, Uni e si ´ eca holiquedeLou ain,B ussels,Belgium Co espondence should be add essed o Ped o Buc Calde on; ped o.buccalde on@uclou ain.be Recei ed 27 May 2016; Re ised 9 Augus 2016; Accep ed 22 Augus 2016 Academic Edi o : Gopi Kollu u Copy igh © 2016 Ja ie Palacios e al. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. The ascula endo helium plays an essen ial ole in he con ol o he blood low. Pha macological agen s like quinone (menadione) a a ious doses modula e his p ocess in a a ie y o ways. In his s udy, Q7, a 2-phenylamino-1,4-naph hoquinone de i a i e, signi ican ly inc eased oxida i e s ess and induced ascula dys unc ion a concen a ions ha we e no cy o oxic o endo helial o ascula smoo h muscle cells. Q7 educed ni ic oxide (NO) le els and endo helial asodila ion o ace ylcholine in a ao a. I also blun ed he calcium elease om in acellula s o es by inc easing he phenyleph ine-induced asocons ic ion when CaCl2was added o a calcium- ee medium bu did no a ec he in lux o calcium om ex acellula space. Q7 inc eased he asocons ic ion o BaCl2(10−3 M), an inwa d ec i ying K+channels blocke , and blocked he asodila ion o KCl (10−2 M) in ao ic ings p econ ac ed wi h BaCl2. This was eco e ed wi h sodium ni op usside (10−8 M), a NO dono . In conclusion, Q7 induced asocons ic ion was h ough a modula ion o cellula mechanisms in ol ing calcium luxes h ough K+channels, and oxida i e s ess induced endo helium damage. These indings con ibu e o he cha ac e iza ion o new quinone de i a i es wi h low cy o oxici y able o pha macologically modula e asodila ion. 1. In oduc ion A unc ional ascula endo helium could ha e majo clinical implica ions in pa hologies such as ca dio ascula disease, diabe es, and cance [1, 2]. P e ious s udies ha e shown ha he ea men wi h quinone- ela ed compounds (i.e., 1,4- naph hoquinone de i a i es) can impai [3, 4] o imp o e ascula unc ions [5]. The unde lying mechanism(s) o quin- oneon ascula unc ionsa es illno ullyunde s ood.Fo ins ance, he obse ed dec eased elaxa ion and inc eased Hindawi Publishing Co po a ion Oxida i e Medicine and Cellula Longe i y Volume 2016, A icle ID 3939540, 12 pages h p://dx.doi.o g/10.1155/2016/3939540 2Oxida i e Medicine and Cellula Longe i y con ac ion o blood essels induced by menadione was pa ly explained by inhibi ion o NO pa hway ia he o - ma ion o eac i e oxygen species (ROS). The hypo ension and aso elaxa ion e ec s o naph hoquinone-oxime we e sugges ed o be due o ac i a ion o soluble guanyla e cyclase (sGC), K+channels, and ia NO pa hway [6]. Quinones a e widely dis ibu ed molecules in na u e, and hey a e ound as endogenous compounds o biolog- ical impo ance (i.e., coenzyme Q in mi ochond ial elec- on chain; i amin K2in blood coagula ion) in humans. Nume ous he apeu ic d ugs, in pa icula an i umo com- pounds, a e quinone-bea ing molecules including an h a- cyclines (doxo ubicin, mi oxan h one, and dauna ubicin), benzoquinones (mi omycin C, geldanamycin), o honaph- hoquinones like 𝛽-lapachone, and se e al syn he ic com- pounds which a e cu en ly unde clinical ials using 1,4- naph hoquinone as pha macopho e g oup [6–9]. They a e epo ed o cause cy o oxic e ec s h ough a ied mech- anisms such as DNA in e cala ion, educ i e alkyla ion o biomolecules, and ROS o ma ion h ough a edox-cycling eac ion [10–12]. Rega ding his la e mechanism, quinone educ ion by 1 o 2 elec ons om NADPH-cy och ome P450 educ ase leads o a semiquinone ee adical which is oxidized back o he o me quinone in he p esence o molecula oxygen, while oxygen is educed o supe oxide anion [13]. This edox-cycling leads o he o ma ion o o he ROS, such as hyd ogen pe oxide and hyd oxyl adicals [14, 15]. Based on his edox-cycling p ope y, se e al quinones ha e been used as he apeu ic agen s agains se e al diseases and pa hologies [16–18]. As such hey induced cell dea h by ei he apop osis o nec osis, inhibi ing cance cells g ow h [19–21]. They also ac i a ed a senescence p og am leading o cell cycle a es [22, 23]. Ou obse a ions we e ha one a ylamino-naph hoquin- one de i a i e, namely, Q7 [2-(4-hyd oxyphenyl)amino-1,4- naph hoquinone], was able o p o oke a d op in ATP cell con en and inhibi cance cell p oli e a ion [24]. This inhibi ion induced cance cells senescence [10], educ ion o DNA damage, and inhibi ion o in i o umo g ow h [11]. I also leads o an inhibi ion o in i o umo p og es- sion by igge ing apop osis, cell cycle a es , supp ession o HIF-1, and uncoupling glycoly ic me abolism [25]. All hese e ec s ha e been a ibu ed o i s abili y o gene a e ROS h ough quinone edox-cycling. Meanwhile, i has been epo ed ha some naph hoquinone de i a i es a e po en inhibi o s o endo helium-dependen asodila ion ia an inhibi ion o endo helial NOS, possibly by in e ac ing wi h he educ ase domain o he enzyme [4, 26]. Indeed, i has been epo ed ha oxida i e s ess enhances ascu- la eac i i y, mos likely by inc easing he o ma ion o ROS and dec easing he a ailabili y o ni ic oxide [27, 28]. Based on hese ac s and due o he c i ical ole o ascula unc ions in di e en pa hologies, we ha e explo ed new biological ac i i ies o Q7 as o be used beyond he con ex o cance , especially as i in e e es wi h oxida i e s ess and ascula unc ion ( eac i i y and endo helium), an in eg al complexin ol edin hesu i alo dea ho hesecance ous cells. 2. Ma e ials and Me hods 2.1. Cell Cul u e. Human umbilical ein endo helial cells (HUVEC)we eisola edbycollagenase0.25mg/mL,Colla- genase Type II om Clos idium his oly icum (Boeh inge , Mannheim, Ge many) diges ion om umbilical co ds ob- ained a bi h om p egnancies and cul u ed (37∘C, 5% CO2) in 1% gela in-coa ed pe i dishes up o passage 2 in medium 199 (M199; Li e Technologies, Ca lsbad, CA, USA) con aining 5 mmol/L D-glucose, 10% new bo n bo ine se um, 10% e al bo ine se um (FBS), 3.2 mmol/L L-glu amine, and 100 U/mL penicillin-s ep omycin (p ima y cul u e medium). A7 5 cells, a ascula smoo h muscle cell (VSMC) line o iginally de i ed om emb yonic a ao a, we e pu chased om heAme icanTypeCul u eCollec ion(ATCC).Theywe e cul u ed in Dulbecco’s Modi ied Eagle Medium (DMEM; In- i ogen, NY, USA, Sigma-Ald ich) supplemen ed wi h 10% FBS and 2 ×10−3 M py u a e. P io o expe imen s, 80–90% con luen A7 5 cells we e se um-s a ed. 2.2. MTS Reduc ion Assay. B ie ly, cells (50–60% con luence) we e seeded in 96-well pla es. Cells we e incuba ed a 37∘Cin a humidi ied 5% CO2/95% ai mix u e. HUVECs and A7 5 ascula smoo h cell line we e incuba ed in he absence o in hep esenceo Q7 (10−7 M, 10−6 M, 10−5 M, and 10−4 M) o 48 h, and cy o oxici y was de e mined using he MTS [3-(4,5-dime hyl hiazol-2-yl)-5-(3ca boxyme hoxyphenyl)- 2-(4-sul ophenyl)-2H- e azolium] educ ion assay. Cell Ti e 96AQueous One Solu ion Cell P oli e a ion Assay, P omega, WI, USA. Cyclophosphamide (10−4 M) was used as nega i e con ol and 10−5 M epi ubicin (an h acycline d ug used o chemo he apy) as posi i e con ol. The MTS and Q7 we e dissol ed in ehicle (DMSO) a inal concen a ion less han 0.1%. All he assays we e pe o med in quin upled and in 3 independen expe imen s. The cy o oxici y was calcula ed in acco dance wi h he o mula: cy o oxici y (%) = (1 −(abso bance sample/abso bance con ol)) ×100. The abso bance o sample was de e mined in he absence ( ehicle; con ol) o in he p esence o Q7.Theabso bance was measu ed wi h a mic opla e eade (In ini e 200 PRO; Tecan, Swi ze land) a 490 nm. 2.3. Animals. Male and emale Sp ague–Dawley a s (4-5 weeks o age, 120–180 g) om he b eeding colony a he An o agas a Uni e si y we e used o his s udy. All a s we e housed in a empe a u e-con olled, ligh -cycled (08:00– 20:00 hou s) oom wi h ad libi um access o d inking wa e and s anda d a chow (Champion, San iago). The assays we e conduc ed acco ding o he Guide o he Ca e and Use o Labo a o y Animals published by he U.S. Na ional Ins i u es o Heal h (NIH Publica ion e ised 2013), and he local animal esea ch commi ee app o ed he expe imen al p ocedu e used in he p esen s udy. 2.4. Isola ion o Ao ic Rings. Ra s we e sac i iced h ough ce ical disloca ion. The ho acic ao a was quickly excised andplacedincold(4 ∘C) physiological K ebs-Ringe bi- ca bona e bu e (KRB) con aining (×10−3 M) 4.2 KCl, 1.19 Oxida i e Medicine and Cellula Longe i y 3 KH2PO4,120NaCl,25Na 2HCO3, 1.2 MgSO4,1.3CaCl 2, and 5 D-glucose (pH 7.4). Rings (3–5 mm and 2–4 mg) we e p epa ed a e connec i e issue was cleaned ou om he ao a, aking special ca e o a oid endo helial damage. Ao ic ings we e equilib a ed o 40 min a 37∘Cbycons an bubbling wi h 95% O2and 5% CO2. 2.5. Vascula Reac i i y Expe imen s. Ao ic ings o na i e animals we e incuba ed (30 min) and pe used acu ely wi h Q7 (10−6 Mand10 −5 M) in he o gan ba h. The ao ic ings om he same animal we e s udied in duplica e, using di e - en asoac i e subs ances (phenyleph ine [PE], ace ylcholine [ACh], sodium ni op usside [SNP], KCl). The ings we e moun ed on wo 25-gauge s ainless s eel wi es; he lowe one wasa ached oas a iona yglass odand heuppe one was a ached o an isome ic ansduce (Radno i, Mon o ia, Cali o nia). The ansduce was connec ed o a Powe Lab 8/35 (Colo ado Sp ings CO) o con inuous eco ding o ascula ension using he LabCha 8 compu e p og am (ADS Ins umen s). A e he equilib a ion pe iod, he ao ic ings we e s abilized by 2 successi e nea -maximum con ac ions wi h KCl (6 ×10−2 M) o 10min.Thepassi e ensiononao a was 1.0 g, which was de e mined o be he es ing ension o ob aining maximum ac i e ension induced by 6 ×10−2 M KCl. Ten min a e con ac ion wi h phenyleph ine (PE; 10−6 M), cumula i e concen a ions o ace ylcholine (ACh) we e added o he medium (10−8 o 10−5 M). Simila p o ocols we e epea ed wi h SNP (10−8 o 10−6 M). To s udy he ole o ex acellula calcium, expe imen s we e pe o med wi h a calcium- ee KRB con aining (×10−3 M) 1.0 EGTA, 4.2 KCl, 1.19 KH2PO4,125NaCl,25Na 2HCO3, 1.2 MgSO4,and 5 D-glucose (pH 7.4). The ao ic ings we e p eincuba ed in a KRB wi h calcium o 30 min; hen he KRB was changed wi h KRB wi hou calcium o 5 min be o e PE (10−6 M) was added. Fi e min a e con ac ion wi h PE (10−6 M), cumula i e concen a ions o CaCl2(0.1 o 1.0 × 10−3 M) we e added o he medium. In o he expe imen s he con ac ion was induced by 10−3 M BaCl2 o 10 min and hen elaxed wi h 10−2 M KCl. BaCl2is used because i inc eases asocons ic ion by blocking o inwa d ec i ying K+channels [29–31], hus depola izing he plasma mem- b ane. 2.6. Lipid Pe oxida ion. Thioba bi u ic acid eac i e sub- s ances (TBARS) we e measu ed in a ao a homogena es. Samples o homogena es (500 𝜇L; 10 ±1.4 mg p o ein/mL) we e incuba ed wi h ehicle and Q7 (10−6–10−4 M) o 30 min and hen cen i uged a 3000 ×g o 20mina 4 ∘C. A 100 𝜇L aliquo o hesupe na an wasmixedwi h200𝜇Lo 10% ichlo oace ic acid (TCA) and 4% bu yla ed hyd oxy oluene. The mix u e was hen cen i uged and 140 𝜇Lo supe na an (in duplica e) was mixed wi h hioba bi u ic acid (0.67%) and hea ed o 1 h a 95∘C. A e cooling o oom empe a u e, 280 𝜇L o bu anol-py idine (15 : 1) was added. A e cen i - uga ion (3000 ×g, 20 min) he abso bance was measu ed a 532 nm. 2.7. Ni i e/Ni a e Assay. The p oduc ion o NO by a seg- men o he a ao a (in na i e a s) was measu ed by ni i e accumula ion using he G iess eac ion me hod [32]. Ao ic ings we e incuba ed in KRB cons an ly bubbled wi h 95% O2and 5% CO2 o 40 min a 37∘C. KRB con aining (×10−3 M) 4.2 KCl, 1.19 KH2PO4,120NaCl,25Na 2HCO3, 1.2 MgSO4,1.3CaCl 2, and 5 D-glucose (pH 7.4). Ao ic ings we e incuba ed a 37∘C o 30minwi hsaline,ACh (10−5 M) o he combina ion o ACh (10−5 M) and Q7 (10−5 M) o ACh (10−5 M) plus Nw-ni o-L-a ginine me hyl es e (L- NAME; 10−4 M). A he end o incuba ion, samples we e collec ed and ni a e educ ion was ca ied ou wi h Zn dus o 30 min a oom empe a u e. To al ni i es in each sample we e de e mined by he addi ion o 1% sul anilamide, ollowed by 0.1% N-(1-naph hyl) e hylenediamine (NED) in 5% phospho ic acid. The abso bance was measu ed wi h a mic opla e eade (In ini e 200 PRO; Tecan, Swi ze land) a 550 nm. Ni i e concen a ion was exp essed as 𝜇M/mg issue and calcula ed om a s anda d cu e wi h sodium ni i e. 2.8. L-Ci ulline Assay. NOS ac i i y was de e mined by incuba ion o HUVECs wi h 10−4 M L-a ginine and 9 ×10−6 Ci/mL L-[3H]a ginine (30 minu es, 37∘C) in he absence o p esence o 10−4 M L-NAME (a NOS inhibi o ). In addi ion, 10−5 MQ7 was used. HUVECs we e incuba ed in HEPES bu e con aining (×10−3 M) 50 HEPES, 100 NaCl, 5 KCl, 2.5 CaCl2, and 1 MgCl2(pH7.4).The ac iono L-[ 3H]ci ulline o ma ion inhibi ed by L-NAME was conside ed NOS ac i - i y [33]. Diges ed cells (95% o mic acid) we e passed h ough an ion-exchange esin Dowex-50W 5 (50X8-200) and L- [3H]ci ulline was de e mined in H2O elua e as desc ibed [34]. NOS ac i i y was calcula ed by sub ac ing L-NAME- insensi i e L-ci ulline om he o al L-ci ulline. 2.9. De e mina ion o In acellula Calcium in Cul u e Cell. A7 5 cells we e cul u ed in 35 mm cul u e dish o con ocal mic oscopy (ibidi, Ge many). The cells we e washed wi h K ebs wi h calcium con aining (×10−3 M) 140 NaCl, 5 KCl, 1 CaCl2,1MgCl 2,10HEPES,and5.6glucose,pH7.4.Then, hey we e loaded wi h 10−5 MFluo-3-AM o 25mina 37 ∘C and hen we e again washed wi h K ebs wi hou calcium con aining (×10−3 M)145NaCl,5KCl,1MgCl 2, 10 HEPES- Na,and5.6glucose,pH7.4.InK ebs ee-calcium5×10−5 M BAPTA-AM was used. The cells we e p eincuba ed in K ebs wi hou calcium o 5minbe o ePE(10 −6 M) was added, and hen 10−3 M o CaCl2was added o he medium. The in ensi y o Ca2+ luo escence was measu ed wi h a lase scanning con ocal mic oscope (Leica TCS SP8, Conco d, ON, Canada) and luo escence was eco ded e e y 5 seconds. Analysis in ol ed de e mina ion o pixels assigned o each cell using Image J so wa e. The a e age pixel alue alloca ed o each cell was ob ained wi h exci a ion a 506 nm and co ec ed o backg ound. 2.10. Chemicals. The ollowing d ugs we e used in his s udy: L-phenyleph ine hyd ochlo ide (PE; Sigma-Ald ich, S . Louis, Mo), ace ylcholine chlo ide (ACh; Sigma-Ald ich, 4Oxida i e Medicine and Cellula Longe i y NH OH O O Figu e 1: Chemical s uc u e o 2-[(4-hyd oxyphenyl)amino]-1,4-naph hoquinone (Q7). ∗ 0 20 40 60 80 100 % cy o oxici y (MTS) Con ol 10 −6 M 10 −5 M 10 −7 M Epi u 10 −6 M Cyclo 10 −4 M Q7 (a) ∗∗ ∗∗∗ 10−7 M 10−6 M 10−5 M 10−4 M Con ol Q7 0 20 40 60 80 100 % cy o oxici y (MTS) (b) Figu e 2: Q7 does no induce cell dea h. The esul s show ha Q7 cy o oxici y was low o negligible on a ascula endo helial cell o ascula smoo h muscle cell line, espec i ely. Q7 cy oxici y was de e mined by MTS assay. Human umbilical ein endo helial cells (HUVEC) (a) and A7 5 ascula smoo h cell line (b) we e incuba ed in he absence o in he p esence o Q7 (10−7 o 10−4 M) o 48 h. Cyclophosphamide (Cyclo; 10−4 M)wasusedasnega i econ olandepi ubicin(Epi ub;10 −5 M) as posi i e con ol. The cells we e seeded a 50% densi y. Da a a e he a e age ±SEM o 4 independen expe imen s; ∗𝑝< 0.05;∗∗𝑝;∗∗∗𝑝< 0.001 e sus con ol. Munich, Ge many), sodium ni op usside (SNP; Me ck, Da ms ad , Ge many), ba ium chlo ide dihyd a e (BaCl2; Sigma-Ald ich, S . Louis, MO), sul anilamide (Sigma-Al- d ich, S . Louis, MO), N-(1-naph hyl)e hylenediamine(NED; Sigma-Ald ich, S . Louis, MO), e ame hoxyp opane (Sig- ma-Ald ich, S . Louis, Mo), hioba bi u ic acid (Me ck, Da ms ad , Ge many), bu yla ed hyd oxy oluene (Me ck, Da ms ad , Ge many), Nw-ni o-L-a ginine me hyl es e (L-NAME; Sigma-Ald ich, S . Louis, MO), and BAPTA- AM (In i ogen, USA). D ugs we e dissol ed in dis illed deionized wa e . The solu ions in K ebs-Ringe bica - bona e (KRB) we e eshly p epa ed be o e each expe imen . Figu e 1 shows chemical s uc u e o 2-[(4-hyd oxyphen- yl)amino]-1,4-naph hoquinone (Q7; 265.07 g/mol); i was syn hesized by amina ion o 1,4-naph hoquinone wi h 4-hyd oxyphenylamine, unde ae obic condi ions, using CeCl3⋅7H2Oas heLewisacidca alys asp e iously epo ed [35]. 2.11. S a is ical Analysis. Values a e exp essed as mean ± s anda d e o o he mean; 𝑛deno es he numbe o animals s udied. One- o wo-way analysis o a iance (ANOVA) was ca ied ou o de ec signi ican di e ences, ollowed by Bon e oni pos - es s o compa e all g oups. A 𝑝 alue o <0.05 was conside ed s a is ically signi ican . 3. Resul s 3.1. Cy o oxici y o Q7 on Vascula Endo helial Cell Line and Vascula Smoo h Muscle. Because oxida i e s ess is asso- cia ed wi h cell dea h, he e ec o Q7 on he iabili y o HUVECand he ascula smoo hmusclecelllineA7 5was assessed (Figu e 2). A e 48 h o incuba ion, he cy o oxici y in p esence o 10−5 MQ7 did no signi ican ly inc ease in endo helial cells (1.00 ±0.05% in con ol e sus 7.39 ± 6.14% wi h 10−5 MQ7) and inc eased signi ican ly in ascula smoo h muscle cells (0.97 ±0.03% in con ol e sus 32.20 ± 4.04% wi h 10−5 MQ7;𝑝 < 0.01). As expec ed, no signi ican cy o oxici y was obse ed wi h he chemo he apy cyclophos- phamide (10−4 M), which is ac i a ed only a e me abolism in heli e and husisinac i eincellcul u e. 3.2. Q7 Induces Oxida i e S ess in Ra Ao a and Dec eases Endo helial NO P oduc ion in Ra Ao a. We conduc ed expe imen s o e alua e oxida i e s ess e ealed by Q7- media ed lipid oxida ion in a ao a issue. TBARS assay Oxida i e Medicine and Cellula Longe i y 5 Con ol 10−6 10−5 [Q7], M 60 80 100 120 140 % TBARS ∗ (a) ∗∗∗ ### ### 0 2 4 6 8 10 Ni i e (𝜇M/mg issue) ACh Q7 +ACh L-NAME +ACh Con ol (b) ∗ L-[3H]Ci ulline inhibi ed Con ol Q7 (pmol/𝜇g p o ein/30min) 0 5 10 15 20 (c) Figu e 3: Q7 p oduces oxida ion o lipids and dec eases endo helial NO. Depic ing oxida i e s ess e ealed by Q7-media ed lipid oxida ion and a change o endo helial NO elease in a ao a issue. The a ao a homogena e de e mina ion o TBARS in p esence o Q7 (10−6 and 10−5 M) was measu ed as % wi h espec o con ol (a). Signi ican di e ences we e ound be ween 10−5 MQ7 e sus con ol o 10−6 Mo Q7 (∗𝑝< 0.05). The p oduc ion o NO by a segmen o he a ao a was measu ed by he accumula ion o ni i e using G iess eac ion me hod (b): he ao ic ings we e incuba ed wi h ehicle (con ol), 10−5 MACh,10 −5 MQ7 plus 10−5 MACh,and10 −5 MAChplus10 −4 M L-NAME. Da a a e he a e age ±SEM o 5 independen expe imen s. ∗∗∗𝑝< 0.001 e sus con ol; ###𝑝< 0.001 e sus ACh; e ec o Q7 on eNOS ac i i y and p oduc ion o endo helial NO (c). The p oduc ion o NO was de e mined indi ec ly by he o ma ion o L-ci ulline in he eac ion ca alyzed by eNOS in HUVEC. The esul s a e mean s anda d e o o he mean. As e isk indica es s a is ically signi ican di e ences compa ed o he con ol (∗𝑝< 0.05;𝑛=3). was used as an index o lipid pe oxida ion. As shown in Figu e 3(a), Q7 (10−5 M) inc eased he o ma ion o TBARS by 114 ±5% as compa ed o con ol ( ehicle). To un a el he modula o y e ec s on mechanisms in ol ed in asodila ion, NO o ma ion in a ao a was assessed by measu ing he p oduc ion o ni i es using he G iess eac ion. The ao ic ings we e incuba ed wi h ehicle, ACh (10−5 M), and Q7 (10−5 M) + ACh (10−5 M) o 30 min. Figu e 3(b) shows ha he p eincuba ion wi h Q7 signi i- can ly dec eased he o ma ion o endo helial NO (8.1 ±1.2 ni i e 𝜇M/mg issue wi h ACh e sus 3.7 ±0.5 ni i e 𝜇M/mg issue wi h Q7 +ACh,𝑝 < 0.01). The p eincuba ion wi h L- NAME also dec eased signi ican ly he p oduc ion o ni i es (2.9 ±0.4 ni i e 𝜇M/mg issue wi h L-NAME + ACh, 𝑝< 0.01). We measu ed eNOS ac i i y in p esence o Q7 by he o ma ion o L-ci ulline in he eac ion ca alyzed by eNOS in HUVEC (Figu e 3(c)). The concen a ion o L-ci ulline o med is di ec ly p opo ional o he NO concen a ion o med and eNOS ac i i y. We showed ha Q7 (10−5 M) inc eased he concen a ion o L-ci ulline in HUVEC (4.29 ±2.46 pmol/𝜇g p o ein con ol, 12.99 ±2.22 pmol/𝜇gp o ein wi h 10−5 MQ7;𝑝 < 0.05). To es whe he dec eased o inc eased NO migh ha e an e ec on asodila ion, we es ed he modula o y e ec o Q7 on ACh-induced asodila ion and PE-induced asocons ic- ion (Figu es 4 and 5). 3.3. Endo helial Vasodila ion o Ra Ao ic Rings Induced by ACh Is Impai ed by Q7.To con i m ha Q7 impai s endo he- lial dependen asodila ion ia a NO pa hway, u he expe - imen s we e conduc ed in PE (10−6 M) p econ ac ed ao as and doses o ei he ACh (10−8–10−5 M; Figu es 5(a)–5(c)) o 6Oxida i e Medicine and Cellula Longe i y O gan ba h: (i) Bu e : pH 7.4 PE T ansduc o PE Ao ic ing Vasocons ic ion o PE Vasocons ic ion Compu e S eel wi e Con ol 98 7 6 (ii) 95%O 2and 5%CO 2 (iii) 37∘C 200 mg 200 mg 5min Figu e 4: Vascula eac i i y expe imen al se up. Ao ic ing was moun ed on wo s eel wi es; he uppe one was a ached o an isome ic ansduce . An equilib a ion pe iod and s abiliza ion wi h KCl we e allowed, and he issue was washed wo imes wi h esh KRB. Then, we pe o med he p o ocol o he expe imen ; we added di e en concen a ions o PE, which caused asocons ic ion o ao ic ing. To inish, da a we e egis e ed in he compu e . The inc ease o he ace o he sc een indica es asocons ic ion o PE, while he dec ease o he ace indica es asodila a ion o he ao ic ing. SNP (10−10–10−6 M; Figu e 5(d)) we e inc eased. As shown in Figu es 5(b) and 5(c), p eincuba ion o 30 min wi h Q7 (10−5 M) esul s in a signi ican dec ease o ACh-media ed asodila ion o in ac ao ic ings: 115 ±2% con ol e sus 73 ±3% wi h 10−5 MQ7 (10−5 MACh;𝑝 < 0.05). This inding sugges s ha Q7 dec eases he endo helial ni ic oxide, ACh-induced, which ag ees wi h dec eased p oduc ion o ni i es obse ed in p e ious expe imen s. Ne e heless, by applying he same expe imen al p o ocol and using a NO- dono compound, namely, SNP, a asodila ion o 100% was obse ed e en in he p esence o 10−5 MQ7 (Figu e 5(d)). This indica es ha he educ ion o asodila ion can be o e come by NO; he e o e, Q7 only impai s he endo helial esponse and he e was no esponse o ascula smoo h muscle. In he nex expe imen s, we s udied i he dec eased asodila ion should be accompanied by educed in acellula calcium in p econ ac ed ao a wi h PE. 3.4. E ec o Q7 on he Calcium Homeos asis in A7 5 Cells. To in es iga e i he con ac ile esponse o PE in p esence o Q7 could be modula ed by calcium eleased om in acellula s o es, u he expe imen s we e conduc ed wi h PE in A7 5 cells in a calcium- ee medium. Figu e 6 shows ha his si ua ion, 10−5 MQ7, d as ically blun ed he elease o calcium omin acellula s o esin esponse o10 −6 MPE,bu i howe e , did no blun he inc ease o in acellula calcium when 1 mM CaCl2was added o ex acellula space. In o de o gain insigh in o po en ial ole o Q7 on ascula eac i i y, we epea a simila p o ocol in esponse o PE in ao ic ings. 3.5. E ec o Q7 on Ao ic Rings Vasocons ic ion Induced by PE. The e ec o Q7 p eincuba ion on he con ac ili y esponse o PE was explo ed. As shown in Figu e 7(a), Q7 a 10−5 M did no po en ia e he con ac ile e ec induced by PE when es ed in he p esence o no mal KRB (1.3 ×10−3 M ex acellula calcium). Howe e , Q7 enhanced he con ac ile esponse o PE when calcium was added o ex acellula space in a calcium- ee medium. No change in he basal ension o ao ic ings was obse ed by Q7 pe se.Wein es iga edi hecon ac ioninducedbyPEinp esenceo Q7 could be modula ed by calcium in low om he ex acellula space. Thus, we added inc easing concen a ions o CaCl2 o a calcium- ee medium. Figu e 7(b) shows ha in he p esence o 10−3 M CaCl2,Q7 (10−5 M) signi ican ly inc eased he con ac ile esponse o PE (10−6 M) om 128 ±1% con ol o 158 ±9% (𝑝 < 0.05). Pe cen ages we e de e mined wi h espec o submaximal con ac ion wi h 6 ×10−2 MKCl. This da a sugges s ha Q7 inc eased calcium in lux hus enhancing PE-induced asocons ic ion. 3.6. Role o Po assium Channels in he Vascula Response o Q7 in Ra Ao ic Rings. The pu a i e ole o po assium channels on ascula con ac ile esponse was u he s udied. Fo his Oxida i e Medicine and Cellula Longe i y 7 98 7 687 6 5 Con ol 5min 200 mg 9 8 7 5 C on o l (a) 98 7 687 65 Q7 PE (−log M)ACh (−log M) 9 8 7 7 5min 200 mg (b) 140 120 100 80 60 40 20 0 % ACh-induced elaxa ion 876594 −log [ACh], M ∗ 10−5 MQ7 10−6 MQ7 ∗ Con ol (c) 140 120 100 80 60 40 20 0 % SNP-induced elaxa ion 8 910 76511 −log [SNP], M 10−5 MQ7 Con ol (d) Figu e 5: Q7 impai s ACh-induced asodila ion in an NO-dependen mechanism. O iginal ace showing he ime cou se o he concen a ion– esponse cu es o PE (10−9–10−6 M) and ACh (10−8–10−5 M) in in ac ao ic ings o a s: con ol (a) and in he p esence o 10−5 MQ7 o 30 min (b). A e an equilib a ion pe iod and be o e PE, he ao ic ings we e s abilized by wo successi e nea -maximal con ac ions wi h 6 ×10−2 M KCl. ACh- esponse cu es in endo helium-in ac a ao a in he p esence o absence (con ol) o Q7 (10−6 M, 10−5 M) (c). A e ies we e p econs ic ed wi h 10−6 M PE. SNP- esponse cu es in a ao a in he p esence o absence (con ol) o 10−5 MQ7 (d). Da a a e he a e age ±SEM o 4-5 independen expe imen s. ∗𝑝< 0.05 e sus con ol. pu pose, BaCl2was used, as i inc eases asocons ic ion by blocking ol age-dependen Ca2+-sensi i e K+channels. Figu e 8(a) shows ha p eincuba ion wi h Q7 signi ican ly enhanced he con ac ile esponse o BaCl2(10−3 M): 125 ± 3% con ol e sus 161 ±15% wi h 10−5 MQ7 (𝑝 < 0.05). In e es ingly, low concen a ions o KCl caused epola iza- ion and dec eased he asocons ic ion, bu , in he p esence o Q7,10 −2 M KCl did no dec ease he asocons ic ion p o oked by BaCl2.OnlywhenaNOdono (10 −8 MSNP) was added o he p epa a ion, he ao ic ings we e able o eco e 100% o i s asodila ion, eaching alues simila o con ol condi ions (Figu e 8(b)). These da a sugges ha po assium channels a e equi ed o he modula o y e ec o Q7 on ascula con ac ile esponse and in consequence, he compound migh a ec he memb ane po en ial. 4. Discussion The modula ion o blood low h ough he ascula endo- helium in luences he p og ession o se e al pa hologies 8Oxida i e Medicine and Cellula Longe i y CaCl 2 050 150 200 250 300100 Time (s) 0.0 0.5 1.0 1.5 2.0 Ca2+signal ( luo-3 luo escence) PE (a) CaCl 2 PE 500 150 200 250 300100 Time (s) 0.0 0.5 1.0 1.5 2.0 Ca2+signal (Fluo-3 luo escence) (b) Figu e 6: Quinone e ec on in acellula Ca2+ le els in A7 5 cells. Q7 blun ed he elease o calcium om in acellula s o es, bu no he in lux o calcium om ex acellula space. Rep esen a i e plo s o ela i e changes in Ca2+ signal ( luo-3 luo escence) o e ime on A7 5 cells in calcium- ee medium wi h 10−6 M phenyleph ine (PE) in absence o Q7 (con ol) (a) o p esence o 10−5 MQ7 (b). Da a a e he a e age ± SEM (𝑛=3). 10−5 MQ7 Con ol 9810 7 6 −log [PE], M 0 50 100 150 200 % con ac ion (a) 10−5 MQ7 Con ol ∗ ∗ ∗∗ 0 50 100 150 200 % con ac ion 0.2 0.4 0.6 0.8 1.00.0 [CaCl2], ×10−3 M (b) Figu e 7: Q7 inc eases PE-dependen asocons ic ion in a Ca2+-dependen manne . PE- esponse cu es in endo helium-in ac a ao a in he p esence o absence (con ol) o 10−5 MQ7 (a). The ascula issue was p eincuba ed in a KRB bu e wi hou calcium o 10 min be o e 10−6 M PE was added; and hen, he CaCl2(0.1, 0.3, 0.6, and 1.0 ×10−3 M) was added o he ba h (b). Da a a e he a e age ±SEM o 4-5 independen expe imen s. ∗𝑝< 0.05 and ∗∗𝑝< 0.01 e sus con ol. [36, 37]. Al hough s udies ha e shown ha quinone ela ed compounds can impai asodila ion by endo helial dys unc- ion [3, 4, 38], he e ec o quinones on endo helial unc ion a low concen a ions is no well cha ac e ized. We ound ha noncy o oxic doses o Q7 induces oxida- i e s ess and educes he o ma ion o endo helial NO in a ao a, hus dec easing endo helial NO-dependen asodila ion. Ou indings a e consis en wi h a p obable mechanism in which oxida i e s ess and dec ease o NO may pa ially block po assium channels, hus depola izing he cell memb ane leading in consequence o he opening o calcium channels, inc eased calcium in lux, and hus inc easing calcium-dependen asocons ic ion. He ein, we show ha Q7 cy o oxici y a e 48 h on as- cula endo helial cell was negligible and on ascula smoo h muscle cell was low. Conside ing ha he incuba ion ime in expe imen s conduc ed o de e mine ascula eac i i y and oxida i e s ess in a ao ic ings las ed 30 min, i appea s unlikely ha such e ec s a e he consequence o Q7 cy o oxici y. Oxida i e Medicine and Cellula Longe i y 9 ∗ 0 50 100 150 200 % con ac ion Q7 Con ol BaCl2 (a) 0 50 100 150 200 % con ac ion ∗∗∗ Q7Con ol Q7 +SNP BaCl2+KCl (b) Figu e 8: Acu e e ec o Q7 onpo assiumchannelsin a ao a.Q7 signi ican ly dec eases he ascula con ac ile esponse in ol ed po assium channels. The con ac ion wi h 10−3 MBaCl 2(a) and e ec o 10−2 MKCl(b)in a ao ap econ ac edwi h10 −3 MBaCl 2a e shown. Open ba (con ol, ehicle), black ba (10−5 MQ7), and s iped ba (10−5 MQ7 +10 −8 M SNP). Pe cen ages o con ac ion wi h BaCl2 we e de e mined wi h espec o submaximal con ac ion wi h 6 ×10−2 M KCl (a), and he pe cen ages o con ac ion in p esence o 10−2 M KCl we e de e mined wi h espec o maximal con ac ion wi h 10−3 MBaCl 2(b). Da a a e he a e age ±SEM o 4-5 independen expe imen s. ∗𝑝< 0.05 and ∗∗∗𝑝< 0.001 e sus con ol. In he p esen s udy, we obse ed ha Q7 signi ican ly inc eased lipid pe oxida ion in a ao a homogena es, as shown by he enhanced TBARS o ma ion. This inding was a ibu ed o he capaci y o Q7 o p oduce oxida i e s ess by inc easing o ROS. In p e ious s udies, we showed ha TBARS inc eased in cal - hymus DNA ea ed wi h Q7 o juglone [39]. F ee adicals can a ack DNA a C4 o deoxy ibose gene a ing p oduc s as p openal, which eac wi h 2- hioba bi u ic acid and p oduce he TBARS o ma ion [40]. In bo h T24 and MCF-7 cells, we ound ha Q7 p o oked ele a ed le els o in acellula ROS [23, 25, 35, 39]. Oxida i e s ess p oduced by Q7 is mainly due o ROS gene a ion h ough a edox-cycling mechanism as desc ibed o o he quinones [12]. This migh a ec c i ically NO le els in he a ao a. Q7 inc eased he o ma ion o L-ci ulline in HUVEC, compa ible wi h inc eased NO gene a ion and eNOS ac i i y [41]. This pa adoxical esul can be explained in pa because he ROS p oduced by edox-cycling o Q7 eac apidly wi h gene a ed NO, leading o a educ ion o i s le el [38]. Quinones (i.e., doxo ubicin, menadione) inc ease he gene a ion o ROS (anion supe oxide) leading o sca enging o NO, in ag eemen wi h p e iously desc ibed esul s [42– 44]. In ac , we now ound ha Q7 signi ican ly educed he ACh-media ed NO o ma ion. Acco dingly, ACh-media ed endo helial asodila ion was signi ican ly educed by Q7, bu i was eco e ed when a NO dono (SNP) was added in o in ac a ao a p epa a ions. The e o e, endo helial dys unc ion caused by Q7 may be explained by a dec ease in he bioa ailabili y o endo helial NO. Oxida i e s ess is associa ed wi h dis up ion o in a- cellula calcium homoeos asis [45]. In ac , Q7 blun ed he eleaseo calcium omin acellula s o esin esponse oPE on ascula smoo h muscle cell (A7 5 cells) in a ee-calcium medium. Simila esul s we obse ed in ca diac ib oblas s p eincuba ed wi h Q7 in a ee-calcium medium in p esence o angio ensin II (da a no shown). These indings sugges ha oxida i e s ess induced by Q7 blocked he elease o in acellula calcium o caused calcium leakage om in acellula s o es oex acellula space.Thisisinag eemen wi h he inhibi o y e ec o quinone- ela ed compounds (menadione) on he elease o calcium om in acellula s o es [46], by inhibi ion o sa coplasmic calcium ATPase [47]. I is possible ha he endoplasmic e iculum emp ying o calcium by Q7 enhanced he con ac ile esponse o PE o ao ic ings when calcium was p o ided ex acellula ly. We ound ha Q7 did no dec ease he in lux o calcium om ex acellula space. We ha e ob ained p elimina y da a sugges ing ha Q7 may also inhibi po assium channels in neu ons. Ou esul s show an inc ease in ac ion po en ial i ing ha is accompa- nied by inc eased inpu esis ance (da a no shown), an e ec ha could be media ed by blockade o hype pola iza ion- ac i a ed cyclic nucleo ide ga ed (HCN) channels, ha ca y he Ih cu en [48]. Howe e , mo e expe imen s a e needed o con i m he pa icipa ion o di e en ion channels in his. To s udy a pu a i e ole o po assium channels in NO-dependen asodila ion, we use BaCl2. Ba ium blocks inwa d ec i ying po assium channels such as ATP-sensi i e K+channels a submillimola concen a ions [49, 50] and ol age-dependen Ca2+-sensi i e K+channels a millimola concen a ions [29, 30]. The esul o he blockage o po as- sium channels by ba ium induced con ac ion in ao ic ings. In he p esence o Q7, he addi ion o KCl (10−2 M) was unable o p o oke a o al asodila ion in a ao a p econ- s ic ed wi h BaCl2(10−3 M), an e ec which was howe e ob ained a 100% by adding SNP (10−8 M).Theinc easeo KCl o e 2 ×10−2 M caused epola iza ion and asodila ion,