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

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    orally administered 60 minutes before hepatic ischemia. Nicorandil significantly decreasedplasma levels of alanine aminotransferase and lactate dehydrogenase by about 50% and




    428inhibited 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 becomea therapeutic drug for ischemia reperfusion-related liver injury.

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

    rtly attributable to ischemia-reperfusion (IR) injury. Aspathophysiology of hepatic IR injury has become

    adily apparent, many mediators, including tumor necro-factor-, interleukin-1, platelet activating factor, cellularhesion molecules, and reactive oxygen species, have beenwn to play critical roles in this type of injury.13 Anderstanding of the complex mechanisms involved in thisury is required to improve morbidity.n various organs, initial exposures to brief ischemicriods produce cellular tolerance for a subsequent pro-ged ischemic insult.4 This phenomenon, termed ischemicconditioning, has been studied mainly in the heart andr.2,3,5 In the heart, a number of substances and signalingthways have been proposed to exert the cardioprotectiveects of ischemic preconditioning,5 for example, mito-ndrial KATP (mito KATP) channels rather than sar-emmal KATP channels.

    6,7 Therefore, it has been re-

    ported that KATP channel openers have a pharmacologicalpreconditioning effect on myocardial protection.8

    The KATP channels were discovered by Noma9 in 1983

    in ventricular myocytes isolated from guinea pigs. Inoueet al10 first demonstrated KATP channels in the mitochon-drial inner membrane of isolated rat livers. These chan-nels, which are composed of two proteins, an inwardlyrectifying potassium channel (Kir6.x) and a sulfonylureareceptor (SUR), are expressed in numerous tissues,including heart, brain, skeletal muscle, and pancreas.11

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

    From the Department of Hepato-Biliary-Pancreatic Surgery,Graduate School of Medicine, Osaka City University, Osaka,Japan.

    Address reprint requests to Seikan Hai, MD, Department ofHepato-Biliary-Pancreatic Surgery, Graduate School of Medi-cine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka545-8585, Japan. E-mail: KATP Channel Openerchemia-Reperfusion Injury in Rat

    Hai, S. Takemura, Y. Minamiyama, K. Yamasaki, Sd S. Suehiro


    Ischemia-reperfusion injury is responsible founder total vascular exclusion or after liver trathat mitochondrial KATP channel openers hapharmacological preconditioning action. Howchannel openers can reduce ischemia-reperfuwas to determine the effects of the mitochoischemia-reperfusion injury in the rat liver.ischemia for 45 minutes followed by 120 minu1-1345/05/$see front matter:10.1016/j.transproceed.2004.12.112eventser

    mamoto, S. Kodai, S. Tanaka, K. Hirohashi,

    morbidity associated with liver surgerylantation. Recently, it has been reportedn effect on myocardial protection via ar, it remains unclear as to whether KATPinjury in the liver. The aim of this studyal KATP channel opener, nicorandil, onle Wistar rats were subjected to 73%of reperfusion. Nicorandil (3 mg/kg) was 2005 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

    Transplantation Proceedings, 37, 428431 (2005)

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    MITOCHONDRIAL CHANNEL OPENER 429NAs were expressed in the intact rat liver as well as inlated primary rat hepatocytes. Although the liver alsotains KATP channels, it remains unclear as to whetherTP channel openers can reduce IR injury in the liver.his study used nicorandil, a mito KATP channel opener,ich has been used clinically for the treatment of anginatoris. Nicorandil has been shown in rat myocardial mito-ndria13 to produce nitric oxide (NO), which plays anportant role in ischemic preconditioning of rat livers.14


    orandil (SG-75) was purchased from Chugai Pharmaceutical. Ltd.

    imals and Hepatic Ischemia

    le 8-week-old Wistar rats (190 to 220 g body weight) purchasedm SLC (Shizuoka, Japan) had free access to food and water. Allmals were anesthetized with urethane (5 mg/kg intraperitone-) and placed in a supine position. To induce the hepaticemia, a laparotomy was performed. The blood supply to the leftmedian lobes of the liver was interrupted by placement of an

    aumatic vascular clamp to produce 73% hepatic ischemia for 45utes. The body temperature was maintained at 3637C by ating lamp. Reperfusion was initiated by removal of the vascularmp. The animals were sacrificed at 120 minutes after reperfu-n; SG-75 (3 mg/kg) was orally administered 60 minutes beforehepatic ischemia.

    perimental Protocol

    the initial series of experiments, the protective effects of SG-75re tested in the following groups: Group 1: sham: animalsjected to anesthesia and laparotomy. Group 2: SG-75sham:mals subjected to anesthesia and laparotomy but treated with-75 at 60 minutes before hepatic ischemia. Group 3: ischemia-erfusion (IR): animals subjected to 45 minutes of hepaticemia, followed by 120 minutes of reperfusion. Group 4: SG-75R: animals subjected to 45 minutes of hepatic ischemia treatedh SG-75 60 minutes before hepatic ischemia.


    parinized blood samples collected from the aorta were sepa-ed to plasma by centrifugation at 12,000g for 5 minutes andred at 20C until assayed for alanine aminotransferase (ALT)lactate dehydrogenase (LDH). After exsanguination, the livers

    re perfused with 50 mL of ice-cold saline via the abdominalta. The postischemic hepatic lobes were collected, includingtion immediately frozen under liquid nitrogen and stored at0C.

    paration of Hepatic Mitochondria

    osolic and mitochondrial-enriched fractions obtained from por-s of the liver were rinsed in 0.15 mol/L KCl, scissor-minced, andogenized in 3 volumes of 0.25 mol/L sucrose. Low-speedtrifugation was performed to remove cellular debris and obtainwhole cell lysates. For preparation of the mitochondrial-

    iched fractions, whole cell lysates were centrifuged at 500g for

    minutes. The supernate was spun at 2600g for 10 minutes. The Sulting pellet was resuspended in a buffer consisting ofmmol/L Tris-HCl (pH 7.4), 0.25 mol/L sucrose, and 5.4 mmol/Lylenediaminetetraacetic acid.

    rameter of Hepatic Injury

    e degree of hepatic injury was assessed by ALT and LDH plasmaels, as measured by SRL Co., Osaka, Japan. The hematologiclyses were performed using an autoanalyzer system (Hitachi0).


    er samples fixed in 10% formaldehyde were embedded inaffin. Replicate sections (4-m) were stained with hematoxylin-in for the evaluation of apoptosis on the basis of morphologicaleria, such as cell shrinkage, chromatin condensation, and mar-ation.

    NEL Assay

    optosis was determined by staining with the terminal deoxynu-otidyl transferase-mediated dUTP nick end labeling (TUNEL)ay (ApopTag Peroxidase In Situ Apoptosis Detection Kit,ergen, Purchase, NY). The number of apoptotic hepatocytes wasnted in 20 high-power (400) fields using a microscope.

    stern Blot Analysis for Cytochrome-c, Bax, Bcl-2, andspase-3

    ual amounts of total protein loaded onto 15% polyacrylamides were transferred to PVDF membranes. Thereafter, the mem-nes were treated with anti-cytochrome-c (BD Biosciencesrmingen, Franklin Lakes, NJ, USA), anti-Bax (BD Biosciencesrmingen), anti-Bcl-2 (BD Biosciences Pharmingen), or anti-pase-3 (Cell Signaling Technology, Beverly, Mass, USA) ac-ding to the manufacturers instructions. Mean density of thed area was measured using the Scion Image Beta 4.02 (Scion,derick, Md, USA) after obtaining black and white images asF files by an image scanner.

    tistical Analysis

    ta were analyzed with the Tukey-Kramer test; the results aresented as mean values SE. Significance was declared whenP value was less than .05.

    SULTSects of SG-75 on Hepatic Ischemia-Reperfusion Injury

    e effects of SG-75 on ALT plasma levels are shown inble 1. Plasma levels of ALT and LDH in the IR groupre markedly increased to 6128 370 IU/L and 24,486 6 IU/L, respectively. Administration of SG-75 signifi-tly decreased the levels to 2633 309 and 12,288 1572/L, respectively.

    Table 1. Plasma Levels of Alanine Aminotransferase

    Sham IR

    -75 () 133 33 6128 370P .05

    -75 () 119 11 2633 309

    esults are expressed as mean SE (IU/L).

    G-75, nicorandil; IR, ischemia reperfusion.

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    stological studies revealed that IR markedly elicitedoptotic cells, retained erythrocytes in the sinusoids, andulated infiltration of lymphocytes and neutrophils.

    ese observations, which were scarcely seen in the sham-erated groups, were strongly reduced by administrationSG-75.

    e TUNEL Assay

    e TUNEL-positive hepatocytes, only a few of which weren in the sham-operated groups, remarkably increased tout 30% of total hepatocytes in the IR group. In the-75 group, the increase in TUNEL-positive hepatocytess significantly inhibited to about 7% (Table 2).

    ects of SG-75 on Cytochrome-c Release, and thepression of Bax, Bcl-2, and Caspase-3

    ile levels of mitochondrial cytochrome-c did not differween any group, the cytosol showed an increased con-t in the IR group; administration of SG-75 inhibited theease of cytochrome-c from the mitochondria, althoughre was no significant difference (Fig 1A). SG-75 signifi-tly inhibited the activation of caspase-3 evidenced by thenificant increase in the cytosol in the IR group (Fig 1B).e mitochondria expression of Bax and Bcl-2 was signifi-tly increased in the IR group and only slightly inhibitedthe administration of SG-75 (Fig 1C, D).


    r results indicate that nicorandil (SG-75) given orally atose of 3 mg/kg confers dramatic protection against IRury in the rat liver via inhibition of apoptosis withoutotension (data on blood pressure measurements was notwn). In hepatic IR, necrosis was not an importantture of hepatocyte injury, because only a few necroticpatocytes were observed after 60 minutes of ischemia andhours of reperfusion, although livers subjected to 90nutes or more of ischemia were associated with largeas of necrosis. Therefore, apoptosis of hepatocytes is atical mechanism of cell death after IR in the liver.15

    optotic signaling pathways in hepatic IR injury includese via death receptors, through the mitochondria involv-p53-dependent gene expression.16 In this present study,examined the state of some mediators associated withoptotic signaling pathways through the mitochondriaause it had been reported that SG-75, given orally tos, was preferentially distributed into heart mitochon-a17 and SG-75 primarily activates mito KATP rather thancolemmal KATP channels in rabbit-isolated myocytes.


    Table 2. TUNEL-Positive Hepatocytes/Total Hepatocytes

    Sham IR

    -75 () 0.9 0.2 31.0 8.9P .05

    -75 () 0.6 0.1 7.10 1.4

    esults are expressed as mean SE (%).

    leaG-75, nicorandil; IR, ischemia reperfusion.itochondria have been suggested to play an impor-t role in hepatic IR injury. In the liver, IR first causestochondrial Ca2 loading, which promotes mitochon-al permeability transition (MPT). Mitochondrialelling caused by the onset of MPT leads to outermbrane rupture and cytochrome-c release into theosol. Cytochrome-c activates downstream caspases


    1. Western blot analysis. (A) Levels of cytochrome-c in theresponding mitochondrial fraction of the liver did not differween any of the groups. Cytochrome-c level in the cytosolreased in the IR group and SG-75 inhibited the release ofochrome-c toward the cytosol. (B) The cytosolic level ofpase-3 increased in the IR group and SG-75 inhibited theivation of caspase-3. The expression of Bax (C) and Bcl-2 (D)the mitochondrial fraction significantly increased in the IRup and was slightly inhibited by the administration of SG-75. .05 vs sham groups.ding to apoptosis. It has been reported that mito

  • KATP channel openers reduce mitochondrial Ca2 over-

    loading during IR in isolated rat hearts.20 This may be atrigger to prevent an apoptotic signaling pathway throughthe mitochondria. Although this point of view is notobvious in the liver, our results, namely that administra-tion of SG-75 inhibited the release of cytochrome-c fromthe mitochondria and the increase activated caspase-3 inIRpreBcprolinapmechoindtraI







    4. Ishida T, Yarimizu K, Gute DC, et al: Mechanisms ofischemic preconditioning. Shock 8:86, 19975. Baxter GF, Ferdinandy P: Delayed preconditioning of myo-

    cardium: current perspectives. Basic Res Cardiol 96:329, 20016. Fryer RM, Eells JT, Hsu AK, et al: Ischemic preconditioning

    in rats: role of mitochondrial KATP channel in preservation ofmitochondrial function. Am J Physiol Heart Circ Physiol 278:H305,20007




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    MITOCHONDRIAL CHANNEL OPENER 431, indicate that mito KATP channel openers mightvent mitochondrial Ca2 overloading in the liver. Thel-2 family of proteins, consisting of anti-apoptotic and-apoptotic members, regulates cell death by control-g mitochondrial membrane permeability. Bax, pro-optotic proteins, translocate to the mitochondrialmbrane, stimulating cytochrome-c efflux from mito-ndria, events that are induced in IR.16,21 Our dataicate that a mito KATP channel opener inhibits Baxnslocation to the mitochondrial membrane.t has also been suggested that Bax directly modulates thetochondrial voltage-dependent anion channel (VDAC),ying a role in MPT during apoptosis,22 although thechanisms responsible for Bax insertion into the mito-ndrial membrane and the localization of Bax within thetochondria are not fully understood. Therefore, ourults may relate to the possibility of inhibiting Bax fromding to the VDAC by administering mito KATP channeleners. The expressio...


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