Cell-permeable Peptide Antioxidants Targeted to Inner Mitochondrial Membrane inhibit Mitochondrial Swelling, Oxidative Cell Death, and Reperfusion Injury

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress. Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress. The mitochondrial respiratory chain on the inner mitochondrial membrane (IMM) 1The abbreviations used are: IMM, inner mitochondrial membrane; ROS, reactive oxygen species; 3NP, 3-nitropropionic acid; OMM, outer mitochondrial membrane; MPT, mitochondrial permeability transition; tBHP, t-butylhydroperoxide; Dmt, 2′,6′-dimethyltyrosine; LDL, low density lipoprotein; FCCP, carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; CLSM, confocal laser scanning microscopy; TMRM, tetramethylrhodamine methyl ester. is a major intracellular source of reactive oxygen species (ROS). ROS cause nonspecific damage to lipids, proteins, and DNA, leading to alteration or loss of cellular function. Mitochondria are continuously exposed to ROS and accumulate oxidative damage more rapidly than the rest of the cell, especially because ROS are highly reactive and shortlived (1Kowaltowski A.J. Vercesi A.E. Free Radic. Biol. Med. 1999; 26: 463-471Crossref PubMed Scopus (706) Google Scholar). 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The inhibition of this complex seems to be related to neuronal death similar to those occurring in Huntington's disease (6Beal M.F. Brouillet E. Jenkins B.G. Ferrante R.J. Kowall N.W. Miller J.M. Storey E. Srivastava R. Rosen B.R. Hyman B.T. J. Neurosci. 1993; 13: 4181-4192Crossref PubMed Google Scholar), and antioxidants can attenuate the neurochemical changes and some behavioral disturbances caused by 3NP in animals (5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar, 7Matthews R.T. Yang L. Browne S. Baik M. Beal M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8892-8897Crossref PubMed Scopus (515) Google Scholar). Mitochondrial Ca2+ is another powerful signal for ROS production. Calcium is taken up into mitochondria via a uniporter in the IMM, and elevation of mitochondrial Ca2+ and ROS production is thought to play an important part in cell death associated with ischemia-reperfusion as well as 3NP (4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar). Increasing evidence suggests that ROS play a key role in promoting cytochrome c release from the mitochondria (8Petrosillo G. Ruggiero F.M. Pistolese M. Paradies G. FEBS Lett. 2001; 509: 435-438Crossref PubMed Scopus (204) Google Scholar, 9Nishimura G. Proske R.J. Doyama H. Higuchi M. FEBS Lett. 2001; 505: 399-404Crossref PubMed Scopus (39) Google Scholar, 10Petrosillo G. Ruggiero F.M. Paradies G. FASEB J. 2003; 17: 2202-2208Crossref PubMed Scopus (307) Google Scholar, 11Galindo M.F. Jordan J. Gonzalez-Garcia C. 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It is now believed that cytochrome c release from mitochondria proceeds by a two-step process: dissociation of cytochrome c from cardiolipin in the IMM, followed by release of cytochrome c through the outer mitochondrial membrane (OMM) (15Ott M. Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1259-1263Crossref PubMed Scopus (792) Google Scholar). Cardiolipin is rich in unsaturated fatty acids, and peroxidation of cardiolipin induces the dissociation of cytochrome c from mitochondria into the cytosol (16Shidoji Y. Hayashi K. Komura S. Ohishi N. Yagi K. Biochem. Biophys. Res. Commun. 1999; 264: 343-347Crossref PubMed Scopus (222) Google Scholar). However, the mechanism by which cytochrome c is released through the OMM is not clear. One mechanism may involve ROS-induced promotion of Ca2+-dependent mitochondrial permeability transition (MPT), with swelling of the mitochondrial matrix and rupture of the OMM (17Byrne A.M. Lemasters J.J. 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This mechanism seems likely in 3NP toxicity and ischemia-reperfusion injury, where increased intracellular Ca2+ and ROS are both present (4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar, 22Crompton M. Andreeva L. Basic Res. Cardiol. 1993; 88: 513-523Crossref PubMed Scopus (80) Google Scholar, 23Paradies G. Petrosillo G. Pistolese M. Di Venosa N. Serena D. Ruggiero F.M. Free Radic. Biol. Med. 1999; 27: 42-50Crossref PubMed Scopus (205) Google Scholar). However, there is also evidence showing that cytochrome c can be released through the OMM in an MPT-independent manner (24Jurgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1373) Google Scholar, 25Doran E. Halestrap A.P. Biochem. 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A. 2002; 99: 1259-1263Crossref PubMed Scopus (792) Google Scholar, 25Doran E. Halestrap A.P. Biochem. J. 2000; 348: 343-350Crossref PubMed Google Scholar, 29Madesh M. Hajnoczky G. J. Cell Biol. 2001; 155: 1003-1015Crossref PubMed Scopus (441) Google Scholar). Given the many ways by which cytochrome c may be released through the OMM, the most efficient approach to inhibit ROS-induced cytochrome c release and cell death would be prevention of lipid peroxidation of the IMM. Unfortunately, none of the available antioxidants specifically targets mitochondria, let alone the IMM. In addition, most of the antioxidants are poorly cell-permeable, requiring concentrations in excess of 100 μm to prevent oxidative cell death. One approach used to target antioxidants such as coenzyme Q and vitamin E to mitochondria has involved conjugation of these lipid-soluble molecules to lipophilic cations such as triphenylalkylphosphonium ions, which are rapidly taken up into the mitochondrial matrix because of the potential gradient across the IMM (30Smith R.A. Porteous C.M. Coulter C.V. Murphy M.P. Eur. J Biochem. 1999; 263: 709-716Crossref PubMed Scopus (416) Google Scholar, 31Kelso G.F. Porteous C.M. Coulter C.V. Hughes G. Porteous W.K. Ledgerwood E.C. Smith R.A. Murphy M.P. J. Biol. Chem. 2001; 276: 4588-4596Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). The introduction of cations into the mitochondrial matrix, however, leads to dissipation of IMM potential, and this was observed in isolated mitochondria with concentrations of triphenylalkylphosphonium ion-conjugated antioxidants greater than 20 μm (30Smith R.A. Porteous C.M. Coulter C.V. Murphy M.P. Eur. J Biochem. 1999; 263: 709-716Crossref PubMed Scopus (416) Google Scholar, 31Kelso G.F. Porteous C.M. Coulter C.V. Hughes G. Porteous W.K. Ledgerwood E.C. Smith R.A. Murphy M.P. J. Biol. Chem. 2001; 276: 4588-4596Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). Furthermore, dissipation of the IMM potential would ultimately limit further drug uptake. We have developed a series of peptide antioxidants that are taken up by mitochondria and concentrate in the IMM. These peptide antioxidants are cell-permeable and are very potent at reducing intracellular ROS and preventing cell death caused by the oxidant t-butylhydroperoxide (tBHP). We have used these IMM-targeted antioxidants to investigate the role of mitochondrially generated ROS in mitochondrial dysfunction in cells exposed to 3NP. To investigate the mechanisms by which these peptide antioxidants protect against mitochondrial dysfunction, we used isolated mitochondria to determine their ability to prevent MPT and cytochrome c release caused by Ca2+ overload and 3NP. In addition, because ROS have been implicated in contractile dysfunction associated with reperfusion of ischemic hearts, we determined the efficacy of these peptide antioxidants in preventing myocardial stunning in an ex vivo perfused heart model. Finally, to prove that the effects of these peptide antioxidants are caused by their ability to scavenge ROS, we designed a peptide analog that lacked antioxidant properties. Our results suggest that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT and rupture of the OMM. These results also confirm a major role for ROS in mitochondrial dysfunction and reperfusion injury and demonstrate the therapeutic potential of these peptide antioxidants in ischemia-reperfusion injury and neurodegeneration. Chemicals and Reagents—The SS peptides are tetrapeptides with alternating aromatic residues and basic amino acids. SS-02 (Dmt-d-Arg-Phe-Lys-NH2; Dmt = 2′,6′-dimethyltyrosine), SS-20 (Phe-d-Arg-Phe-Lys-NH2), SS-31 (d-Arg-Dmt-Lys-Phe-NH2), and [3H]SS-02 were synthesized as described previously (32Schiller P.W. Nguyen T.M. Berezowska I. Dupuis S. Weltrowska G. Chung N.N. Lemieux C. Eur. J. Med. Chem. 2000; 35: 895-901Crossref PubMed Scopus (147) Google Scholar, 33Zhao G.M. Qian X. Schiller P.W. Szeto H.H. J. Pharmacol. Exp. Ther. 2003; 307: 947-954Crossref PubMed Scopus (55) Google Scholar). A fluorescent analog (SS-19; Dmt-d-Arg-Phe-atnDap-NH2) containing β-anthraniloyl-l-α,β-diaminopropionic acid in place of the Lys4 residue in SS-02 was prepared for mitochondrial and cellular uptake studies (34Berezowska I. Chung N.N. Lemieux C. Zelent B. Szeto H.H. Schiller P.W. Peptides. 2003; 24: 1195-1200Crossref PubMed Scopus (25) Google Scholar). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Measurement of Antioxidant Properties of SS Peptides in Vitro—The ability of SS peptides to scavenge H2O2in vitro was determined using luminol chemiluminescence (35Li Y. Zhu H. Trush M.A. Biochim. Biophys. Acta. 1999; 1428: 1-12Crossref PubMed Scopus (128) Google Scholar). H2O2 (4.4 nmol) was incubated with 1 to 100 μm of various peptides in 0.5 ml of phosphate buffer, pH 8.0, for 30 s. Luminol (25 μm) and horseradish peroxidase (0.7 IU) were then added to the solution, and chemiluminescence was monitored with an aggregometer (Chronolog, Havertown, PA) for 20 min at 37 °C. Antioxidant properties of SS peptides were further established by inhibition of fatty acid peroxidation and low density lipoprotein (LDL) oxidation. Linoleic acid peroxidation was initiated with 2,2′-azobis(2-amidinopropane) and the formation of conjugated dienes was monitored spectrophotometrically at 234 nm (36Longoni B. Pryor W.A. Marchiafava P. Biochem. Biophys. Res. Commun. 1997; 233: 778-780Crossref PubMed Scopus (44) Google Scholar). Freshly prepared human LDL (0.1 mg/ml in phosphate-buffered saline) was oxidized catalytically by the addition of 10 μm CuSO4, and the formation of conjugated dienes was monitored at 234 nm for 5 h at 37 °C (37Moosmann B. Behl C. Mol. Pharmacol. 2002; 61: 260-268Crossref PubMed Scopus (103) Google Scholar). Mitochondrial Preparation—Male CD1 mice were sacrificed by decapitation, and the livers immediately excised and homogenized in ice-cold isolation buffer (10 mm sucrose, 200 mm mannitol, 5 mm HEPES, and 1 mm EGTA, pH 7.4) containing 1 mg/ml fatty acid-free bovine serum albumin. The homogenate was centrifuged for 10 min at 900 × g, and the supernatant was centrifuged again at 13,800 × g for 10 min. The mitochondrial pellets were washed twice, centrifuged at 11,200 × g, and re-suspended in the same buffer (no EGTA). All experiments were conducted in accordance with guidelines approved by the Institution for the Care and Use of Animals at Weill Medical College of Cornell University. Mitochondrial Uptake Studies—Uptake of SS-19 by isolated mitochondria was examined by fluorescence quenching upon addition of a mitochondrial suspension (0.35 mg) (Hitachi F-4500 fluorescence spectrophotometer; excitation/emission = 320/420 nm). For mitochondrial uptake of [3H]SS-02, mitochondria (0.8 mg) were suspended in buffer (70 mm sucrose, 230 mm mannitol, 3 mm HEPES, 5 mm succinate, 5 mm KH2PO4, and 0.5 μm rotenone, pH 7.4) containing [3H]SS-02 and 1 μm SS-02 at room temperature. Uptake was stopped by centrifugation (16,000 × g for 5 min at 4 °C), the mitochondrial pellet was washed twice and resuspended in 0.2 ml of 1% SDS/0.2 N NaOH, and radioactivity was determined. Mitochondrial uptake of SS-19 and [3H]SS-02 were also determined in the presence of 1.5 μm carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupler that results in mitochondrial depolarization. To determine the localization of the peptide within mitochondria, three cycles of freeze-thaw treatment were used to isolate inner and outer membranes (38Vardanis A. J. Biol. Chem. 1977; 252: 807-813Abstract Full Text PDF PubMed Google Scholar). Treatment with 0.2% digitonin was used to disrupt the outer membrane to determine peptide distribution to the IMM and matrix (39Greenawalt J.W. Methods Enzymol. 1974; 31: 310-323Crossref PubMed Scopus (245) Google Scholar). Cell Culture—Caco-2 cells (American Type Culture Collection, Manassas, VA) and N2A cells (provided by Dr. Gunnar Gouras, Department of Neurology, Weill Medical College of Cornell University) were cultured as described previously (40Zhao K. Luo G. Zhao G.M. Schiller P.W. Szeto H.H. J. Pharmacol. Exp. Ther. 2003; 304: 425-432Crossref PubMed Scopus (129) Google Scholar, 41Gouras G.K. Xu H. Gross R.S. Greenfield J.P. Hai B. Wang R. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1202-1205Crossref PubMed Scopus (275) Google Scholar). Cell culture supplies were obtained from Invitrogen. Cellular Uptake and Intracellular Localization of Peptide Antioxidants—Peptide uptake into Caco-2 cells was carried out as described previously (40Zhao K. Luo G. Zhao G.M. Schiller P.W. Szeto H.H. J. Pharmacol. Exp. Ther. 2003; 304: 425-432Crossref PubMed Scopus (129) Google Scholar). Cells (106/well) were incubated with [3H]SS-02 at 37 °C for 60 min, and radioactivity was determined in the medium and in cell lysate. To determine intracellular peptide localization, Caco-2 cells were incubated with SS-19 (0.1 μm) for 15 min at 37 °C, and confocal laser scanning microscopy (CLSM) was carried out with living cells using a C-Apochromat 63×/1.2 W Corr objective (Nikon, Tokyo, Japan) with excitation and emission wavelengths set at 320 and 420 nm, resepctively. To demonstrate localization of SS-19 to mitochondria, Caco-2 cells were incubated with SS-19 and Mitotracker tetramethylrhodamine methyl ester (TMRM; Molecular Probes, Portland, OR; excitation/emission = 550/575 nm) for 30 min at 37 °C and then examined by CLSM. Intracellular ROS and Cell Viability—N2A cells were plated in 96-well plates at a density of 1 × 104/well and allowed to grow for 2 days before treatment with tBHP (0.5 or 1 mm) for 40 min. Cells were washed twice and replaced with medium alone or medium containing varying concentrations of SS-02 or SS-31 for 4 h. Intracellular ROS was measured by 5-(and 6)-carboxy-2′,7′-dichlorohydro-fluorescein diacetate (Molecular Probes). Cell death was assessed by a cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay; Promega, Madison, WI). Intracellular Mitochondrial Potential—Caco-2 cells were treated with 3NP (10 mm) in the absence or presence of SS-02 (0.1 μm) for 4 h and then incubated with TMRM and examined under CLSM as described above. Mitochondrial H2O2Production—0.1 mg of mitochondrial protein was added to 0.5 ml of potassium phosphate buffer (100 mm, pH 8.0) containing 5 mm succinate; 25 μm luminol and 0.7 IU of horseradish peroxidase were added, and chemiluminescence was monitored continuously for 20 min at 37 °C. The amount of H2O2 produced was determined by area under the curve. Mitochondrial Oxygen Consumption—Mitochondrial protein (1 mg) was added to 2.0 ml of respiration buffer (70 mm sucrose, 230 mm mannitol, 2 mm HEPES, 5 mm KH2PO4, 5 mm MgCl2, and 0.5 mm EDTA, pH 7.4). Oxygen consumption was measured with a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK). Respiration was measured in the presence of 5 mm succinate, and state 3 respiration was initiated with the addition of 0.35 mm ADP. Mitochondrial Membrane Potential—Mitochondrial potential was qualitatively assessed using TMRM fluorescence intensity (excitation/emission = 550/575 nm). Isolated mitochondria (0.3 mg) were added to 2.0 ml of buffer (70 mm sucrose, 230 mm mannitol, 3 mm HEPES, 2 mm Tris-phosphate, 5 mm succinate, and 1 μm rotenone) containing TMRM (0.4–2 μm), and potential was assessed by quenching of the fluorescent signal. Mitochondrial Swelling Assays—Isolated mitochondria (0.1 mg) were added to 0.2 ml of buffer (70 mm sucrose, 230 mm mannitol, 3 mm HEPES, 2 mm Tris-phosphate, 5 mm succinate, and 1 μm rotenone) and swelling was measured by decrease in absorbance at 540 nm using a 96-well plate reader (Molecular Devices, Sunnyvale, CA). Mitochondrial Cytochrome c Release—Isolated mitochondria (0.75 mg/2 ml) were incubated in the absence or presence of SS-02 for 100 s before addition of Ca2+ to induce swelling. Swelling was measured by light scattering at 610 nm. Alamethicin (7 μg/ml) was added to induce maximal swelling, and the magnitude of swelling induced by Ca2+ was expressed as a percentage of maximal swelling. After incubation for 400 s, the mitochondrial pellet was collected by centrifugation. Cytochrome c content in the pellet and supernatant was determined using a commercial rat/mouse cytochrome c immunoassay kit (R & D Systems, Minneapolis, MN). Ischemia-Reperfusion Studies—Details of the isolated perfused guinea pig heart model have been published previously (42Wu D. Soong Y. Zhao G.M. Szeto H.H. Am. J. Physiol. 2002; 283: H783-H791Crossref PubMed Scopus (41) Google Scholar). Isolated hearts were perfused continuously with either Krebs-Henseleit solution or Krebs-Henseleit solution containing various SS peptides and allowed to stabilize for 30 min. Contractile force was measured with a small hook inserted into the apex of the left ventricle, and the silk ligature tightly connected to a Grass force-displacement transducer. Global ischemia was then induced by complete interruption of coronary perfusion for 30 min. Reperfusion was carried out for 90 min after ischemia. Antioxidant Properties of SS Peptides—The antioxidant properties of SS peptides were demonstrated by their ability to scavenge H2O2 and inhibit the oxidation of linoleic acid and LDL in vitro. The prototype peptide, SS-02, dose-dependently reduced the luminol-derived chemiluminescence produced by H2O2 in the presence of horseradish peroxidase (Fig. 1A). SS-02 also dose-dependently inhibited the oxidation of fatty acids (Fig. 1B) and LDL in vitro (Fig. 1C). The antioxidant activity of SS-02 was not dependent on the specific order of the four amino acids in that SS-31 showed similar antioxidant activity (Fig. 1, D and E). However, substitution of Dmt1 by Phe1 (SS-20) eliminated antioxidant activity (Fig. 1, D and E). Cellular Uptake of SS-02—To demonstrate that the SS peptides are cell-permeable, we incubated Caco-2 cells with [3H]SS-02 at 37 °C for 60 min and measured the amount of radioactivity in cell lysate. [3H]SS-02 was readily taken up into Caco-2 cells. The amount of [3H]SS-02 in cell lysate and media averaged 6152 ± 128 cpm and 229,622 ± 2199, respectively (mean ± S.E.; n = 6). Based on a cell volume of ∼3.3 μl/mg protein (43Terada T. Sawada K. Saito H. Hashimoto Y. Inui K. Am. J. Physiol. 1999; 276: G1435-G1441PubMed Google Scholar) and 200 μl of media, the intracellular concentration of [3H]SS-02 can be estimated to be 9.8 ± 0.26 times higher than extracellular concentration. Intracellular Targeting of SS Peptides—The fluorescent analog SS-19 was used to determine cellular uptake and intracellular localization by CLSM. The confocal images showed uptake of SS-19 (blue fluorescence) into Caco-2 cells within 15 min (Fig. 2, left). Fluorescence was detected in the cytoplasm of all cells, but the peptide was entirely excluded from the nucleus. The distribution pattern resembled mitochondrial distribution as shown by Mitotracker TMRM (Fig. 2, middle). The co-localization of SS-19 and TMRM (Fig. 2, right) suggests targeting of SS-19 to mitochondria after cellular uptake. Mitochondrial Uptake of SS Peptides—Mitochondrial uptake of SS peptides was examined using isolated mouse liver mitochondria. Addition of isolated mitochondria to SS-19 resulted in immediate quenching of the fluorescent signal (Fig. 3A). To ensure that the uptake of SS-19 by mitochondria was not an artifact of the fluorophore, we incubated mitochondria with [3H]SS-02 and determined radioactivity in the mitochondrial pellet. Uptake of [3H]SS-02 by mitochondria was rapid with maximal levels reached by 2 min (Fig. 3B). Radioactivity averaged 67,021 ± 2008 cpm in the mitochondrial pellet, and 128,131 ± 2015 cpm in the supernatant (n = 3). Assuming mitochondrial volume of 1 μl/mg protein (44Lim K.H. Javadov S.A. Das M. Clarke S.J. Suleiman M.S. Halestrap A.P. J. Physiol. 2002; 545: 961-974Crossref PubMed Scopus (203) Google Scholar), it can be estimated that [3H]SS-02 accumulates 104.6 ± 1.6-fold in mitochondria. Pretreatment of mitochondria with FCCP only reduced SS-19 quenching or [3H]SS-02 uptake by ∼20% (Fig. 3, A and B), suggesting that only 20% of this cationic peptide was targeted into the mitochondrial matrix in a potential-dependent manner. When mitochondria were incubated with [3H]SS-02 for 5 min and the mitochondrial pellet was subjected to three freeze-thaw cycles, 72% of [3H]SS-02 was retained in the membrane pellet consisting of both IMM and OMM (Fig. 3C). Treatment of the mitochondrial suspension with 1% digitonin to disrupt the OMM allowed us to determine that 85% of the radioactivity was in the mitoplast (IMM and matrix) (Fig. 3C). These results suggest that the peptides are predominantly targeted to the IMM. SS Peptides Reduce Intracellular ROS and Cell Death Caused by tBHP—To show that SS peptides are effective when applied to whole cells, neuronal N2A cells were treated with tBHP (0.5 or 1.0 mm) for 40 min, washed, and then incubated with media containing SS-02 or SS-31, or media alone for 4 h. Incubation with tBHP resulted in dose-dependent increase in intracellular ROS and decrease in cell viability (Fig. 4). Incubation of these cells with either SS-31 or SS-02 dose-dependently reduced intracellular ROS (Fig. 4A) and increased cell survival (Fig. 4, B and C), with EC50 in the nanomolar range. SS-02 Protects against 3NP-induced Mitochondrial Depolarization in Caco-2 Cells—To demonstrate that reduction in mitochondrially generated ROS can protect against mitochondrial dysfunction, we examined the effect of SS-02 on mitochondrial depolarization caused by treatment of cells with 3NP. Caco-2 cells were treated with 10 mm 3NP in the absence or presence of 0.1 μm SS-02, and mitochondrial potential was visualized by confocal mi