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• W1978791657 abstract "The mitochondrial permeability transition pore (PTP) and associated release of cytochrome c are thought to be important in the apoptotic process. Nitric oxide (NO⋅) has been reported to inhibit apoptosis by acting on a variety of extra-mitochondrial targets. The relationship between cytochromec release and PTP opening, and the effects of NO⋅are not clearly established. Nitric oxide, S-nitrosothiols and peroxynitrite are reported to variously inhibit or promote PTP opening. In this study the effects of NO⋅ on the PTP were characterized by exposing isolated rat liver mitochondria to physiological and pathological rates of NO⋅ released from NONOate NO⋅donors. Nitric oxide reversibly inhibited PTP opening with an IC50 of 11 nm NO⋅/s, which can be readily achieved in vivo by NO⋅ synthases. The mechanism involved mitochondrial membrane depolarization and inhibition of Ca2+ accumulation. At supraphysiological release rates (>2 μm/s) NO⋅ accelerated PTP opening. Substantial cytochrome c release occurred with only a 20% change in mitochondrial swelling, was an early event in the PTP, and was also inhibited by NO⋅. Furthermore, NO⋅ exposure resulted in significantly lower cytochrome c release for the same degree of PTP opening. It is proposed that this pathway represents an additional mechanism underlying the antiapoptotic effects of NO⋅. The mitochondrial permeability transition pore (PTP) and associated release of cytochrome c are thought to be important in the apoptotic process. Nitric oxide (NO⋅) has been reported to inhibit apoptosis by acting on a variety of extra-mitochondrial targets. The relationship between cytochromec release and PTP opening, and the effects of NO⋅are not clearly established. Nitric oxide, S-nitrosothiols and peroxynitrite are reported to variously inhibit or promote PTP opening. In this study the effects of NO⋅ on the PTP were characterized by exposing isolated rat liver mitochondria to physiological and pathological rates of NO⋅ released from NONOate NO⋅donors. Nitric oxide reversibly inhibited PTP opening with an IC50 of 11 nm NO⋅/s, which can be readily achieved in vivo by NO⋅ synthases. The mechanism involved mitochondrial membrane depolarization and inhibition of Ca2+ accumulation. At supraphysiological release rates (>2 μm/s) NO⋅ accelerated PTP opening. Substantial cytochrome c release occurred with only a 20% change in mitochondrial swelling, was an early event in the PTP, and was also inhibited by NO⋅. Furthermore, NO⋅ exposure resulted in significantly lower cytochrome c release for the same degree of PTP opening. It is proposed that this pathway represents an additional mechanism underlying the antiapoptotic effects of NO⋅. nitric oxide 2-(N,N-1-diethylamino)-diazenolate-2-oxide (Z)-1-[2-(2-aminoethyl)-N-(ammonio-ethyl)amino]diaze-1-ium-1,2-diolate carbonyl-cyanide-p-(trifluoromethoxy)-phenylhydrazine oxyhemoglobin permeability transition pore tert-butyl hydroperoxide matrix-assisted laser-desorption time-of-flight triphenylmethylphosphonium A recent addition to the signal transduction pathways that can be modulated by nitric oxide (NO⋅)1 is apoptosis, with both pro- and antiapoptotic effects reported depending on both cell type and NO⋅ concentration (reviewed in Refs. 1.Brune B. von Knethen A. Sandau K. Cell Death Differ. 1999; 6: 969-975Crossref PubMed Scopus (263) Google Scholar and 2.Kim Y-M. Bombeck C.A. Billiar T.R. Circ. Res. 1999; 84: 253-2563Crossref PubMed Scopus (381) Google Scholar). Antiapoptotic effects are associated with low levels (10 nmto 1 μm) of exposure from the activation of endogenous NO⋅ synthases and slow release rates from NO⋅ donors (3.Kim Y-M. Kim T-H. Seol D.W. Talanian R.V. Billiar T.R. J. Biol. Chem. 1998; 273: 31437-31441Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 4.Kim Y-M. Talanian R.V. Billiar T.R. J. Biol. Chem. 1997; 272: 31138-31148Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar, 5.Li J. Bombeck C.A. Yang S. Kim Y-M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 6.Mohr S. Zech B. Lapetina E.G. Brune B. Biochem. Biophys. Res. Commun. 1997; 238: 387-391Crossref PubMed Scopus (202) Google Scholar, 7.Melino G. Bernassola F. Knight R.A. Corasaniti M.T. Nistico G. Finazzi-Agro A. Nature. 1997; 388: 432-433Crossref PubMed Scopus (372) Google Scholar, 8.Rossig L. Fichrlscherer B. Breitschopf K. Haendeler J. Zeiher A.M. Mulsch A. Dimmeler S. J. Biol. Chem. 1999; 274: 6823-6826Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 9.Li J. Billiar T.R. Talanian R.V. Kim Y.M. Biochem. Biophys. Res. Commun. 1997; 240: 419-424Crossref PubMed Scopus (472) Google Scholar, 10.Lui L. Stamler J.S. Cell Death Differ. 1999; 10: 937-942Google Scholar, 11.Beauvais F. Michel L. Dubertret L. FEBS Lett. 1995; 361: 229-232Crossref PubMed Scopus (122) Google Scholar, 12.Shen Y.H. Wang X.L. Wilcken D.E. FEBS Lett. 1998; 433: 125-131Crossref PubMed Scopus (155) Google Scholar). Specific molecular targets include inhibition of Bcl-2 cleavage (3.Kim Y-M. Kim T-H. Seol D.W. Talanian R.V. Billiar T.R. J. Biol. Chem. 1998; 273: 31437-31441Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), inactivation of caspases by S-nitrosation (4.Kim Y-M. Talanian R.V. Billiar T.R. J. Biol. Chem. 1997; 272: 31138-31148Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar, 5.Li J. Bombeck C.A. Yang S. Kim Y-M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 6.Mohr S. Zech B. Lapetina E.G. Brune B. Biochem. Biophys. Res. Commun. 1997; 238: 387-391Crossref PubMed Scopus (202) Google Scholar, 7.Melino G. Bernassola F. Knight R.A. Corasaniti M.T. Nistico G. Finazzi-Agro A. Nature. 1997; 388: 432-433Crossref PubMed Scopus (372) Google Scholar, 8.Rossig L. Fichrlscherer B. Breitschopf K. Haendeler J. Zeiher A.M. Mulsch A. Dimmeler S. J. Biol. Chem. 1999; 274: 6823-6826Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 9.Li J. Billiar T.R. Talanian R.V. Kim Y.M. Biochem. Biophys. Res. Commun. 1997; 240: 419-424Crossref PubMed Scopus (472) Google Scholar, 10.Lui L. Stamler J.S. Cell Death Differ. 1999; 10: 937-942Google Scholar), and cGMP-mediated effects (11.Beauvais F. Michel L. Dubertret L. FEBS Lett. 1995; 361: 229-232Crossref PubMed Scopus (122) Google Scholar, 12.Shen Y.H. Wang X.L. Wilcken D.E. FEBS Lett. 1998; 433: 125-131Crossref PubMed Scopus (155) Google Scholar). However, exposure of cells to high NO⋅ concentrations (>1 μm) results in extensive inhibition of mitochondrial ATP synthesis (13.Cleeter M.W.J. Cooper J.M. Darley-Usmar V.M. Moncada S. Schapira A.H.V. FEBS Lett. 1994; 345: 50-54Crossref PubMed Scopus (1141) Google Scholar, 14.Brookes P.S. Bolanos J.P. Heales S.J.R. FEBS Lett. 1999; 446: 261-263Crossref PubMed Scopus (79) Google Scholar, 15.Ereciñska M. Nelson D. Vanderkooi J.M. J. Neurochem. 1995; 65: 2699-2705Crossref PubMed Scopus (37) Google Scholar), and while apoptosis is inhibited, necrotic cell death results (16.Leist M. Single B. Naumann H. Fava E. Simon B. Kuhnle S. Nicotera P. Biochem. Biophys. Res. Commun. 1999; 258: 215-221Crossref PubMed Scopus (55) Google Scholar, 17.Leist M. Single B. Naumann H. Fava E. Simon B. Kuhnle S. Nicotera P. Exp. Cell. Res. 1999; 249: 396-403Crossref PubMed Scopus (245) Google Scholar). This is consistent with the greater ATP dependence of apoptosis compared with necrosis (16.Leist M. Single B. Naumann H. Fava E. Simon B. Kuhnle S. Nicotera P. Biochem. Biophys. Res. Commun. 1999; 258: 215-221Crossref PubMed Scopus (55) Google Scholar, 17.Leist M. Single B. Naumann H. Fava E. Simon B. Kuhnle S. Nicotera P. Exp. Cell. Res. 1999; 249: 396-403Crossref PubMed Scopus (245) Google Scholar). Under some conditions, such as inflammation or neurodegenerative diseases, NO⋅-dependent apoptosis has been observed (18.Bosca L. Hortelano S. Cell. Signal. 1999; 11: 239-244Crossref PubMed Scopus (112) Google Scholar, 19.Brune B. Sandau K. von Knethen A. Biochemistry (Moscow). 1998; 63: 817-825PubMed Google Scholar, 20.Sarih M. Souvannavong V. Adam A. Biochem. Biophys. Res. Commun. 1993; 191: 503-508Crossref PubMed Scopus (351) Google Scholar, 21.Fukuo K. Hata S. Suhara T. Nakahashi T. Shinto Y. Tsujimoto Y. Morimoto S. Ogihara T. Hypertension. 1996; 27: 823-826Crossref PubMed Google Scholar, 22.Estevez A.G. Radi R. Barbeito L. Shin J.T. Thompson J.A. Beckman J.S. J. Neurochem. 1995; 65: 1543-1550Crossref PubMed Scopus (287) Google Scholar, 23.Salgo M.G. Bermudez E. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 500-505Crossref PubMed Scopus (256) Google Scholar, 24.Lin K-T. Xue J-Y. Nomen M. Spur B. Wong P.Y-K. J. Biol. Chem. 1995; 270: 16487-16490Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). In this case, the hypothesis that NO⋅ reacts with superoxide to form peroxynitrite (25.Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6647) Google Scholar) has been suggested as a mechanism leading to apoptotic cell death (22.Estevez A.G. Radi R. Barbeito L. Shin J.T. Thompson J.A. Beckman J.S. J. Neurochem. 1995; 65: 1543-1550Crossref PubMed Scopus (287) Google Scholar, 23.Salgo M.G. Bermudez E. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 500-505Crossref PubMed Scopus (256) Google Scholar, 24.Lin K-T. Xue J-Y. Nomen M. Spur B. Wong P.Y-K. J. Biol. Chem. 1995; 270: 16487-16490Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). It is clear from these observations that a number of molecular mechanisms underlie the effects of NO⋅ on apoptosis. A site for NO⋅ modulation of the apoptotic process that has received little attention is the controlled release of cytochromec from mitochondria. This is important, since it is becoming increasingly apparent that mitochondria are important mediators of apoptosis (26.Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4405) Google Scholar, 27.Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1543Crossref PubMed Scopus (1259) Google Scholar, 28.Zamzami N. Marchetti P. Castedo M. Hirsch T. Susin S.A. Masse B. Kroemer G. FEBS Lett. 1996; 384: 53-574Crossref PubMed Scopus (387) Google Scholar, 29.Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1771) Google Scholar), and is possibly related to recent evidence for a mitochondrial NO⋅ synthase that would produce NO⋅ within the organelle (30.Tatoyan A Guilivi C. J. Biol. Chem. 1998; 273: 11044-11-48Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 31.Ghafourifar P. Richter C. Biol. Chem. 1999; 380: 1025-1028Crossref PubMed Scopus (66) Google Scholar). The release of cytochrome c and apoptosis-inducing factor from mitochondria leads to activation of caspases 9 and 3 (29.Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1771) Google Scholar), and it was recently reported that caspase 3 can activate caspase 8, which can in turn trigger cytochrome crelease (32.Bossy-Wetzel E. Green D.R. J. Biol. Chem. 1999; 274: 17484-17490Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). These findings suggest an amplification role for mitochondria in apoptotic signaling and support the concept that the control of cytochrome c release from the mitochondrion is likely to impact the progression of apoptosis. Mitochondrial cytochrome c release is regulated by the Bcl-2 family of proteins (33.Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (628) Google Scholar), which are targeted at the mitochondrial permeability transition pore (PTP). This multisubunit protein complex includes the mitochondrial voltage-dependent anion channel, the adenine nucleotide translocase, and cyclophilin D (33.Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (628) Google Scholar, 34.Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2097) Google Scholar). Consistent with the molecular composition of the PTP, mitochondrial cytochrome c release is triggered by PTP inducers (Ca2+, phosphate, oxidants) and inhibited by PTP inhibitors (cyclosporin A, bongkrekic acid, EGTA). Recently, it was demonstrated that reconstituted PTP components (voltage-dependent anion channel, adenine nucleotide translocase, cyclophilin D) can regulate cytochrome c release from liposomes (35.Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1898) Google Scholar). The reported effects of NO⋅ on the mitochondrial PTP appear to be inconsistent. In some studies, mitochondria were exposed toS-nitrosothiols or high concentrations of NO⋅(0.6–10 μm), and PTP opening was observed (36.Balakirev M.Y. Khramtsov V.V. Zimmer G. Eur. J. Biochem. 1997; 246: 710-718Crossref PubMed Scopus (96) Google Scholar, 37.Borutaite V. Morkuniene R. Brown G.C. FEBS Lett. 2000; 467: 155-159Crossref PubMed Scopus (65) Google Scholar). It has been suggested that these effects were not mediated by NO⋅but by other reactive nitrogen species such as peroxynitrite (22.Estevez A.G. Radi R. Barbeito L. Shin J.T. Thompson J.A. Beckman J.S. J. Neurochem. 1995; 65: 1543-1550Crossref PubMed Scopus (287) Google Scholar, 23.Salgo M.G. Bermudez E. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 500-505Crossref PubMed Scopus (256) Google Scholar, 24.Lin K-T. Xue J-Y. Nomen M. Spur B. Wong P.Y-K. J. Biol. Chem. 1995; 270: 16487-16490Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 25.Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6647) Google Scholar,38.Packer M.A. Murphy M.P. FEBS Lett. 1994; 345: 237-240Crossref PubMed Scopus (144) Google Scholar) or by S-nitrosation of mitochondrial proteins (37.Borutaite V. Morkuniene R. Brown G.C. FEBS Lett. 2000; 467: 155-159Crossref PubMed Scopus (65) Google Scholar). In contrast, it was also suggested that NO⋅ may inhibit PTP in a subpopulation of mitochondria, but the effects on cytochromec release were not reported (36.Balakirev M.Y. Khramtsov V.V. Zimmer G. Eur. J. Biochem. 1997; 246: 710-718Crossref PubMed Scopus (96) Google Scholar). Two major sites of interaction of NO⋅ with mitochondria in the submicromolar concentration range have been identified at complex III and the oxygen binding site of cytochrome c oxidase (13.Cleeter M.W.J. Cooper J.M. Darley-Usmar V.M. Moncada S. Schapira A.H.V. FEBS Lett. 1994; 345: 50-54Crossref PubMed Scopus (1141) Google Scholar, 39.Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Through inhibition of electron transport at these sites and a decrease in mitochondrial membrane potential, it has been observed that NO⋅stimulates mitochondrial Ca2+ efflux (40.Schweizer M. Richter C. Biochem. Biophys. Res. Commun. 1994; 204: 169-175Crossref PubMed Scopus (334) Google Scholar). In addition, inhibition of the putative mitochondrial NOS facilitates Ca2+ accumulation, consistent with either NO⋅-dependent prevention of Ca2+ uptake or enhanced release (31.Ghafourifar P. Richter C. Biol. Chem. 1999; 380: 1025-1028Crossref PubMed Scopus (66) Google Scholar). These observations are significant in the context of control of cytochrome c release, since the PTP is initiated by an increase in the intramitochondrial Ca2+concentration, and predict that NO⋅ should inhibit PTP opening (34.Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2097) Google Scholar). From the current literature, it is evident that the effects of NO⋅ per se, and of varying NO⋅ concentrations at the level of the isolated mitochondrion, on PTP and cytochromec release are uncertain. It was hypothesized that low rates of NO⋅ formation could inhibit Ca2+ accumulation and thus inhibit subsequent PTP opening and cytochrome c release from isolated mitochondria. This hypothesis was tested by exposing isolated rat liver mitochondria to NO⋅ released from NONOate compounds at rates encompassing both the physiological and pathological ranges, and the effects on PTP opening and cytochrome crelease were determined. Male Harlan Sprague-Dawley rats, 250–300 g in weight, were handled in accordance with recommendations in Ref. 48.National Institutes of HealthThe Guide for the Care and Use of Laboratory Animals, DHEW Publication NIH85–23. National Institutes of Health, Bethesda, MD1996Google Scholar. Food and water were available ad libitum. All biochemicals were from Sigma except cyclosporin A and NONOate compounds (Alexis, San Diego CA), antibodies (Pharmingen, San Diego CA), and chemiluminescence reagents (Amersham Pharmacia Biotech). Stock solutions of NONOates were prepared in 10 mm NaOH and stored frozen. Their degradation, assayed spectrophotometrically at 251 nm, was not significant over the course of 1 week. Liver mitochondria were isolated according to standard procedures (41.Rickwood D. Wilson M.T. Darley-Usmar V.M. Darley Usmar V.M. Rickwood D. Wilson M.T. Mitochondria: A Practical Approach. IRL Press, Oxford1987: 3-5Google Scholar) in buffer containing sucrose (250 mm), Tris (10 mm), and EGTA (2 mm), pH 7.4, at 4 °C. The final centrifugation step and resuspension were performed in EGTA-free medium. Protein was determined using the Folin-phenol reagent (42.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) against a standard curve constructed using bovine serum albumin. Mitochondrial PTP opening was assayed essentially as described by Packer and Murphy (38.Packer M.A. Murphy M.P. FEBS Lett. 1994; 345: 237-240Crossref PubMed Scopus (144) Google Scholar). Opening of the PTP causes mitochondrial swelling that is conveniently assayed as a decrease in the light scattering (and thus absorbance) of a mitochondrial suspension. Absorbance at 540 nm was measured using a Gilford ResponseTM spectrophotometer with a 37 °C thermostatic chamber. Mitochondria (1 mg of protein) were suspended in polystyrene cuvettes in 1 ml of buffer containing HEPES (40 mm), mannitol (195 mm), sucrose (25 mm), succinate (5 mm), and rotenone (1 μm), pH 7.2. After a 2-min equilibration period, CaCl2 (60 μm) was added, and absorbance was monitored for 20 min. NO⋅ donors (1–5 μl of stock) were added 20 s before mitochondria unless otherwise indicated. The addition of NO⋅donors in NaOH did not significantly alter the pH of the buffer, and 5 μl NaOH alone had no effect on swelling (result not shown). Half-lives of NONOate compounds in the buffer system used in these experiments were as follows: DEA NONOate, 4.29 min; spermine NONOate, 202 min; DETA NONOate, 1600 min. These values differ from those published by the manufacturer and highlight the importance of determining decomposition characteristics of these compounds in each experimental system studied. Rates of NO⋅ release were determined by calculation from half-lives and polarographically using an NO⋅ electrode (WPI, Sarasota, FL), both methods yielding indistinguishable results. In the open cuvette system employed in these studies, a linear relationship was observed between the rate of NO⋅ release and the steady state concentration achieved, such that 15 nm NO⋅/s equated to a steady state concentration of 1 μm. Beyond 100 nmNO⋅/s, this relationship became nonlinear. At various time points during swelling experiments, cyclosporin A (5 μm) and EGTA (1 mm) were added to mitochondrial suspensions to prevent further swelling, and 900-μl aliquots were centrifuged at 14,000 × g for 15 min. Supernatants were snap-frozen (liquid N2) and later analyzed by Western blotting. Samples were run on 15% SDS-polyacrylamide gels (43.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) and electroblotted to nitrocellulose membranes (44.Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4534Crossref PubMed Scopus (44644) Google Scholar). Cytochrome c was detected using a mouse anti-cytochrome c IgG (Pharmingen), followed by a horseradish peroxidase-linked secondary antibody and ECL detection (both from Amersham Pharmacia Biotech). Supernatant samples were also analyzed by MALDI-TOF mass spectrometry in the mass spectrometry core facility at the University of Alabama at Birmingham, following a 10-fold concentration step using CentriconTM filtration devices (Millipore Corp., Bedford MA; 3000 molecular weight cut-off). The mass spectrometer was calibrated using apomyoglobin. Cytochromec was not detectable by spectrophotometric methods (reduced/oxidized A 550) in these supernatants but was detectable in samples only centrifuged for 3 min, highlighting the importance of complete centrifugation in such experiments to avoid artifacts from nonpelleted mitochondria. Mitochondria were incubated as for swelling experiments but at a lower protein concentration (0.25 mg/ml) and in the presence of the non-membrane-permeant Ca2+-sensitive dye Arsenazo III (100 μm), which measures extramitochondrial Ca2+(38.Packer M.A. Murphy M.P. FEBS Lett. 1994; 345: 237-240Crossref PubMed Scopus (144) Google Scholar). The difference in absorbance between 675 and 685 nm was measured using a Beckman DU700 diode array spectrophotometer. Optionally present were spermine NONOate (2.2 mm) or ruthenium red (10 μm). Readings were taken every 1 s, and data were smoothed using a 4-s moving integration window. Mitochondria were incubated in the same buffer used for swelling experiments, in an open chamber fitted with an electrode sensitive to the lipophilic cation triphenylmethylphosphonium (TPMP+) (45.Brand M.D. Brown G.C. Cooper C.E. Bioenergetics: A Practical Approach. IRL Press, Oxford1995: 39-62Google Scholar). Nigericin was present at 100 nm. Four TPMP+ additions (final concentration 4 μm) were made to calibrate the electrode, followed by succinate (5 mm) to energize mitochondria and then FCCP to uncouple and correct for electrode drift. Membrane potential was calculated from the accumulation ratio of TPMP+ using the Nernst equation and a previously determined TPMP+ binding correction (45.Brand M.D. Brown G.C. Cooper C.E. Bioenergetics: A Practical Approach. IRL Press, Oxford1995: 39-62Google Scholar). Readings were taken every 1 s, and data were smoothed using a 4-s moving integration window. Fig. 1 A shows the effects of a variety of NO⋅ donors on the Ca2+-induced swelling of rat liver mitochondria. Swelling was inhibited by NO⋅ and also by cyclosporin A and was completely abolished by omission of Ca2+ from the incubation, indicating that the phenomenon being observed was indeed PTP opening. Fig. 1 Bshows a dose response of the PTP to the effects of NO⋅ released from the 3 NONOates. The maximal inhibition of PTP by NO⋅ was comparable with or greater than that achievable by cyclosporin A (Fig.1 A). The NO⋅ release rate that gave 50% inhibition of PTP was ∼11 nm NO⋅/s, equating to a steady state concentration of ∼0.7 μm. Data from all three NO⋅ donors when normalized for NO⋅ release rate were indistinguishable, indicating that the effects were specific to NO⋅ and not due to the parent compounds. Fig. 2 A shows the effects of the NO⋅ donor spermine NONOate on mitochondrial Ca2+uptake measured by the Arsenazo III method (38.Packer M.A. Murphy M.P. FEBS Lett. 1994; 345: 237-240Crossref PubMed Scopus (144) Google Scholar). The addition of CaCl2 to the incubation caused a rapid increase in the Ca2+ signal that progressively decreased as the Ca2+ was taken up by the mitochondria and was reversed by the uncoupler FCCP. This phenomenon was ruthenium red-sensitive, indicating Ca2+ entry to the mitochondrial matrix via the Ca2+ uniporter. The presence of spermine NONOate at a concentration that inhibits PTP opening also completely prevented Ca2+ uptake. In a second series of experiments, the effects of NO⋅ on Δψ were determined using TPMP+ (45.Brand M.D. Brown G.C. Cooper C.E. Bioenergetics: A Practical Approach. IRL Press, Oxford1995: 39-62Google Scholar). Development of Δψ results in accumulation of TPMP+ by mitochondria and a decreased signal from the TPMP+electrode. Consistent with the literature (40.Schweizer M. Richter C. Biochem. Biophys. Res. Commun. 1994; 204: 169-175Crossref PubMed Scopus (334) Google Scholar), Fig. 2 Bshows that the presence of NO⋅ prevented development of Δψ upon the addition of succinate as substrate. These data support the hypothesis that NO⋅ inhibits the PTP by preventing membrane potential-driven Ca2+ accumulation. To establish that inhibition of PTP opening required the continuous generation of NO⋅, the effects of oxyhemoglobin (oxyHb) were examined. Fig. 3 A shows that the addition of oxyHb to mitochondria incubated with Ca2+plus NO⋅ results in a complete and immediate reversal of the NO⋅-dependent inhibition of PTP opening. Oxyhemoglobin alone had no effect on the PTP. In a further series of experiments, the ability of NO⋅ to inhibit PTP triggered by oxidants was examined. Fig. 3 B shows that the well characterized induction of the PTP by Ca2+ plustert-butyl hydroperoxide can also be inhibited by NO⋅. Similar results were obtained when Ca2+ plus phosphate was used to induce PTP (not shown). Fig. 4 (A and B) shows the results of a Western blot for cytochrome c in supernatants from PTP experiments. Calcium-treated mitochondria (lane 1) released cytochrome c, and NO⋅ inhibited this by 80% (lane 2). Untreated mitochondria (lane 3) did not release cytochrome c; nor did those treated with Ca2+plus cyclosporin-A (not shown). To examine the release of proteins from mitochondria that cannot be detected by Western blotting, concentrated supernatants were also analyzed by MALDI-TOF mass spectrometry. Fig.4 C shows typical mass spectra from the same postmitochondrial supernatants as in Fig. 4 A. In agreement with the Western blot data, these spectra indicate that NO⋅inhibited cytochrome c release (12.1-kDa peak). The release of other proteins upon PTP opening (e.g. the peak at 10.8 kDa) was also inhibited by NO⋅. The amount of protein released from untreated mitochondria was very low, indicating minimal nonspecific leakage of proteins during the time course of these experiments. In a cell system, complete inhibition of the PTP in all mitochondria within a cell could only occur if high concentrations of NO⋅were present. However, this could also result in inhibition of ATP synthesis and would ultimately be cytotoxic. In a cytoprotective role, a partial inhibition of the PTP by NO⋅ is envisaged, but it is unclear whether this would significantly impact on cytochromec release. This was examined in the next series of experiments. Fig. 5 A shows the time course of cytochrome c release during Ca2+-induced PTP opening. Superimposed is the swelling profile for these mitochondria. It is immediately apparent that ∼80% of cytochrome c is released within ∼1 min of Ca2+ addition and prior to the rapid phase of swelling. This suggests that cytochrome c release is an early event in Ca2+-induced PTP. To further determine the effects of NO⋅ on the relationship between PTP and cytochrome c release, the effect of adding NO⋅ at different times during swelling was examined. Fig.5 B shows that the addition of spermine NONOate before mitochondria or at the same time as Ca2+ inhibited the PTP, whereas the addition on the verge of the rapid phase of swelling was ineffective. A partial inhibition of swelling resulted if NO⋅was added 2 min after Ca2+, at a point after which essentially all cytochrome c had been released (Fig.5 A). This is confirmed in the inset to Fig.5 B, which shows the effect of time of the addition of NO⋅ on cytochrome c release. The relationship between the degree of swelling and cytochromec release in the presence and absence of NO⋅ is shown in Fig. 6. These data again demonstrate that in the absence of NO⋅ most cytochrome c release occurs with only moderate (25%) swelling. However, when NO⋅partially inhibits the PTP, this relationship is modified such that a much greater degree of swelling (∼80%) is required to elicit substantial release of cytochrome c. Release of mitochondrial cytochrome c to the cytosol through mechanisms related to opening of the PTP has been intensively investigated. It is well recognized that the PTP can play a central role in apoptosis (27.Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1543Crossref PubMed Scopus (1259) Google Scholar, 28.Zamzami N. Marchetti P. Castedo M. Hirsch T. Susin S.A. Masse B. Kroemer G. FEBS Lett. 1996; 384: 53-574Crossref PubMed Scopus (387) Google Scholar, 33.Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (628) Google Scholar, 35.Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1898) Google Scholar) and that NO⋅ has specific interactions with mitochondria at the level of both cytochromec oxidase and complex III (13.Cleeter M.W.J. Cooper J.M. Darley-Usmar V.M. Moncada S. Schapira A.H.V. FEBS Lett. 1994; 345: 50-54Crossref PubMed Scopus (1141) Google Scholar, 14.Brookes P.S. Bolanos J.P. Heales S.J.R. FEBS Lett. 1999; 446: 261-263Crossref PubMed Scopus (79) Google Scholar, 15.Ereciñska M. Nelson D. Vanderkooi J.M. J. Neurochem. 1995; 65: 2699-2705Crossref PubMed Scopus (37) Google Scholar, 39.Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Since it is also known that NO⋅ can modulate apoptosis, it was hypothesized that the mechanism may be operative at the level of the mitochondrial PTP and cytochrome c release. However, the literature is far from clear on the basic elements of this hypothesis, including an examination of the effects of NO⋅ release rate on the PTP and the temporal relationship between cytochrome c release and PTP opening. This is important, since in a cellular setting gradients of NO⋅ and varying rates and sites of formation are likely to result in partial modulatory effects on mitochondrial function. These issues were addressed in isolated mitochondria using biologically relevant rates of NO⋅ formation, and effects on PTP and cytochrome c release were determined. When administered at the start of incubations (∼20 s before mitochondria), NO⋅ was able to inhibit PTP opening at rates of NO⋅ release compatible with the physiologic range achievable by NO⋅ synthases (Fig. 1) (46.Patel R.P. McAndrew J. Sellak H. White C.R. Jo H. Freeman B.A. Darley-Usmar V.M. Biochim. Biophys. Acta. 1999; 1411: 385-400Crossref PubMed Scopus (396) Google Scholar). This suggests that the PTP may be a target for the antiapoptotic effects of physiological levels of NO⋅. At high release rates (>2 μm/s), NO⋅restored PTP opening to control levels and, in agreement with a previous study (36.Balakirev M.Y. Khramtsov V.V. Zimmer G. Eur. J. Biochem. 1997; 246: 710-718Crossref PubMed Scopus (96) Google Scholar), shortened the delay to the onset of PTP following the Ca2+ addition (not shown). However, such concentrations of NO⋅ are in excess of those expected to be encounteredin vivo, even under pathologic conditions with maximal stimulation of iNOS (46.Patel R.P. McAndrew J. Sellak H. White C.R. Jo H. Freeman B.A. Darley-Usmar V.M. Biochim. Biophys. Acta. 1999; 1411: 385-400Crossref PubMed Scopus (396) Google Scholar). It is likely that at such high concentrations NO⋅ may form S-nitrosating species or react with superoxide to form ONOO− (25.Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6647) Google Scholar). Both of these reactive nitrogen species have been shown to induce PTP opening when added to Ca2+-loaded mitochondria (37.Borutaite V. Morkuniene R. Brown G.C. FEBS Lett. 2000; 467: 155-159Crossref PubMed Scopus (65) Google Scholar, 38.Packer M.A. Murphy M.P. FEBS Lett. 1994; 345: 237-240Crossref PubMed Scopus (144) Google Scholar), and ONOO−is capable of inducing apoptosis (22.Estevez A.G. Radi R. Barbeito L. Shin J.T. Thompson J.A. Beckman J.S. J. Neurochem. 1995; 65: 1543-1550Crossref PubMed Scopus (287) Google Scholar, 23.Salgo M.G. Bermudez E. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 500-505Crossref PubMed Scopus (256) Google Scholar, 24.Lin K-T. Xue J-Y. Nomen M. Spur B. Wong P.Y-K. J. Biol. Chem. 1995; 270: 16487-16490Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). To elucidate which stage of the PTP was inhibited by NO⋅, experiments were performed in which NO⋅ was added at different time points after the addition of Ca2+. Fig. 5 Bshows that the time of the NO⋅ addition to mitochondria is critical in determining its effects on the PTP. The requirement for the presence of NO⋅ within 1–2 min after the initiation of Ca2+-induced swelling (traces B–D) suggests that NO⋅ is exerting its effects at an early stage in PTP development such as Ca2+ entry into the mitochondrial matrix. Fig. 2 shows that NO⋅ does indeed inhibit the ability of mitochondria to accumulate Ca2+, and consistent with the literature (40.Schweizer M. Richter C. Biochem. Biophys. Res. Commun. 1994; 204: 169-175Crossref PubMed Scopus (334) Google Scholar) the likely mechanism is by inhibiting the development of a membrane potential upon substrate addition to mitochondria. Consistent with Ca2+ accumulation as a target for the inhibitory effects of NO⋅ on the PTP, Fig. 3 B shows that NO⋅ inhibits t-BuOOH-dependent PTP opening, which is also Ca2+-dependent. In addition, NO⋅ inhibited Ca2+ plus phosphate-induced PTP. Together these data support the hypothesis that NO⋅inhibits the PTP by inhibiting membrane potential-driven Ca2+ accumulation. The addition of oxyHb to bind NO⋅ completely reverses its effects on PTP opening (Fig. 3 A). These data indicate that NO⋅ may be decreasing mitochondrial membrane potential through reversible inhibition of electron transport, possibly by binding to heme groups such as those in cytochrome c oxidase (13.Cleeter M.W.J. Cooper J.M. Darley-Usmar V.M. Moncada S. Schapira A.H.V. FEBS Lett. 1994; 345: 50-54Crossref PubMed Scopus (1141) Google Scholar) or complex III (39.Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The binding of NO⋅ to cytochrome coxidase is competitive with oxygen (13.Cleeter M.W.J. Cooper J.M. Darley-Usmar V.M. Moncada S. Schapira A.H.V. FEBS Lett. 1994; 345: 50-54Crossref PubMed Scopus (1141) Google Scholar), and thus if this enzyme is the molecular target for NO⋅ inhibition of the PTP, the concentrations of NO⋅ required to inhibit PTP opening at physiological oxygen tensions (1–5 μm) would be much lower than those observed here (at saturating, 240 μmO2). The release of apoptogenic proteins including cytochrome cfrom mitochondria is mechanistically linked to PTP opening. In agreement with this, Fig. 4 shows that inhibition of the PTP by NO⋅ also inhibits cytochrome c release. This suggests that the PTP may be a target for the antiapoptotic effects of NO⋅. In a cytoprotective role, complete inhibition of PTP opening by NO⋅ is unlikely and would be detrimental through inhibition of ATP synthesis (16.Leist M. Single B. Naumann H. Fava E. Simon B. Kuhnle S. Nicotera P. Biochem. Biophys. Res. Commun. 1999; 258: 215-221Crossref PubMed Scopus (55) Google Scholar, 17.Leist M. Single B. Naumann H. Fava E. Simon B. Kuhnle S. Nicotera P. Exp. Cell. Res. 1999; 249: 396-403Crossref PubMed Scopus (245) Google Scholar). In order to more precisely understand the cytoprotective role of NO⋅, a series of experiments were performed to determine the temporal relationship between PTP opening and cytochrome c release and the effects of varying rates of NO⋅ on these parameters. Cytochrome c release is an early event in PTP opening, and consistent with this observation, the addition of NO⋅ to mitochondria 2 min after Ca2+ is moderately effective at preventing swelling but completely ineffective at inhibiting cytochromec release (Fig. 5, B and inset). In the absence of NO⋅, the relationship between cytochromec release and swelling is hyperbolic in character, such that very little swelling is required for almost complete cytochromec release. This result may partly explain some observations that cytochrome c release can occur in cells without appreciable signs of PTP opening (47.Eskes R. Antonsson B. Osen-Sand A. Montessuit S. Richter C. Sadoul R. Mazzei G. Nichols A. Martinou J.C. J. Cell Biol. 1998; 143: 217-224Crossref PubMed Scopus (580) Google Scholar). In the presence of NO⋅, the curve in Fig. 6 is shifted significantly to the right, indicating that a far greater degree of swelling is required to elicit cytochromec release. This has significant implications for the role of NO⋅ in apoptosis and suggests that acute control of cytochromec release is an antiapoptotic target for NO⋅. In summary, we have identified inhibition of mitochondrial PTP opening as a novel site of action for NO⋅ signaling in apoptosis. The mechanism involves depolarization of the mitochondrial membrane and inhibition of Ca2+ accumulation. Clearly, in the cellular setting the effects of NO⋅ on mitochondrial function are not expected to result in complete inhibition of respiration but favor partial inhibition of mitochondria. In turn, it is postulated that this would result in NO⋅ lowering the concentration of cytochromec available to initiate apoptosis. These experiments suggest that a fine balance exists between the pro- and antiapoptotic properties of NO⋅ at the level of the mitochondrion. We thank Rakesh Patel for providing purified oxyHb." @default.
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• W1978791657 title "Concentration-dependent Effects of Nitric Oxide on Mitochondrial Permeability Transition and Cytochrome cRelease" @default.
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