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Page 1: Mitochondrial KATP channel opener prevents ischemia-reperfusion injury in rat liver

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itochondrial KATP Channel Opener Preventsschemia-Reperfusion Injury in Rat Liver

. Hai, S. Takemura, Y. Minamiyama, K. Yamasaki, S. Yamamoto, S. Kodai, S. Tanaka, K. Hirohashi,nd S. Suehiro

ABSTRACT

Ischemia-reperfusion injury is responsible for the morbidity associated with liver surgeryunder total vascular exclusion or after liver transplantation. Recently, it has been reportedthat mitochondrial KATP channel openers have an effect on myocardial protection via apharmacological preconditioning action. However, it remains unclear as to whether KATPchannel openers can reduce ischemia-reperfusion injury in the liver. The aim of this studywas to determine the effects of the mitochondrial KATP channel opener, nicorandil, onischemia-reperfusion injury in the rat liver. Male Wistar rats were subjected to 73%ischemia for 45 minutes followed by 120 minutes of reperfusion. Nicorandil (3 mg/kg) wasorally administered 60 minutes before hepatic ischemia. Nicorandil significantly decreasedplasma levels of alanine aminotransferase and lactate dehydrogenase by about 50% andinhibited the remarkably increased TUNEL-positive hepatocytes after reperfusion. Somemediators associated with apoptosis were analyzed by Western blotting. Cytochrome-c andcaspase-3 levels in the cytosol increased after reperfusion; nicorandil inhibited the releaseof cytochrome-c and activation of caspase-3. The expression of Bax and Bcl-2 wassignificantly increased after reperfusion, being slightly inhibited by the administration ofnicorandil. These results suggest that the protective effects of nicorandil against hepaticischemia-reperfusion injury correlate with the inhibition of mitochondrial cytochrome-crelease and caspase-3 activation. These findings demonstrate that nicorandil may become

a therapeutic drug for ischemia reperfusion-related liver injury.

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HE morbidity associated with hepatic resections undertotal vascular exclusion and liver transplantation is

artly attributable to ischemia-reperfusion (IR) injury. Ashe pathophysiology of hepatic IR injury has becometeadily apparent, many mediators, including tumor necro-is factor-�, interleukin-1, platelet activating factor, cellulardhesion molecules, and reactive oxygen species, have beenhown to play critical roles in this type of injury.1–3 Annderstanding of the complex mechanisms involved in this

njury is required to improve morbidity.In various organs, initial exposures to brief ischemic

eriods produce cellular tolerance for a subsequent pro-onged ischemic insult.4 This phenomenon, termed ischemicreconditioning, has been studied mainly in the heart andiver.2,3,5 In the heart, a number of substances and signalingathways have been proposed to exert the cardioprotectiveffects of ischemic preconditioning,5 for example, mito-hondrial K (mito K ) channels rather than sar-

ATP ATP

olemmal KATP channels.6,7 Therefore, it has been re- 5

041-1345/05/$–see front matteroi:10.1016/j.transproceed.2004.12.112

28

orted that KATP channel openers have a pharmacologicalreconditioning effect on myocardial protection.8

The KATP channels were discovered by Noma9 in 1983n ventricular myocytes isolated from guinea pigs. Inouet al10 first demonstrated KATP channels in the mitochon-rial inner membrane of isolated rat livers. These chan-els, which are composed of two proteins, an inwardlyectifying potassium channel (Kir6.x) and a sulfonylureaeceptor (SUR), are expressed in numerous tissues,ncluding heart, brain, skeletal muscle, and pancreas.11

ecently, Malhi et al12 reported that Kir6.1 and SUR1

From the Department of Hepato-Biliary-Pancreatic Surgery,raduate School of Medicine, Osaka City University, Osaka,apan.Address reprint requests to Seikan Hai, MD, Department ofepato-Biliary-Pancreatic Surgery, Graduate School of Medi-ine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka

45-8585, Japan. E-mail: [email protected]

© 2005 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 37, 428–431 (2005)

Page 2: Mitochondrial KATP channel opener prevents ischemia-reperfusion injury in rat liver

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MITOCHONDRIAL CHANNEL OPENER 429

RNAs were expressed in the intact rat liver as well as insolated primary rat hepatocytes. Although the liver alsoontains KATP channels, it remains unclear as to whetherATP channel openers can reduce IR injury in the liver.This study used nicorandil, a mito KATP channel opener,

hich has been used clinically for the treatment of anginaectoris. Nicorandil has been shown in rat myocardial mito-hondria13 to produce nitric oxide (NO), which plays anmportant role in ischemic preconditioning of rat livers.14

ATERIALS AND METHODSrugs

icorandil (SG-75) was purchased from Chugai Pharmaceuticalo. Ltd.

nimals and Hepatic Ischemia

ale 8-week-old Wistar rats (190 to 220 g body weight) purchasedrom SLC (Shizuoka, Japan) had free access to food and water. Allnimals were anesthetized with urethane (5 mg/kg intraperitone-lly) and placed in a supine position. To induce the hepaticschemia, a laparotomy was performed. The blood supply to the leftnd median lobes of the liver was interrupted by placement of antraumatic vascular clamp to produce 73% hepatic ischemia for 45inutes. The body temperature was maintained at 36�37°C by a

eating lamp. Reperfusion was initiated by removal of the vascularlamp. The animals were sacrificed at 120 minutes after reperfu-ion; SG-75 (3 mg/kg) was orally administered 60 minutes beforehe hepatic ischemia.

xperimental Protocol

n the initial series of experiments, the protective effects of SG-75ere tested in the following groups: Group 1: sham: animals

ubjected to anesthesia and laparotomy. Group 2: SG-75�sham:nimals subjected to anesthesia and laparotomy but treated withG-75 at 60 minutes before hepatic ischemia. Group 3: ischemia-eperfusion (IR): animals subjected to 45 minutes of hepaticschemia, followed by 120 minutes of reperfusion. Group 4: SG-75

IR: animals subjected to 45 minutes of hepatic ischemia treatedith SG-75 60 minutes before hepatic ischemia.

amples

eparinized blood samples collected from the aorta were sepa-ated to plasma by centrifugation at 12,000g for 5 minutes andtored at �20°C until assayed for alanine aminotransferase (ALT)nd lactate dehydrogenase (LDH). After exsanguination, the liversere perfused with 50 mL of ice-cold saline via the abdominalorta. The postischemic hepatic lobes were collected, includingortion immediately frozen under liquid nitrogen and stored at80°C.

reparation of Hepatic Mitochondria

ytosolic and mitochondrial-enriched fractions obtained from por-ions of the liver were rinsed in 0.15 mol/L KCl, scissor-minced, andomogenized in 3 volumes of 0.25 mol/L sucrose. Low-speedentrifugation was performed to remove cellular debris and obtainhe whole cell lysates. For preparation of the mitochondrial-nriched fractions, whole cell lysates were centrifuged at 500g for

0 minutes. The supernate was spun at 2600g for 10 minutes. The

esulting pellet was resuspended in a buffer consisting of0 mmol/L Tris-HCl (pH 7.4), 0.25 mol/L sucrose, and 5.4 mmol/Lthylenediaminetetraacetic acid.

arameter of Hepatic Injury

he degree of hepatic injury was assessed by ALT and LDH plasmaevels, as measured by SRL Co., Osaka, Japan. The hematologicnalyses were performed using an autoanalyzer system (Hitachi170).

istology

iver samples fixed in 10% formaldehyde were embedded inaraffin. Replicate sections (4-�m) were stained with hematoxylin-osin for the evaluation of apoptosis on the basis of morphologicalriteria, such as cell shrinkage, chromatin condensation, and mar-ination.

UNEL Assay

poptosis was determined by staining with the terminal deoxynu-leotidyl transferase-mediated dUTP nick end labeling (TUNEL)ssay (ApopTag Peroxidase In Situ Apoptosis Detection Kit,ntergen, Purchase, NY). The number of apoptotic hepatocytes wasounted in 20 high-power (�400) fields using a microscope.

estern Blot Analysis for Cytochrome-c, Bax, Bcl-2, andaspase-3

qual amounts of total protein loaded onto 15% polyacrylamideels were transferred to PVDF membranes. Thereafter, the mem-ranes were treated with anti-cytochrome-c (BD Biosciencesharmingen, Franklin Lakes, NJ, USA), anti-Bax (BD Biosciencesharmingen), anti-Bcl-2 (BD Biosciences Pharmingen), or anti-aspase-3 (Cell Signaling Technology, Beverly, Mass, USA) ac-ording to the manufacturer’s instructions. Mean density of theand area was measured using the Scion Image Beta 4.02 (Scion,rederick, Md, USA) after obtaining black and white images asIFF files by an image scanner.

tatistical Analysis

ata were analyzed with the Tukey-Kramer test; the results areresented as mean values � SE. Significance was declared whenhe P value was less than .05.

ESULTSffects of SG-75 on Hepatic Ischemia-Reperfusion Injury

he effects of SG-75 on ALT plasma levels are shown inable 1. Plasma levels of ALT and LDH in the IR groupere markedly increased to 6128 � 370 IU/L and 24,486 �656 IU/L, respectively. Administration of SG-75 signifi-antly decreased the levels to 2633 � 309 and 12,288 � 1572U/L, respectively.

Table 1. Plasma Levels of Alanine Aminotransferase

Sham IR

G-75 (�) 133 � 33 6128 � 370P � .05

G-75 (�) 119 � 11 2633 � 309

Results are expressed as mean � SE (IU/L).SG-75, nicorandil; IR, ischemia reperfusion.

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430 HAI, TAKEMURA, MINAMIYAMA ET AL

istology

istological studies revealed that IR markedly elicitedpoptotic cells, retained erythrocytes in the sinusoids, andtimulated infiltration of lymphocytes and neutrophils.hese observations, which were scarcely seen in the sham-perated groups, were strongly reduced by administrationf SG-75.

he TUNEL Assay

he TUNEL-positive hepatocytes, only a few of which wereeen in the sham-operated groups, remarkably increased tobout 30% of total hepatocytes in the IR group. In theG-75 group, the increase in TUNEL-positive hepatocytesas significantly inhibited to about 7% (Table 2).

ffects of SG-75 on Cytochrome-c Release, and thexpression of Bax, Bcl-2, and Caspase-3

hile levels of mitochondrial cytochrome-c did not differetween any group, the cytosol showed an increased con-ent in the IR group; administration of SG-75 inhibited theelease of cytochrome-c from the mitochondria, althoughhere was no significant difference (Fig 1A). SG-75 signifi-antly inhibited the activation of caspase-3 evidenced by theignificant increase in the cytosol in the IR group (Fig 1B).he mitochondria expression of Bax and Bcl-2 was signifi-antly increased in the IR group and only slightly inhibitedy the administration of SG-75 (Fig 1C, D).

ISCUSSION

ur results indicate that nicorandil (SG-75) given orally atdose of 3 mg/kg confers dramatic protection against IR

njury in the rat liver via inhibition of apoptosis withoutypotension (data on blood pressure measurements was nothown). In hepatic IR, necrosis was not an importanteature of hepatocyte injury, because only a few necroticepatocytes were observed after 60 minutes of ischemia and

hours of reperfusion, although livers subjected to 90inutes or more of ischemia were associated with large

reas of necrosis. Therefore, apoptosis of hepatocytes is aritical mechanism of cell death after IR in the liver.15

poptotic signaling pathways in hepatic IR injury includehose via death receptors, through the mitochondria involv-ng p53-dependent gene expression.16 In this present study,e examined the state of some mediators associated withpoptotic signaling pathways through the mitochondriaecause it had been reported that SG-75, given orally toats, was preferentially distributed into heart mitochon-ria17 and SG-75 primarily activates mito KATP rather thanarcolemmal KATP channels in rabbit-isolated myocytes.18

Table 2. TUNEL-Positive Hepatocytes/Total Hepatocytes

Sham IR

G-75 (�) 0.9 � 0.2 31.0 � 8.9P � .05

G-75 (�) 0.6 � 0.1 7.10 � 1.4

lResults are expressed as mean � SE (%).SG-75, nicorandil; IR, ischemia reperfusion.

Mitochondria have been suggested to play an impor-ant role in hepatic IR injury. In the liver, IR first causesitochondrial Ca2� loading, which promotes mitochon-

rial permeability transition (MPT). Mitochondrialwelling caused by the onset of MPT leads to outerembrane rupture and cytochrome-c release into the

ytosol. Cytochrome-c activates downstream caspases

ig 1. Western blot analysis. (A) Levels of cytochrome-c in theorresponding mitochondrial fraction of the liver did not differetween any of the groups. Cytochrome-c level in the cytosol

ncreased in the IR group and SG-75 inhibited the release ofytochrome-c toward the cytosol. (B) The cytosolic level ofaspase-3 increased in the IR group and SG-75 inhibited thectivation of caspase-3. The expression of Bax (C) and Bcl-2 (D)

n the mitochondrial fraction significantly increased in the IRroup and was slightly inhibited by the administration of SG-75.P � .05 vs sham groups.

eading to apoptosis.16,19 It has been reported that mito

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MITOCHONDRIAL CHANNEL OPENER 431

ATP channel openers reduce mitochondrial Ca2� over-oading during IR in isolated rat hearts.20 This may be arigger to prevent an apoptotic signaling pathway throughhe mitochondria. Although this point of view is notbvious in the liver, our results, namely that administra-ion of SG-75 inhibited the release of cytochrome-c fromhe mitochondria and the increase activated caspase-3 inR, indicate that mito KATP channel openers mightrevent mitochondrial Ca2� overloading in the liver. Thecl-2 family of proteins, consisting of anti-apoptotic andro-apoptotic members, regulates cell death by control-

ing mitochondrial membrane permeability. Bax, pro-poptotic proteins, translocate to the mitochondrialembrane, stimulating cytochrome-c efflux from mito-

hondria, events that are induced in IR.16,21 Our datandicate that a mito KATP channel opener inhibits Baxranslocation to the mitochondrial membrane.

It has also been suggested that Bax directly modulates theitochondrial voltage-dependent anion channel (VDAC),

laying a role in MPT during apoptosis,22 although theechanisms responsible for Bax insertion into the mito-

hondrial membrane and the localization of Bax within theitochondria are not fully understood. Therefore, our

esults may relate to the possibility of inhibiting Bax frominding to the VDAC by administering mito KATP channelpeners. The expression of Bcl-2, an anti-apoptotic protein,aralleled Bax in the present study. Using Bcl-2 transgenicice, Selzner et al23 reported that Bcl-2 overexpression

rotects against an hepatic IR injury by inhibiting apoptosis.herefore, Bcl-2 may play an important role to reduce IR

njury, a result that illustrates the balance among pro-ersus anti-apoptotic molecules.

In conclusion, these results suggest that the protectiveffects of SG-75 against hepatic IR injury correlate with thenhibition of mitochondrial cytochrome-c release and acti-ation of caspase-3. Thus, SG-75 may become a therapeuticrug for pharmacological preconditioning against IR-elated liver injury. However, further investigations areequired to clarify this role, because the hepatic protectiveechanisms associated with mito KATP channels are not yet

learly defined.

EFERENCES

1. Serracino-Inglott F, Habib NA, Mathie RT: Hepatic ischemia-eperfusion injury. Am J Surg 181:160, 2001

2. Jaeschke H: Molecular mechanisms of hepatic ischemia-eperfusion injury and preconditioning. Am J Physiol Gastrointestiver Physiol 284:G15, 20033. Cutrn JC, Perrelli MG, Cavalieri B, et al: Microvascular

ysfunction induced by reperfusion injury and protective effect of

schemic preconditioning. Free Radic Biol Med 33:1200, 2002 r

4. Ishida T, Yarimizu K, Gute DC, et al: Mechanisms ofschemic preconditioning. Shock 8:86, 1997

5. Baxter GF, Ferdinandy P: Delayed preconditioning of myo-ardium: current perspectives. Basic Res Cardiol 96:329, 2001

6. Fryer RM, Eells JT, Hsu AK, et al: Ischemic preconditioningn rats: role of mitochondrial KATP channel in preservation of

itochondrial function. Am J Physiol Heart Circ Physiol 278:H305,0007. Gross GJ, Fryer RM: Sarcolemmal versus mitochondrialTP-sensitive K� channels and myocardial preconditioning. Circes 84:973, 19998. Grover GJ: Pharmacology of ATP-sensitive potassium chan-

el (KATP) openers in models of myocardial ischemia and reper-usion. Can J Physiol Pharmacol 75:309, 1997

9. Noma A: ATP-regulated K� channels in cardiac muscle.ature 305:147, 198310. Inoue I, Nagase H, Kishi K, et al: ATP-sensitive K� channel

n the mitochondrial inner membrane. Nature 352:244, 199111. Babenko AP, Aguilar-Bryan L, Bryan J: A view of SUR/

IR6.X, KATP channels. Annu Rev Physiol 60:667, 199812. Malhi H, Irani AN, Rajvanshi P, et al: KATP channels

egulate mitogenically induced proliferation in primary rat hepa-ocytes and human liver cell lines. Implications for liver growthontrol and potential therapeutic targeting. J Biol Chem 275:26050,00013. Sakai K, Akima M, Saito K, et al: Nicorandil metabolism in

at myocardial mitochondria. J Cardiovasc Pharmacol 35:723, 200014. Peralta C, Hotter G, Closa D, et al: Protective effect of

reconditioning on the injury associated to hepatic ischemia-eperfusion in the rat: role of nitric oxide and adenosine. Hepatol-gy 25:934, 199715. Kohli V, Selzner M, Madden JF, et al: Endothelial cell and

epatocyte deaths occur by apoptosis after ischemia-reperfusionnjury in the rat liver. Transplantation 67:1099, 1999

16. Jaeschke H, Lemasters JJ: Apoptosis versus oncotic necrosisn hepatic ischemia/reperfusion injury. Gastroenterology 125:1246,00317. Sakai K, Tsuchiya Y, Kitajima S, et al: Myocardial distribu-

ion and biotransformation in vitro and in vivo of nicorandil in rats,ith special reference to mitochondria. J Cardiovasc Pharmacol3:163, 199918. Sato T, Sasaki N, O’Rourke B, et al: Nicorandil, a potent cardio-

rotective agent, acts by opening mitochondrial ATP-dependent potas-ium channels. J Am Coll Cardiol 35:514, 2000

19. Jassem W, Fuggle SV, Rela M, et al: The role of mitochon-ria in ischemia/reperfusion injury. Transplantation 73:493, 200220. Wang L, Cherednichenko G, Hernandez L, et al: Precondi-

ioning limits mitochondrial Ca2� during ischemia in rat hearts:ole of KATP channels. Am J Physiol Heart Circ Physiol 280:H2321,00121. Eskes R, Desagher S, Antonsson B, et al: Bid induces the

ligomerization and insertion of Bax into the outer mitochondrialembrane. Mol Cell Biol 20:929, 200022. Shimizu S, Ide T, Yanagida T, et al: Electrophysiological

tudy of a novel large pore formed by Bax and the voltage-ependent anion channel that is permeable to cytochrome c. J Biolhem 275:12321, 200023. Selzner M, Rudiger HA, Selzner N, et al: Transgenic mice

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eperfusion. J Hepatol 36:218, 2002

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