浙江大学 文本 Activation of Mitochondrial ATP-Dependent Potassium Channels by Nitric Oxide_生理学

Activation of Mitochondrial ATP-Dependent Potassium Channels by Nitric Oxide Norihito Sasaki.MD.PhD:Toshiaki Sato,MD.PhD:Andreas Ohler.MD. Brian O'Rourke,PhD:Eduardo Marban.MD,PhD Bacrun-Nitric oxide (NO)hes been implicated as amdor of"scndwindischemic prexxceditioning and minochondrial ATR-depender K'(mitoKn)chanels are the likely effbces The lrks between N and michannels mre unknown Mether and Remkes-We me:red minochoedrial redox putertial an index uf miK channel opering in rabbit vemneular myocytes.The NO donor S-ritroso-V-ocetyl-DL-pencillamine (SNAP.0.I %o I mmol/L)oxidized the matochon drial matrix dooe-dependently without activating sarcnlemmal Kchannels SNAP-induced axidation wus blocked by the selective mitok channel blocker 5-hydroxydecamnole and by the NO scavenger 2-(4-carboxypheryl4.4.5.5'. tetramehnlimidszole-l-uxyl-3-cide.SNAP-induced mitochoodrial axidation was detecthle cher by phutumultiplier tube recordings of flawvoproein mucrescence or by confocal imaging SNAP also enhancod the oative elTects of dizoxide when both agents were applied together.Exposure to 1 mmnoL 8Br-cGMP failed to mimic the efects of SNAP. ConclusiomNO directly activaes mitoKchnnels and potentiaes the ability of dipoide to open these channels.Thes resits prode novel mechneste links between MXinduced crdoproectkom nnd mitok chsrncls (Cimwacun. 2000:101:439-44线) Key Words:ischemic preconditioningm nitric oxide m myccytes m milochondria N No Methods The investiratine cmfimx with the G地for fiv Care m时ws erated during the axidation of L arginine to citrulline.not Labnratory Amnealr puhliched hy the Natinral Iraditutes of lkalf only in the cytosol but also in mitochondria During NI日pie.emS231be1955 ischemia,the endogenous production of NO in the heart is Materials inereased3口is also common th写eul交Eeeo Cnlagzrome (pe ll)was puirchtood fom Warthingh DXazrcide administer exogenous nitrates in acute ischemic sy 2,4-diratrophenol [DPLSoum eyanide (CNI,SNAP.and 8-bcom dromes,further boosling the conceniration of NO.Al- GMP (BBr-GMP]were obuane Somn Siama Chenical Co.SHD. though NO has been reported to afford cardioprotection Ciroodil and 2-ld-carbooyphem11,4'5_s'-tetramethylinidaooole. L-axy 3-ode (carboxy-PTTOI were purchosed fhom Reseerch om reperfiusion ijury‘e role ofNO during里he carly Hiochemieal Intrrrottional [haoxide.SNAP.pinacidil and phase of ischemic preconditioning is comroversial. oboxy-PTIO weie disolved a DMSO befot:they wee added Conversely.several lines of evidence comincingly impli- experimental solutioes.The final coneentrution of DMSO eme NO as a mediator of the Inte phase (second window) 压<0.% of ischemic preconditioning against infarction and stu Cell Isolation and Measurement of Mitochondrial ning.The cardioprotective effects of NO have been Redox State explained by seweral factors.sch as microwascular e Rahbit ventncular rtyocytes were isolned enzsmorically from adilt feets,ineutrophil action induction of stress protein, raHhit hearts and pbced in peirary caltire as desrrbed pevuus- or modulation of cardisc excitability.'Recertly,Bernardo .s Expenmems were perfocred over the nest day.Mitok et a reported t delged iscmie prxcondaiuning is chuniel octivitywrs monitored aouim asively by measann na- gronen fluore32r落nInk支fm3h3ndnl3xki inhibited by 5-hydroxydecanoule (5HD)in the rabbit heart. Becmuse SHD is&selective blocker of mitochondrial n:eatrd)Celk were superfiprd with exerral solutimn conaen- AIP deperdent potassium (mitoK)channels in rabbit ag tin muoVL)NaCl 140.KCT 5.CoCl,1.Mgcl;1,and HEPES 10 ventricular cells,the ahility uf SHD to gbolish eor- (N1.j时db.4ah目对市m《2n门 Erdepmnous flvognoiin flugrescenog was cxocnd for 100 ms gvery winprocectice motivated us to look fo possible links 6ndb与1 senon ne lmp with中filercemed al4 between NO and msitoK channels nm Emitled flucresrrnce was reconded at 5M nn by a phaerrul Recrived July ?1989;accepted Nagint 4.1999. Font the lotitale of Moleealar Curdibiolegy.Jotro Hepkis Uriversiy.Batrore.M Cuie psdcie l Bluau Marliat MD,PiD,Dictlor,Itoalur ef Moloul Cudiubioley,844 Roo Bld The Julu Hopkit Uaiveraay Schodl of Mediene.Rsmrtore MI 2120%Imail martoneighreieda e209 Anencan Hor Associatior,Inc. Cireaimau女nail山对ttpc/wmw,rrl山isnabo呢 49

Activation of Mitochondrial ATP-Dependent Potassium Channels by Nitric Oxide Norihito Sasaki, MD, PhD; Toshiaki Sato, MD, PhD; Andreas Ohler, MD; Brian O’Rourke, PhD; Eduardo Marba´n, MD, PhD Background—Nitric oxide (NO) has been implicated as a mediator of “second-window” ischemic preconditioning, and mitochondrial ATP-dependent K1 (mitoKATP) channels are the likely effectors. The links between NO and mitoKATP channels are unknown. Methods and Results—We measured mitochondrial redox potential as an index of mitoKATP channel opening in rabbit ventricular myocytes. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP, 0.1 to 1 mmol/L) oxidized the mitochon￾drial matrix dose-dependently without activating sarcolemmal KATP channels. SNAP-induced oxidation was blocked by the selective mitoKATP channel blocker 5-hydroxydecanoate and by the NO scavenger 2-(4-carboxyphenyl)-4,49,5,59- tetramethylimidazole-1-oxyl-3-oxide. SNAP-induced mitochondrial oxidation was detectable either by photomultiplier tube recordings of flavoprotein fluorescence or by confocal imaging. SNAP also enhanced the oxidative effects of diazoxide when both agents were applied together. Exposure to 1 mmol/L 8Br-cGMP failed to mimic the effects of SNAP. Conclusions—NO directly activates mitoKATP channels and potentiates the ability of diazoxide to open these channels. These results provide novel mechanistic links between NO-induced cardioprotection and mitoKATP channels. (Circulation. 2000;101:439-445.) Key Words: ischemic preconditioning n nitric oxide n myocytes n mitochondria Nitric oxide is a key signaling molecule that figures prominently in ischemic preconditioning. NO is gen￾erated during the oxidation of L-arginine to citrulline, not only in the cytosol but also in mitochondria.1–3 During ischemia, the endogenous production of NO in the heart is increased.1 It is also common therapeutic practice to administer exogenous nitrates in acute ischemic syn￾dromes, further boosting the concentration of NO. Al￾though NO has been reported to afford cardioprotection from reperfusion injury,4–6 the role of NO during the early phase of ischemic preconditioning is controversial.7–10 Conversely, several lines of evidence convincingly impli￾cate NO as a mediator of the late phase (second window) of ischemic preconditioning against infarction and stun￾ning.11–13 The cardioprotective effects of NO have been explained by several factors, such as microvascular ef￾fects,4 antineutrophil action,4 induction of stress protein,14 or modulation of cardiac excitability.7 Recently, Bernardo et al15 reported that delayed ischemic preconditioning is inhibited by 5-hydroxydecanoate (5HD) in the rabbit heart. Because 5HD is a selective blocker of mitochondrial ATP-dependent potassium (mitoKATP) channels in rabbit ventricular cells,16 the ability of 5HD to abolish second￾window protection motivated us to look for possible links between NO and mitoKATP channels. Methods The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised 1985). Materials Collagenase (type II) was purchased from Worthington. Diazoxide, 2,4-dinitrophenol (DNP), sodium cyanide (CN), SNAP, and 8-bromo cGMP (8Br-cGMP) were obtained from Sigma Chemical Co. 5HD, pinacidil, and 2-(4-carboxyphenyl)-4,49,5,59-tetramethylimidazole- 1-oxyl 3-oxide (carboxy-PTIO) were purchased from Research Biochemical International. Diazoxide, SNAP, pinacidil, and carboxy-PTIO were dissolved in DMSO before they were added to experimental solutions. The final concentration of DMSO was ,0.1%. Cell Isolation and Measurement of Mitochondrial Redox State Rabbit ventricular myocytes were isolated enzymatically from adult rabbit hearts and placed in primary culture as described previous￾ly.16,17 Experiments were performed over the next day. MitoKATP channel activity was monitored noninvasively by measuring fla￾voprotein fluorescence as an index of mitochondrial redox state with or without simultaneous whole-cell membrane current recordings (as indicated).16–18 Cells were superfused with external solution contain￾ing (in mmol/L) NaCl 140, KCl 5, CaCl2 1, MgCl2 1, and HEPES 10 (pH adjusted to 7.4 with NaOH) at room temperature ('22°C). Endogenous flavoprotein fluorescence was excited for 100 ms every 6 seconds by a xenon arc lamp with a bandpass filter centered at 480 nm. Emitted fluorescence was recorded at 530 nm by a photomul￾Received July 7, 1999; accepted August 4, 1999. From the Institute of Molecular Cardiobiology, Johns Hopkins University, Baltimore, Md. Correspondence to Eduardo Marba´n, MD, PhD, Director, Institute of Molecular Cardiobiology, 844 Ross Bldg, The Johns Hopkins University School of Medicine, Baltimore MD 21205. E-mail marban@jhmi.edu © 2000 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org 439

440 Circulanion February 1,2000 A ONP tiplier tube and digtooed The redox signal was avcraped dunng the excitation window aed calibrated at the end of each expenment by 10 DIAZD SNAP 0620 esporsure lo DNP.which enemug:les respratin from ATP synthesis and induces maximal axidation Therefure,the values of frvopobein fluorescence are expressed as a peroentape of the DNP-induoed flu3re实cnee.Individul5 ocytes were obeerve时wha×40ob✉ tive lo monie Deorescence I cell al a lime. Confocal Imaging of Flavoprotein Fluorescence 20 Confocal images were obtrined with a Diaphot 300 imverled fluo escence mtiereocope with a PCM-2000 ensocal seanring altach- ter (Nicon He].I Flucrescence win eveited hy the 488-nm lite od an argon laser.and the cmission at 305 to 335 nm wrs recorded 50 60 belne.dacoide,SNAP,SNAP+SHD,CN.and DNP imges Time (minl were erhanced by averging of 7 sequential inages huving stable B mean Duorescence intensities dunng expoesure to coch agem.Imapes ere andyned oe。persorl compiter with the sollwire世gam 100 Sple3边(Compis,Ine 80 SNAP Data Analysis li3gccg,the skpe of relalive chan吧Emhe鱼orscerce dunng 40 dnug applicotion was calculaled by a least-squures method.The best-fit lne is indicated by a dotted line in Figure 1 (A.B,and CL 20 Fe3p队and Figire4《A ard B)Pooled :bit are presered eun士SM,nd the ru■cer of cells or experiments is shown as n Statistical companson was evulaaned by lwzy ANOVA.with a walue of Pc0.05 considerrd sigraficant. 20 1C0 Electrophysiological Recordings In some expenments (Fiaure 3)uhole-cell cumenes and tlavopeocein C DNP fluorescence were measured simultaneo.sly.The imemal ppette oliion cortaned (in mmoli.)polassiumn gutamate 120.KO 25. 0 8NAP MgCl:0.5,K-EGTA 10,HEPES 10,and MgATP I (pH ad usted to 80- 020 5HD 7.2 wih KOHI.Whole-cell cumenes were elicited every 6 seoonds fmm4hsli吧Neaf-0mVy工consecuive pto一40 由V(fe1De)el0mV《fe30s.nd Pnoprreein fluorescence wus excited d.ring the 100-ms step to -40 mV.To quantify feAT curer店al0■V4 cre measured200snoe Fulse Results Effects of SNAP on mitoKarr Channels Figure IA shows the time course of flavoprotcin fluoresoence 40 in a cell exposed first to diazoxide,then to SNAP,and finally E apain to disoxide.In the first application,diazoide revers- bly oxklizcd the llavoproleins.Subsoqucrt exposue to DIAZOC1 SNAP alne yradually increasod faoprolin uxidation with 39 a slope of 0.59%/min.Afler a 15-minute exposure to SNAP. DAZ002 mitochondrial oxidation persisted even minutes after wash- SNAP out.Although NO has been reported to inhibit respim- tion..152 the present changes are in the opposite diredtion to 02010012 Figun 1.Eleeta nf SNAP on fapretain cocidation.A Tima course of favaprotein fuorescence in a cell exposed twice to dazoxdde (DAZO,100 umoVL)with an intervening exposure to BNAP (100 mo].B nnd C show representatie daca indicat- ing efocta cf 5HD [1 mmolL)on SNAP-induced oxidation.Bars indicale perods whon cels were exposed 1o esch drug.Uotted and E Summartzod data for peroontage of diazoxide induced mcasured in frst [DVZO(1)]and second cxpcsure after SNAP SNAF1.'Pe0.06 vs DIAZO().F,SNAP-induoed fa vnpmh件exictalion的irad in ab9G满ninf-n4 righf]af 1 mmoM 5HD

tiplier tube and digitized. The redox signal was averaged during the excitation window and calibrated at the end of each experiment by exposure to DNP, which uncouples respiration from ATP synthesis and induces maximal oxidation. Therefore, the values of flavoprotein fluorescence are expressed as a percentage of the DNP-induced fluorescence. Individual myocytes were observed with a 340 objec￾tive to monitor fluorescence 1 cell at a time. Confocal Imaging of Flavoprotein Fluorescence Confocal images were obtained with a Diaphot 300 inverted fluo￾rescence microscope with a PCM-2000 confocal scanning attach￾ment (Nikon, Inc).17,18 Fluorescence was excited by the 488-nm line of an argon laser, and the emission at 505 to 535 nm was recorded. A time series of images was collected at intervals of 10 seconds, and baseline, diazoxide, SNAP, SNAP15HD, CN, and DNP images were enhanced by averaging of 7 sequential images having stable mean fluorescence intensities during exposure to each agent. Images were analyzed on a personal computer with the software program Simple32 (Compix, Inc). Data Analysis To evaluate the effects of pharmacological agents on flavoprotein fluorescence, the slope of relative change in the fluorescence during drug application was calculated by a least-squares method. The best-fit line is indicated by a dotted line in Figure 1 (A, B, and C), Figure 3 (top), and Figure 4 (A and B). Pooled data are presented as mean6SEM, and the number of cells or experiments is shown as n. Statistical comparison was evaluated by 1-way ANOVA, with a value of P,0.05 considered significant. Electrophysiological Recordings In some experiments (Figure 3), whole-cell currents and flavoprotein fluorescence were measured simultaneously. The internal pipette solution contained (in mmol/L) potassium glutamate 120, KCl 25, MgCl2 0.5, K-EGTA 10, HEPES 10, and MgATP 1 (pH adjusted to 7.2 with KOH). Whole-cell currents were elicited every 6 seconds from a holding potential of 280 mV by 2 consecutive steps to 240 mV (for 100 ms) and 0 mV (for 380 ms), and flavoprotein fluorescence was excited during the 100-ms step to 240 mV. To quantify IK,ATP, currents at 0 mV were measured 200 ms into the pulse. Results Effects of SNAP on mitoKATP Channels Figure 1A shows the time course of flavoprotein fluorescence in a cell exposed first to diazoxide, then to SNAP, and finally again to diazoxide. In the first application, diazoxide revers￾ibly oxidized the flavoproteins. Subsequent exposure to SNAP alone gradually increased flavoprotein oxidation with a slope of 0.59%/min. After a 15-minute exposure to SNAP, mitochondrial oxidation persisted even 5 minutes after wash￾out. Although NO has been reported to inhibit respira￾tion, 2,19,20 the present changes are in the opposite direction to Figure 1. Effects of SNAP on flavoprotein oxidation. A, Time course of flavoprotein fluorescence in a cell exposed twice to diazoxide (DIAZO, 100 mmol/L) with an intervening exposure to SNAP (100 mmol/L). B and C show representative data indicat￾ing effects of 5HD (1 mmol/L) on SNAP-induced oxidation. Bars indicate periods when cells were exposed to each drug. Dotted line indicates best fit for changes in flavoprotein fluorescence. D and E, Summarized data for percentage of diazoxide-induced flavoprotein oxidation and latency to mitoKATP channel activation measured in first [DIAZO(1)] and second exposure after SNAP [DIAZO(2) SNAP]. *P,0.05 vs DIAZO(1). F, SNAP-induced fla￾voprotein oxidation measured in absence (left) and in presence (right) of 1 mmol/L 5HD. 440 Circulation February 1, 2000

Sasaki et al Activation of mitoKre Channels hy NO 44 Control DIAZO Wash SNAP Figure 2 Corfoodl imaging contima mlochordra-cxrzing efect of SNAP A ate rdlatie Inorcase in mbochondral avoprenein,nyi州d im线ol cal8 hsodina [CamenL Al,Rer 7 minue exposure to dazoxdde (100 gmolL.Bl. 12 minuea'cpasure to SNAP SNAP+5HD 200 wmolL,Ul.and adcitional applca- tion of 1 mmoVL 5HD SNAP+5HU.El. Dynamio ronge of favoprotein fuores cenoe is indicaled by exposuras to cy #性rcdL,月nd0NP (100 molL,G)as minimum and max m.m,respectivey.H.transmtted light P州Gh物的#往 DNP Transmitted those that would be expected from such an eflect (in which ously shonn tht repeatod exposures to dacode akne do not case the mitochondrial matrix would have been reduced,as it produce polentiation7 Figure IE ummariees the lstercy t mi- is with CN-).Noce also that the second exposure to Kchannel activution,measured as the time required to increase diaznxide after SNAP increased flavoprotein oocidation above flavopootein fluocescence to 20%of its maximal value afer wiching the levels renched in the first application,with less of a lag in dicide.The lkncy was significantly sbbeviaed during the dring the second exposure (4 minutes,verss 8 to 9 minutes secund cxposure lo disoxide afier SNAP.Figure IF surmirizes dr雪g the first exposure以 the effects of 5HID on the SNAP-induced foresoence changes and To deemmine whether mitoKchornels are ivohed in SNAP. venfies that 5IID significamthy ard corsistenly inhbits SNAP. induced mitochondrial oxicrtion,we applied 5ID,a selective induced miochondrial codstion These resuts inccne thart SNAP. mitoK chanel blocker.Figre 1,Band C,shos thot I mmol induced miondril oxidstin is modisod by adtivatiun of SHD)reversed (H)or preverred [C)the SNAP-induced flvoprotein mnoKup chame点 oxidation Fipure 1D)sumeanzes the implitude of diaxoxide. rduod fvoprdten cxialkn durrg te [DIAZO(I刀 Efects of SNAP on Flavoprotein Fluurescence secand exposres to dirzoxide afer the appliction of SNAP Detected by Confocal Imaging DIAZOZ)SNAP]Pretreatmert with SNAP significantly en- To further confirm the NO-anduced activation of mitoK hced the effects nf dporide-induced oxiditon,we hove previ- channels,the effect of SNAP on flavoprotein fluorescence

those that would be expected from such an effect (in which case the mitochondrial matrix would have been reduced, as it is with CN2).18 Note also that the second exposure to diazoxide after SNAP increased flavoprotein oxidation above the levels reached in the first application, with less of a lag during the second exposure (4 minutes, versus 8 to 9 minutes during the first exposure). To determine whether mitoKATP channels are involved in SNAP￾induced mitochondrial oxidation, we applied 5HD, a selective mitoKATP channel blocker. Figure 1, B and C, shows that 1 mmol/L 5HD reversed (B) or prevented (C) the SNAP-induced flavoprotein oxidation. Figure 1D summarizes the amplitude of diazoxide￾induced flavoprotein oxidation during the first [DIAZO(1)] and second exposures to diazoxide after the application of SNAP [DIAZO(2) SNAP]. Pretreatment with SNAP significantly en￾hanced the effects of diazoxide-induced oxidation; we have previ￾ously shown that repeated exposures to diazoxide alone do not produce potentiation.17 Figure 1E summarizes the latency to mi￾toKATP channel activation, measured as the time required to increase flavoprotein fluorescence to 20% of its maximal value after washing in diazoxide. The latency was significantly abbreviated during the second exposure to diazoxide after SNAP. Figure 1F summarizes the effects of 5HD on the SNAP-induced fluorescence changes and verifies that 5HD significantly and consistently inhibits SNAP￾induced mitochondrial oxidation. These results indicate that SNAP￾induced mitochondrial oxidation is mediated by activation of mitoKATP channels. Effects of SNAP on Flavoprotein Fluorescence Detected by Confocal Imaging To further confirm the NO-induced activation of mitoKATP channels, the effect of SNAP on flavoprotein fluorescence Figure 2. Confocal imaging confirms mitochondria-oxidizing effect of SNAP. A pseudocolor palette was applied to visu￾alize relative increase in mitochondrial flavoprotein, to yield images of cells at baseline (Control, A), after 7 minutes’ exposure to diazoxide (100 mmol/L, B), washing out diazoxide (Wash, C), after 12 minutes’ exposure to SNAP (200 mmol/L, D), and additional applica￾tion of 1 mmol/L 5HD (SNAP15HD, E). Dynamic range of flavoprotein fluores￾cence is indicated by exposures to cya￾nide (4 mmol/L, F) and DNP (100 mmol/L, G) as minimum and maxi￾mum, respectively. H, transmitted light image of the same field. Sasaki et al Activation of mitoKATP Channels by NO 441

442 Circulanion Fchruary 1,2000 E0- mDKa伊 1 mM 5HD A 0.5 mM SNAP 40 phaddll 100 IXAZO SNAP+PTO D1420 0 20 0 20 surface Kare 108 400 0 B Time (min) 0- 100 10 2 30 50 DIAZO BBr-c3MP CIAZO Time (min) 80 Figure 3.Efects af SNP on flavoprotein fluoresoenoe and 0 and icm Tap,015 mmoll SNAP inducad favepralin nxidation 40 witheut preeapesure to dliaznoda.Rara indinane parinds whan ce ls were expooed to esch drug.Note that 1 mmoL HD innibined oxidative aet SNAP nd 100 pmolL pinaciciL 20 Bottem,Time course of whole-cell cument messured at 0 mv: pinscidl activated lt ten in presence of 5D. wus measured by confocal imaging.Fluorescence was low under contral condirions (Figure 2A).hut exposure to dia- zoxide reversibly ineretsal fuorescence (B.washo ine in C).Subexquenl cxposure to SNAP also increased flavopro- tein fluorescence (D),hut SNAP-induced oxidation was inhibited hy ndditional npplicarion of SHD (E)Images were calibrated at the end of the experiment by exposure to cyanade (F)and DNP (G).The patchy distributian of fluorescence in the conficul imges is lypical of mtochondria,confim- ing tha NO oxidizesh mitochondrial malrix bry activation of mitok channels. E Effects of SNAP on mitoKATr and Sarcolemmal Kur Channels To test the scleetivity of'NO on mitoK versus sarcolemmal KAm csnnels,we examinod the elfocts of SNAP on flavopto- 5 tein fluorescence and whole-cell currents simultaneoushy.In M Figure 3,application of 0 5 mmol1.SNAP without preexpo- sure to diazoxide gradually oxidized the miochondrial matrix Figure 4.Changes in faoprotoin axdation induced by coopplioa ton of csrbcoy-PTlC [100 umo)with SNAP (10D pmol Al and with a slope of 0.78%/min.SNAP.induced oxdation was 1mmaL r cGMP (B)in a ocl twice cxpoccd to dodee.C inhibited by coapplication of I mmolL 5IID.In the contin- and D summarlze data for porocntage of damoodde inducod fa ud prescnce of SHD.subsogucr cxposure to 100 umolL mesoured in first [DZO(1 and second expooure afer ooappica- pinacidd (a mixed mioKsurface Ky aggonist1 failed to ton of carboxy-PTO wth SNAP [DAZO SN/P+PTICI.E and F induce mitochondrial oxidarion.In contrast,Figure 3 (bot- aurmerizA AnNogoua dota for BBr-CGMP. tom)shows that SNAP had no effect on sareolemmal KAm channels,becausse pincidil activated sarcolemmal K chan colemmal Km current (5549t829 pA at 0 mV.n=4. nels despite the presence of 511D.These results are represen- P<00 verss before)These results indicate that SNAP tative and reproducible.A 20-minute exposure to SNAP selectively activntes mitoK channels.Furthemmore,Figure (0.5 mmol/L)had no significar effict on whole-cell curremt 3 democstrates that SNAP-induced activation of mioK (before,5.66.I pA versus afier.12.94.8 pA at 0 mV,n-4. channcls does not requre preexpoure o diacoxide.Firally. P=NS).Nevertheless,in the presence of I mmol/SHD,a the finding that SHD suppresses the mitochondnal oxidaton 10-minute expooure to 100 pmoVl prscidil increased sar- indueed by pinaeidil,but not the agonist cffect on

was measured by confocal imaging. Fluorescence was low under control conditions (Figure 2A), but exposure to dia￾zoxide reversibly increased fluorescence (B; washout image in C). Subsequent exposure to SNAP also increased flavopro￾tein fluorescence (D), but SNAP-induced oxidation was inhibited by additional application of 5HD (E). Images were calibrated at the end of the experiment by exposure to cyanide (F) and DNP (G). The patchy distribution of fluorescence in the confocal images is typical of mitochondria,17,18 confirm￾ing that NO oxidizes the mitochondrial matrix by activation of mitoKATP channels. Effects of SNAP on mitoKATP and Sarcolemmal KATP Channels To test the selectivity of NO on mitoKATP versus sarcolemmal KATP channels, we examined the effects of SNAP on flavopro￾tein fluorescence and whole-cell currents simultaneously. In Figure 3, application of 0.5 mmol/L SNAP without preexpo￾sure to diazoxide gradually oxidized the mitochondrial matrix with a slope of 0.78%/min. SNAP-induced oxidation was inhibited by coapplication of 1 mmol/L 5HD. In the contin￾ued presence of 5HD, subsequent exposure to 100 mmol/L pinacidil (a mixed mitoKATP/surface KATP agonist)16 failed to induce mitochondrial oxidation. In contrast, Figure 3 (bot￾tom) shows that SNAP had no effect on sarcolemmal KATP channels, because pinacidil activated sarcolemmal KATP chan￾nels despite the presence of 5HD. These results are represen￾tative and reproducible. A 20-minute exposure to SNAP (0.5 mmol/L) had no significant effect on whole-cell current (before, 5.666.1 pA versus after, 12.964.8 pA at 0 mV, n54, P5NS). Nevertheless, in the presence of 1 mmol/L 5HD, a 10-minute exposure to 100 mmol/L pinacidil increased sar￾colemmal KATP current (554.9682.9 pA at 0 mV, n54, P,0.001 versus before). These results indicate that SNAP selectively activates mitoKATP channels. Furthermore, Figure 3 demonstrates that SNAP-induced activation of mitoKATP channels does not require preexposure to diazoxide. Finally, the finding that 5HD suppresses the mitochondrial oxidation induced by pinacidil, but not the agonist effect on IK,ATP, Figure 4. Changes in flavoprotein oxidation induced by coapplica￾tion of carboxy-PTIO (100 mmol/L) with SNAP (100 mmol/L) (A) and 1 mmol/L 8Br-cGMP (B) in a cell twice exposed to diazoxide. C and D summarize data for percentage of diazoxide-induced fla￾voprotein oxidation and latency of mitoKATP channel activation measured in first [DIAZO(1)] and second exposure after coapplica￾tion of carboxy-PTIO with SNAP [DIAZO(2) SNAP1PTIO]. E and F summarize analogous data for 8Br-cGMP. Figure 3. Effects of SNAP on flavoprotein fluorescence and IK,ATP. Simultaneous measurement of flavoprotein fluorescence and IK,ATP. Top, 0.5 mmol/L SNAP induced flavoprotein oxidation without preexposure to diazoxide. Bars indicate periods when cells were exposed to each drug. Note that 1 mmol/L 5HD inhibited oxidative effects of SNAP and 100 mmol/L pinacidil. Bottom, Time course of whole-cell current measured at 0 mV; pinacidil activated IK,ATP even in presence of 5HD. 442 Circulation February 1, 2000

Suxaki et al Activation of mitoKy Channels hy NO 4d3 B 04- 1 11 4n 4 CD. Cortrol P SMAP AFg-GW甲 2 0.1 mM GHD PTO Fge乐Summa屯e可dsta for slope of1 avoprobein oxids0n e to varo phama gical ageasnr catod.'P0.01 vs Control.Inset,Dose-rospore roationsnip for 04h”tT0 SNAP 10.1,0.2.0.5,and 1 mmol].aP-0.06,#P:0.01 vs 0.1 mmo SNAP. 100. demorestrales that I mmoVL 5HD is a sclective inhibitor of mito channels in mhhit ventricuar cells Mediation by NO Independent of cGMP To verify that the SNAP-induced changes are actually medi- ated by the release of NO.we tested the effects of carboory- PTIO.an NO savengper,on the SNAP-induced flavopeotein oidstion.Figure 4A shows that coapplication of carbooy- Figure 6.Coapplcation of diamoxide and SNAP.A,100 umolL PTIO wih SNAP prevemed the flavoprotein axidatica (slope da2D9d件Hdc8时内war例wn00对ation in addti3Ta平pc ton of 1 mmoVL SNAP.B gummenzes dsta for dazoode- <0min)Decause mamyy (but not all)of the effects of NO induced oxidation in absonce (ft]and presence (right of SNAP occur via a ciMP-dependent pathway,we tested whether (1mmolL).C and E show time course of dazade inducod NO-induced activation of mitoK is mimickod by 8Br- 中hwg#in车lication of SNAP1 mmol)wm carboxy-FTI 1100 umol)and BEr-cGMP (1 mmoVL respeo cGMP.Figure 4B shows that expos.re to this cell-permeable cGMP anslogue did not increase flavoproten oxidation,o daznxida with sh af savral pharaoolngieco agerta a indi- did pretrestment with 8Br cGMP enhance diazoxide induced eated.'P.co.n5 v8 DIAZD. oocidation.The effects of the NO scaverger and 8Br-cGMP were observed reproducibly.Figure 4.C and D.shows thal gous state-dependent changes in the case of'NO,we caboxy-PT1O abolished the enhancing effects of'SNAP on quntified t efficts ofSNAP onchamnels that bd already becn diazoxide-induced oxidaton,comfirming that the SNAP. opened by diazmide.Figure 6A shows that 1 mmol/.SNAP induced change is mediated by release of NO.Figure 4,E and mpiy enhanced dazoxide-induced oxidation when appliad F.suammanzes data for 8Br-cGMP.confirming thut it laik to ana the eflid of'daode hd readod sleady stme.Nole that mimc the elTocts of'SNAP in this cae,the cficts of SNAP were reversible.Figure 6C The pooled data in Figure 5 reveal that SNAP significantly shows that carboxy-PI abolished the enhincing effect of increases the slope of percent change in flavoprotein oxida- SNAP on diacocide-induced cocdation,and Figure hE demon- tion and that the SNAP-indoced effect is inhibied by 5HD snres that 8Rr-cMP Eailed to mimic the effects of SNAP cn and carbaxy-PTO.Te inset shows the dose-esponse rel mitoK in the presence of'dinzoxide.Figure 6.B.D.ind F tionship belwocn SNAP conccrralion and lavoprolein ox- summarizes data toc coodministration of diazoxide with SNAP. dation.Taken togcthct with the results in Figure 4,thes SNAP+carboxy-PTIO,ad 8Br-cGMP,respectively.These re- experimerts support the idea tha.SNAP activales mitoK sults indicnte that NO enhances mitoK channels preactivated channels dose-deperdenthy via a direct effect of NO,not by diode Channels thtare女oen平pear o be moe medated hy eGMP suxceptible to the potentiating actions of NO than channels that are in the cloced stane. Effeets of SNAP in the Presence of Diazoxide We previously reported that procein kinase C (PKC) Discussion activation enhances diazoxide-induced changes without Our data reveal that NO selectively activmes miok affecting hasal flavoprotein fluorescenee.This finding channels hut not sarcolemmal K ehannels.The modulation indicates that the modulation of mitoK by PKC may of mitoK channels by NO is manifested in 2 way's.One is depend on whether the channels are in the open ar closed the gradnl axidation induced by NO alone,and the other is state when the kinase becomes active.To test for analo- potentiation of mitoK channels preopered by diazoocide

demonstrates that 1 mmol/L 5HD is a selective inhibitor of mitoKATP channels in rabbit ventricular cells.16,17 Mediation by NO Independent of cGMP To verify that the SNAP-induced changes are actually medi￾ated by the release of NO, we tested the effects of carboxy￾PTIO, an NO scavenger,21 on the SNAP-induced flavoprotein oxidation. Figure 4A shows that coapplication of carboxy￾PTIO with SNAP prevented the flavoprotein oxidation (slope ,0%/min). Because many (but not all) of the effects of NO occur via a cGMP-dependent pathway,22,23 we tested whether NO-induced activation of mitoKATP is mimicked by 8Br￾cGMP. Figure 4B shows that exposure to this cell-permeable cGMP analogue did not increase flavoprotein oxidation, nor did pretreatment with 8Br-cGMP enhance diazoxide-induced oxidation. The effects of the NO scavenger and 8Br-cGMP were observed reproducibly. Figure 4, C and D, shows that carboxy-PTIO abolished the enhancing effects of SNAP on diazoxide-induced oxidation, confirming that the SNAP￾induced change is mediated by release of NO. Figure 4, E and F, summarizes data for 8Br-cGMP, confirming that it fails to mimic the effects of SNAP. The pooled data in Figure 5 reveal that SNAP significantly increases the slope of percent change in flavoprotein oxida￾tion and that the SNAP-induced effect is inhibited by 5HD and carboxy-PTIO. The inset shows the dose-response rela￾tionship between SNAP concentration and flavoprotein oxi￾dation. Taken together with the results in Figure 4, these experiments support the idea that SNAP activates mitoKATP channels dose-dependently via a direct effect of NO, not mediated by cGMP. Effects of SNAP in the Presence of Diazoxide We previously reported that protein kinase C (PKC) activation enhances diazoxide-induced changes without affecting basal flavoprotein fluorescence.16 This finding indicates that the modulation of mitoKATP by PKC may depend on whether the channels are in the open or closed state when the kinase becomes active. To test for analo￾gous state-dependent changes in the case of NO, we quantified the effects of SNAP on channels that had already been opened by diazoxide. Figure 6A shows that 1 mmol/L SNAP rapidly enhanced diazoxide-induced oxidation when applied after the effect of diazoxide had reached steady state. Note that in this case, the effects of SNAP were reversible. Figure 6C shows that carboxy-PTIO abolished the enhancing effect of SNAP on diazoxide-induced oxidation, and Figure 6E demon￾strates that 8Br-cGMP failed to mimic the effects of SNAP on mitoKATP in the presence of diazoxide. Figure 6, B, D, and F, summarizes data for coadministration of diazoxide with SNAP, SNAP1carboxy-PTIO, and 8Br-cGMP, respectively. These re￾sults indicate that NO enhances mitoKATP channels preactivated by diazoxide. Channels that are already open appear to be more susceptible to the potentiating actions of NO than channels that are in the closed state. Discussion Our data reveal that NO selectively activates mitoKATP channels but not sarcolemmal KATP channels. The modulation of mitoKATP channels by NO is manifested in 2 ways. One is the gradual oxidation induced by NO alone, and the other is potentiation of mitoKATP channels preopened by diazoxide. Figure 6. Coapplication of diazoxide and SNAP. A, 100 mmol/L diazoxide-induced flavoprotein oxidation in additional applica￾tion of 1 mmol/L SNAP. B summarizes data for diazoxide￾induced oxidation in absence (left) and presence (right) of SNAP (1 mmol/L). C and E show time course of diazoxide-induced change in additional application of SNAP (1 mmol/L) with carboxy-PTIO (100 mmol/L) and 8Br-cGMP (1 mmol/L), respec￾tively. D and F summarize data for effects of coapplication of diazoxide with each of several pharmacological agents as indi￾cated. *P,0.05 vs DIAZO. Figure 5. Summarized data for slope of flavoprotein oxidation during exposure to various pharmacological agents as indi￾cated. *P,0.01 vs Control. Inset, Dose-response relationship for SNAP (0.1, 0.2, 0.5, and 1 mmol/L). #P,0.05, ##P,0.01 vs 0.1 mmol/L SNAP. Sasaki et al Activation of mitoKATP Channels by NO 443

444 Circulanion Fehruary 1,2000 The later effect resembles that of phorbol esters that turn on Acknowledgments PKC and enhance diazoxide-induced oxidaton,but unlike This sy wis spport时y the NllI (grart R34,-7知D大tm NO.phorbol esters alone do not suffice to activate the ad R01-54598 to Dr CRoukel Jopn Heart Foudion and Baver channels.It is possible that the 2 ohservations share a Yolchn Reearch Cirt Abrood (to Dr Srduit a Bau Felovehp in common pathwuy.inasmuch a reactive axygen species (such Lpid Mtahuisn and Athenescleroe来Dr Satol an时the Dartshe To时chunpamins九i Dr Ck s止e NO product p民roxynitnte)Ieo%n to activate PKC.Shinbo and lijima reported that the application References of the NO donor NOR-3 increased the open prohabelity of L.Z起L Warg P,Kappuan野P.D6oac2 of ninc0k agonist-activated surface K.channels in carduc myocytes whereas NOR-3 had no effect n the abeence of charnel 0xu640ey.JBo Cher.199527304-301. agonists.We have found tha.PKC primes ventricular surface K chanmels to cpen in response to agonists ar to metabolic ha.JA思1居2：1I0sg-043 3.(urvi C.Funetusal implicatirers of nirie rocide produoed by milo- inhibition,but basal ctivity s unaffected.These findings again resemhle the raped enhancement of mitoKw by NO in 63-队. the presence of diacoxide but differ in the inability of the 4.nmiK,Vmm-Jea人Lefer D球.Zho乙，Fowler WC I, modulator to aler basal activity.Although the structural McGee S.Joheson WE Iracoosary Larginne during reperdasion on and relationship between surface and mitoK channels is un 19922行11列-1165限 aom#止e results sugges.t比ate2 channels may空arg 5. Wiliyns MW.Taf CS.S.7ho 7Q.Vien-Iohareer 1. regulatary pathways sensitive to NO,as is the case for Eax0nna1Ek转uccb年uro.bcheni-op0量n 起1Cnx.3079-. PKC.7 In parcrealic B cells.NO hs been argued to 6.HantmanX,ure GM H山waTG,Wall TM,swR线s中uk inhibit glycolysis,leading to a secoodary activation of surface R.Irhblion.ef mltic onide s家s色ocandial procton b与 KAu chamels However,SNAP did not increase whale-cell n平ad/%nncol Mrp Thv.142:I07l-l张 KAcurrent in the present study,consistent with the finding J.Vegh A,Sndkeres 1.Pret J.Preerndtiering n the igchaznic ryncar- daat NO akme did nt activule single KAm cfunnels in PAaneuow.192 17-2 cell-attached mode in guinca pig vertricuar myocytes.The s.Taa由A.Mulk N,上leGI,Hiaeg I Eagchren K3 1 12 mitoKar channel is网espy现nsitive to changes in Precondirionine ofsat Feant w th monophosphorl lipd A:a sele for nnrio 0we.J%wpTw.159%285:12T4-127丙 bulk [ATPL remnzining closed even at 0.5 mmolL [ATP] 9.Woslfson RG.Fxdl VC.Nenld Gll Yelloe DM Irhhitios of netng ocide Considering these observations,it seems unlikely that the 号Thss6dt5ngse的an3e3 sine dependert nechonisn efects of NO on mitoK channels teflect inhibition of Corwsns.1959I513-1551. lycolysi队 I0.ockoagh BO,BA到chP.Gouver C.lhbl山ono mlrie uxide hk3nkxn线13 hanic proousdlinitg in bdlal1cd国 Both NO and mitok channels have been implicated in 2上6.w/P0以.19952542H24从 the delayed phase of preconditioning known a the"second 11.Takin H.Tarts XL Qiu Y,Gao Y,French BA.Bolli R.Niuric coude window"of prokection MitoK channel opening is es iduce lal:prexonlra1gfo世H1utgd cadioprocelive during ischemia.whereas blockade of 年Ng1g3:7-4 miloK cannels abolishes both clssic and scond-window 12.Holli K,Marchkalpedi 5 Tang XL,Takano H,Qu Y.Cuo Y.Zhang protection.The present study establishes NO i an enduge- ladoon Ak.The prrectnve effact af le preoandtinag nous mitoKAu channel cpener that may be able to recruit mocastial starnine in conscious rabbnts is modiored by airo ooode eandoprocection in the second window.NO miry play a 号thase:evidence th第amio oskde形bh5ar经dsd nedioroe of the lne phase of ischemc precondionine.Cie fer.1997. particulurly prominent rle in the secand window ee of 91:309-107. chunges in gen expression noably the upregulaln of ntr 13.Bolli R.Dav B.Tang XT.Qis Y.Ping P.Xin XT.Jones WK.Takzno oxide synthase that oocurs within 24 bours of conditioning Bane fer Cando.1993325-323. ischem Although the relationships between mitok chan 14.Besjrnn L.NeMilin D Sueo (het shak)pruxira:ndlocaka nel aetivation and cardioprotection remain elusive.the open- hpeanes in c出u■bdu的d dicex.O元红.5883 ing of charrels in the inner memhrane may desipale the 117-132. mitochondril polemial established by the proon pump. I5ar向T.D'Angdlo M.ko等.mA.kkm中RC:Delyed ischertic peacardtionne is medated hy aperine ndf ATRaersive perhaps blunting the Ca overload tht would otherwis occur as a result of the large driving force for Caentry into 1i12214 mitochondrin during ischemia It was recenrly reported 16.Sats T.O'Rourke H.Mrhin E.of ritocherdal AT. that mitok channel openers release Ca'from Ca'-londed depesdert K'charnels by pronein kitase C.Cve Res.199883:110-114. 17.Luu Y.Sete T.O'Rourke B.Morbn E.Mnochoserial ATP-dependen mitoch电T刀c unopling by diacoxie3sox petssum charels novel efecors of cardopronection?Cieslaton. much geneler than that which can be induced hy agents soch 597263-65. as DNP;indeed,severe uncoupling should he harmful to 14.Rorashio DN.Marbin E.O'Route B.Sabcelufar nesbolo masents myocytes,heeause energy production ts critcally reduced 1底5161i-623. We speculme that NO.firctioning 8s an erdogenous mi- 19.Podkro J.Cuento MC,Ladeo C,Iticbo N,Schupfet F.Buvert A. toK chsreel upener.may titale the couplirg level of the mitochondria to an optimumn that blunts mitochondrial cal- dtion n r hent nimtacng an anal pomalee Ari cium overlond without significantly undermining ATP syn- 21twrW.ooper It.rley-llenr VM.Mon由发.hra thetic capocity

The latter effect resembles that of phorbol esters that turn on PKC and enhance diazoxide-induced oxidation,16 but unlike NO, phorbol esters alone do not suffice to activate the channels. It is possible that the 2 observations share a common pathway, inasmuch as reactive oxygen species (such as the NO product peroxynitrite) are known to activate PKC.13,24,25 Shinbo and Iijima26 reported that the application of the NO donor NOR-3 increased the open probability of agonist-activated surface KATP channels in cardiac myocytes, whereas NOR-3 had no effect in the absence of channel agonists. We have found that PKC primes ventricular surface KATP channels to open in response to agonists or to metabolic inhibition, but basal activity is unaffected.27,28 These findings again resemble the rapid enhancement of mitoKATP by NO in the presence of diazoxide but differ in the inability of the modulator to alter basal activity. Although the structural relationship between surface and mitoKATP channels is un￾known,29 the results suggest that the 2 channels may share regulatory pathways sensitive to NO, as is the case for PKC.16,27,29 In pancreatic b cells, NO has been argued to inhibit glycolysis, leading to a secondary activation of surface KATP channels.30 However, SNAP did not increase whole-cell KATP current in the present study, consistent with the finding that NO alone did not activate single KATP channels in cell-attached mode in guinea pig ventricular myocytes.26 The mitoKATP channel is not especially sensitive to changes in bulk [ATP], remaining closed even at 0.5 mmol/L [ATP].31 Considering these observations, it seems unlikely that the effects of NO on mitoKATP channels reflect inhibition of glycolysis. Both NO and mitoKATP channels have been implicated in the delayed phase of preconditioning known as the “second window” of protection.11–13 MitoKATP channel opening is cardioprotective during ischemia,17,32 whereas blockade of mitoKATP channels abolishes both classic and second-window protection. The present study establishes NO as an endoge￾nous mitoKATP channel opener that may be able to recruit cardioprotection in the second window. NO may play a particularly prominent role in the second window because of changes in gene expression, notably the upregulation of nitric oxide synthase that occurs within 24 hours of conditioning ischemia. Although the relationships between mitoKATP chan￾nel activation and cardioprotection remain elusive, the open￾ing of channels in the inner membrane may dissipate the mitochondrial potential established by the proton pump, perhaps blunting the Ca21 overload that would otherwise occur as a result of the large driving force for Ca21 entry into mitochondria during ischemia.16,17 It was recently reported that mitoKATP channel openers release Ca21 from Ca21-loaded mitochondria.33 The uncoupling by diazoxide appears to be much gentler than that which can be induced by agents such as DNP34; indeed, severe uncoupling should be harmful to myocytes, because energy production is critically reduced. We speculate that NO, functioning as an endogenous mi￾toKATP channel opener, may titrate the coupling level of the mitochondria to an optimum that blunts mitochondrial cal￾cium overload without significantly undermining ATP syn￾thetic capacity. Acknowledgments This study was supported by the NIH (grant R37-HL-36957 to Dr Marba´n and ROI-54598 to Dr O’Rourke), a Japan Heart Foundation and Bayer Yakuhin Research Grant Abroad (to Dr Sasaki), a Banyu Fellowship in Lipid Metabolism and Atherosclerosis (to Dr Sato), and the Deutsche Forschungsgemeinschaft (to Dr Ohler). References 1. Zweier JL, Wang P, Kuppusamy P. Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy. J Biol Chem. 1995;270:304–307. 2. Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mito￾chondria. J Biol Chem. 1998;273:11038–11043. 3. Giulivi C. Functional implications of nitric oxide produced by mito￾chondria in mitochondrial metabolism. Biochem J. 1998;332: 673–679. 4. Nakanishi K, Vinten-Johansen J, Lefer DJ, Zhao Z, Fowler WC III, McGee S, Johnston WE. Intracoronary L-arginine during reperfusion improves endothelial function and reduces infarct size. Am J Physiol. 1992;263:H1650–H1658. 5. Williams MW, Taft CS, Ramnauth S, Zhao ZQ, Vinten-Johansen J. Endogenous nitric oxide protects against ischemia-reperfusion injury in the rabbit. Cardiovasc Res. 1995;30:79–86. 6. Hartman JC, Kurc GM, Hullinger TG, Wall TM, Sheehy RM, Shebuski RJ. Inhibition of nitric oxide synthase prevents myocardial protection by ramiprilat. J Pharmacol Exp Ther. 1994;270:1071–1076. 7. Vegh A, Szekeres L, Parratt J. Preconditioning of the ischaemic myocar￾dium: involvement of the L-arginine nitric oxide pathway. Br J Pharmacol. 1992;107:648–652. 8. Tosaki A, Maulik N, Elliott GT, Blasig IE, Engelman RM, Das DK. Preconditioning of rat heart with monophosphoryl lipid A: a role for nitric oxide. J Pharmacol Exp Ther. 1998;285:1274–1279. 9. Woolfson RG, Patel VC, Neild GH, Yellon DM. Inhibition of nitric oxide synthesis reduces infarct size by an adenosine-dependent mechanism. Circulation. 1995;91:1545–1551. 10. Weselcouch EO, Baird AJ, Sleph P, Grover GJ. Inhibition of nitric oxide synthesis does not affect ischemic preconditioning in isolated perfused rat hearts. Am J Physiol. 1995;268:H242–H249. 11. Takano H, Tang XL, Qiu Y, Guo Y, French BA, Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res. 1998;83:73–84. 12. Bolli R, Manchikalapudi S, Tang XL, Takano H, Qiu Y, Guo Y, Zhang Q, Jadoon AK. The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase: evidence that nitric oxide acts both as a trigger and as a mediator of the late phase of ischemic preconditioning. Circ Res. 1997; 81:1094–1107. 13. Bolli R, Dawn B, Tang XL, Qiu Y, Ping P, Xuan XT, Jones WK, Takano H, Guo Y, Zhang J. The nitric oxide hypothesis of late preconditioning. Basic Res Cardiol. 1998;93:325–328. 14. Benjamin IJ, McMillan DR. 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