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Mitochondrial Permeability Transition Dynamics: An Indicator of Mitochondrial Potassium Channel Opener Fang Shen, Li-ping Wu, Yuan Lu, Hua-wei Liang, Iain C. Bruce, Qiang Xia Abstract-Mitochondrial permeability transition (MPT) is an intracellular event that is closely related to apoptosis and necrosis. However, whether this process underlies the recently reported neuroprotective potency of mitochondrial potassium channel openers applied in vivo remains uncertain. This study aims to clarify this issue by determining the effects of potassium channel openers on MPT dynamics in vitro along with their in vivo effects. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 90 min, followed by reperfusion. 30μl of diazoxide, an opener of the mitochondrial adenosine triphosphate-sensitive K+ channel (mitoKATP), or NS1619, an opener of the mitochondrial Ca2+-activated potassium channel (mitoKCa) (2 mM and 0.1 mM respectively), was infused into the right lateral cerebral ventricle 15 min before the induction of ischemia. Neurological scores were assessed 24 h after MCAO and then infarct area was determined by standard 2,3,5-triphenyltetrazolium chloride staining techniques. To further clarify the capacity of diazoxide and NS1619 to protect mitochondria from Ca2+-induced MPT, we isolated brain-derived non-synaptosomal mitochondria and evaluated the effects of diazoxide and NS1619 on Ca2+-induced MPT dynamics through measurement of spectrophotometric alterations in light scattering at 520 nm. Neurological scores and infarct size were improved in animals pretreated with diazoxide and NS1619. In isolated mitochondria, MPT was readily induced by 200 μM Ca2+ and was effectively inhibited by diazoxide and NS1619. The specific MPT pore opener atractyloside abolished the inhibitory effects. According to time-constant analysis, MPT dynamics was in accordance with the neuroprotective effects of channel openers in vivo. Therefore, measuring MPT dynamics provides a new means of predicting the neuroprotective effects of mitochondrial potassium channel openers. I. INTRODUCTION ITOCHONDRIA exist in virtually all cell types and, as the main producers of ATP, are essential for life. The inner mitochondrial membrane is normally impermeable to solutes except those with specific transport systems. As a result of Ca2+ accumulation and oxidative stress, mitochondria may undergo a sudden permeability increase of the inner membrane, the mitochondrial permeability transition (MPT). The rapid change of permeability causes membrane depolarization, uncoupling of oxidative phosphorylation, release of mitochondrial ions and metabolites, and mitochondrial swelling. MPT is also accompanied by increased production of reactive oxygen species and the release of apoptogenic proteins such as cytochrome C, Smac/DIABLO and apoptosis inducing factor. F. Shen, L-P. Wu, Y. Lu, H-W Liang and Q. Xia are with the Department of Physiology, Zhejiang University School of Medicine, Hangzhou, China (phone: 86-571-87217146; fax: 86-571-87217147; e-mail: xiaqiang@zju.edu.cn). I.C. Bruce is with the Department of Physiology, the University of Hong Kong, Hong Kong, China (e-mail: hrmybic@hkucc.hku.hk). K+ channel openers such as diazoxide are of interest because of their use in ischemia/reperfusion injury in the heart and are currently under investigation in the brain [1]. Evidence indicates that the opening of the mitochondrial ATP-sensitive K+ channel (mitoKATP) is responsible for this protective effect [2] and depolarization induced by increased K+ conductance across the mitochondrial membrane may be the central event in this process. Siemen et al. [3] reported finding a Ca2+-activated K+ channel with high conductance (mitoBK) in the inner mitochondrial membrane in a human glioma cell line. This channel has gating properties similar to the large potassium channel (BK) of the plasma membrane. NS1619 is a specific BK channel opener and Xu et al. [4] found that activation of myocardial mitoBK by NS1619 is cardioprotective. However, to our knowledge, the effects of NS1619 on neuronal mitochondria have not yet been investigated. In the present study, we investigated diazoxide and NS1619 for their potential to protect brain during in vivo ischemia/reperfusion injury and further determined their direct in vitro effects on brain-derived non-synaptosomal mitochondria exposed to high Ca2+. Furthermore, the relationship between in vitro MPT dynamics and in vivo neuroprotective effects by the mitochondrial potassium channel openers was investigated. II. METHODOLOGY Experiments were carried out in male Sprague-Dawley rats (240-300 g) obtained from the Animal Centre of Zhejiang academy of Medical Sciences. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals of Zhejiang University. A. In vivo Experiments After anaesthesia with 4% chloral hydrate (10 ml/kg, i.p.), the head was fixed in a stereotactic frame, and 30 μl diazoxide (2 mM, n=6) or NS1619 (0.1 mM, n=6) was infused through a craniotomy into the right lateral ventricle 15 min before the induction of ischemia. Transient focal cerebral ischemia was induced on the right side by MCAO under anesthesia [5]. In brief, through a midline neck incision, the external (ECA) and internal carotid M Limited circulation. For review only. Preprint submitted to 27th IEEE EMBS Annual International Conference. Received May 1, 2005
Limited circulation.For review only. CC o。aa7 nd the FCA stump wis then tightened Th IIL RESULTS The ch nate was diluled with 200
arteries (ICA) were dissected from surrounding connective tissue. The branches of the ECA were ligated and cut. The ECA was then ligated in two places and divided. Two microvascular clips were placed across the common carotid artery and the ICA. A 4-0 monofilament nylon suture was introduced into the ICA via the ECA stump. The suture was inserted 2 cm until some resistance was felt and a slight curving of the suture was observed within the ICA lumen. The suture around the ECA stump was then tightened. The proximal microvascular clip was removed, and the incision was closed. After 90 min of MCAO, the suture was removed to allow reperfusion, and the stump of the ECA was ligated with 5-0 silk sutures. Neurological evaluation was performed 24 h after reperfusion by evaluators blind as to the treatment as described by Bederson et al. [6]. Specifically, no spontaneous activity was graded as 0, spontaneous circling as 1, circling if pulled by the tail as 2, lowered resistance to lateral push without circling as 3, contralateral forelimb flexion as 4, and no apparent deficit as 5. After neurological evaluation, all animals were anesthetized and decapitated. Brains were sliced into 2 mm sections. Each slice was incubated for 20 min in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; Sigma) at room temperature and then fixed in 10% buffered formaldehyde. Infarct size was determined with image analysis software (Image J 1.32) and compared among different treatments. B. In vitro Experiments The procedure for isolation of brain mitochondria was that of Kosenko et al. [7] with small modifications. Rats were killed by stunning and cervical dislocation. The brain was removed quickly (typically, within 20 s) into ice-cold isolation medium (0.25 M sucrose, 1 mM K-EDTA and 10 mM Tris-HCl, pH 7.4). Brains were chopped with scissors and frequently washed with isolation medium to remove blood. The chopped tissue was homogenized in 7.5 ml of isolation medium. The homogenate was diluted with isolation medium to a final volume of 15 ml and homogenized again. The homogenate was centrifuged at 2000 g for 3 min, the supernatant was centrifuged again at 2000 g for 3 min, and the second supernatant was centrifuged at 12,000 g for 8 min to obtain the mitochondrial pellet. The pellet was suspended in isolation medium without EDTA and centrifuged at 12,000 g for 10 min. The pellet was resuspended in the same medium to obtain a protein concentration in the range of 20-25 mg/ml. All procedures were carried out at 2-4°C, and the isolation took about 70 min. Mitochondrial permeability transition was measured as the decrease in light scattering at 520 nm (A520) using a Spectrometer (UV-4802). The decrease in light scattering is closely paralleled by the percentage of the mitochondrial population undergoing permeability transition [8]. Experiments were initiated adding diazoxide or NS1619 (with or without atractyloside) and a fixed amount of mitochondria (20 mg/ml) to isotonic swelling buffer (150 mM KCl, 20 mM MOPS and 10 mM Tris-HCl, pH 7.4). CaCl2 was added at 2 min to induce swelling. Relative A520 (A’520) was calculated by dividing every A520 value by the initial (maximum) A520 value (A’520= A520/ A520max). A’520 decrease was compared among different treatments. The time constant of A520 reduction was calculated and analyzed by Clampfit software (Axon Co., USA). C. Statistical Analysis Data were expressed as mean ± SD and were compared by one-way ANOVA followed by Newman-Keuls post hoc analysis. A P value<0.05 was regarded as statistically significant. III. RESULTS A. In vivo Experiments 24 h after reperfusion, animals pretreated with 2 mM diazoxide or 0.1 mM NS1619 had significantly improved neurological scores compared with untreated animals (Fig. 1). These treatments also decreased infarct size after MCAO (Fig. 2). B. In vitro Experiments Relative to basal conditions (no high Ca2+), 200 μM Ca2+ induced marked decreases in mitochondrial A520 and A’520, showing this was sufficient to induce mitochondrial swelling. 30 μM diazoxide and 10 μM NS1619 alleviated the mitochondrial swelling induced by Ca2+. 100 μM atractyloside abolished their protective effects (Figs. 3 and 4). MPT dynamics were in accordance with the in vivo results (Fig. 5). Limited circulation. For review only. Preprint submitted to 27th IEEE EMBS Annual International Conference. Received May 1, 2005
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Control NS1619 Diazoxide 0 1 2 3 * * Neurological score Fig. 1. Effect of 0.1 mM NS1619 and 2 mM diazoxide on neurological scoring after focal brain ischemia/reperfusion injury. Values are expressed as mean ± SD, n=5-6. * P<0.05 vs. Control Control Vehicle NS1619 Diazoxide 0 5 10 15 20 25 30 35 ** ** Infarct volume (% of total) Fig. 2. Effect of 0.1 mM NS1619 and 2 mM diazoxide on infarct area after focal brain ischemia/reperfusion injury. Vehicle: 0.1 N NaOH. Values are expressed as mean ± SD, n=2-5. ** P<0.01 vs. Control 1min A520 0.025 Ca2+ Basal 30μM Diazoxide CaCl2 Atractyloside+Diazoxide A Basal Atr+DE CaCl2 Diazoxide 0.00 0.02 0.04 0.06 0.08 ## ** B A'520Max-A'520Min ** Fig. 3. Typical tracings (A) and statistics (B) for the effect of 30 μM diazoxide on 200 μM Ca2+- induced A520 change. Basal: no high Ca2+; Atractyloside: 100 μM. Values are expressed as mean ± SD, n=6. ** P<0.01 vs. diazoxide; ## P<0.01 vs CaCl2 1min A520 0.025 Ca2+ Basal CaCl2 10μM NS1619 30μM NS1619 Atractyloside+NS1619 A Basal CaCl2 10uM 30uM Atr+NS1619 0.00 0.02 0.04 0.06 0.08 NS1619 ** ## B A'520Max-A'520Min ** Fig. 4. Typical tracings (A) and statistics (B) for the effect of 10 μM or 30 μM NS1619 on 200 μM Ca2+-induced A520 change. Basal: no high Ca2+; Atractyloside: 100 μM. Values are expressed as means ± SD, n=6. ** P<0.01 vs.NS1619; ## P<0.01 vs CaCl2 Ca Tau Control NS1619 Tau 10 μM NS1619 DE Tau 30 μM DE NS1619+Atr Tau 10 μM NS1619+100 μM Atr DE+Atr Tau 30 μM DE+100 μM Atr 0 10 20 30 0.0 0.5 1.0 MPT Dynamics In vivo infarct (% of total) Fig. 5. Relationship between MPT dynamics and in vivo effect on infarct area. TauCa: time constant of A520 reduction induced by 200 μM Ca2+; TauNS1619: effect of 10 μM NS1619 on time constant of A520 reduction induced by 200 μM Ca2+; TauDE: effect of 30 μM DE on time constant of A520 reduction induced by 200 μM Ca2+; TauNS1619+Atr: effect of 10 μM NS1619 + 100 μM Atr on time constant of A520 reduction induced by 200 μM Ca2+; TauDE+Atr: effect of 30 μM DE + 100 μM Atr on time constant of A520 reduction induced by 200 μM Ca2+; Control: no 200 μM Ca2+. Limited circulation. For review only. Preprint submitted to 27th IEEE EMBS Annual International Conference. Received May 1, 2005
Limited circulation.For review only. IV.DISCUsSION The maior finding of the present study is thst pretrestmen with the mituKc channel opener NS1619.just as with the mitoK channel opener diaoxide,had a neruprutective effeet agsinst trarsien ficnl cerebeal ischem i the rat Additionally,data from isolated mitochordna provided evidence that these two potassium channel openers can directly net on mitochondria to exert a protective effect Swvelling idced by high(Adding atractyloside dimmated these proteetive cfloets,suggesting n close link between croes-mitochondrial memhrane K"conductance ad mitochondral membmane permenbilization Thus,our results show that sclective targeting of the mitoke.nnd mitok channels can.by modulnting K conductnnce and thus preserving mitochondria membrane integrity,lessen the consequences of ischemic insults on the brain.In vitro MPT dynamics can be used to predict the effects of potassium channel openers in vivo. REFERENCES T1】G.人.Gio"A-etsliee potessiunt che凸d ayocailial cosaitimig"loic Res Crdiol.vol 9.85-March 194 D.GaB.Puceh.V Yatn-Yuurs,N.B. larheronn a al."Cardioprorectwe ersecr or dixnoside and ts intersctinz with miuchusdral ATP-waptive K chaarch Puoible mechenisn of canfoproection"Cing Res vol 81.pp.1072.1042. Decemhe:1997. ]D.Sicmen,C.Loapel山es,上.EBua,E.Gi线adF.1La 2 cfivalnd K"charn加Ik-teitrer mtechondn可 awuu了u lutta liau vll E Bchen Boptys.vl 757.pp 515-354.Apnl 1999 4w.XY.1山uS.wu联T.Ded日ud.E5kd mitochondrial merbrane”Scimce,vol28.p1位-133 K.Shinizu R.Veluung,uN D.W.Buiin'Clutclelitisufinduol R5,d.新1，e316-324412000. 周J.H.ederson,L.Ls,MTsL.MC.Nhmr同3ndR.Ls d,"Kat rridde co女al artrry ocxiain:eval的n of the model and develognent ofa neurologk exninoton"Stoke,vol.17.pp.472.476. E Kownio,N Veredlknu Y.Kutitoly,C.Muuliu uaV.Felips, "Pegnn and handing of hean mnochondo gofal to shaty uptice ul idraa of clciur,"Bnin ReaBuin ReaPitoc.wol.7.Ep. 24-251,uh2001. 国D.度Harter and R A.He“TzCa'uood memhane ro1o自moochend鱼I.T几e protective mochanbsns Arch Bicchxm.Bieplrys,vul.195,pe.453-459,July 1979. Preprint submitted to 27th IEEE EMBS Annual International Conferenoe. Received May 1,2005
IV. DISCUSSION The major finding of the present study is that pretreatment with the mitoKCa channel opener NS1619, just as with the mitoKATP channel opener diazoxide, had a neuroprotective effect against transient focal cerebral ischemia in the rat. Additionally, data from isolated mitochondria provided evidence that these two potassium channel openers can directly act on mitochondria to exert a protective effect against swelling induced by high Ca2+. Adding atractyloside eliminated these protective effects, suggesting a close link between cross-mitochondrial membrane K+ conductance and mitochondrial membrane permeabilization. Thus, our results show that selective targeting of the mitoKCa and mitoKATP channels can, by modulating K+ conductance and thus preserving mitochondria membrane integrity, lessen the consequences of ischemic insults on the brain. In vitro MPT dynamics can be used to predict the effects of potassium channel openers in vivo. REFERENCES [1] G. J. Gross, "ATP-sensitive potassium channels and myocardial preconditioning," Basic Res.Cardiol., vol. 90, pp. 85-88, March 1995. [2] K. D. Garlid, P. Paucek, V. Yarov-Yarovoy, H. N. Murray and R. B. Darbenzio et al., "Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection," Circ.Res., vol. 81, pp. 1072-1082, December 1997. [3] D. Siemen, C. Loupatatzis, J. Borecky, E. Gulbins, and F. Lang, "Ca2+-activated K+ channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line," Biochem.Biophys.Res.Commun., vol. 257, pp. 549-554, April 1999. [4] W. Xu, Y. Liu, S. Wang, T. McDonald and J. E. Van Eyk et al., "Cytoprotective role of Ca2+- activated K+ channels in the cardiac inner mitochondrial membrane," Science, vol. 298, pp. 1029-1033, November 2002. [5] K. Shimizu, R. Veltkamp, and D. W. Busija, "Characteristics of induced spreading depression after transient focal ischemia in the rat," Brain Res., vol. 861, pp. 316-324, April 2000. [6] J. B. Bederson, L. H. Pitts, M. Tsuji, M. C. Nishimura and R. L. Davis et al., "Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination," Stroke, vol. 17, pp. 472-476, May 1986. [7] E. Kosenko, N. Venediktova, Y. Kaminsky, C. Montoliu, and V. Felipo, "Preparation and handling of brain mitochondria useful to study uptake and release of calcium," Brain Res.Brain Res.Protoc., vol. 7, pp. 248-254, July 2001. [8] D. R. Hunter and R. A. Haworth, "The Ca2+-induced membrane transition in mitochondria. I. The protective mechanisms," Arch.Biochem.Biophys., vol. 195, pp. 453-459, July 1979. Limited circulation. For review only. Preprint submitted to 27th IEEE EMBS Annual International Conference. Received May 1, 2005