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Originally published In Press as doi:10.1074/jbc.M001077200 on April 28, 2000

J. Biol. Chem., Vol. 275, Issue 27, 20474-20479, July 7, 2000
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Concentration-dependent Effects of Nitric Oxide on Mitochondrial Permeability Transition and Cytochrome c Release*

Paul S. BrookesDagger §, Emmanuel Padilla SalinasDagger ||, Kenta Darley-Usmar**, Jason P. Eiserich§**, Bruce A. Freeman§**DaggerDagger, Victor M. Darley-UsmarDagger §§§, and Peter G. AndersonDagger §¶¶

From the Departments of Dagger  Pathology and ** Anesthesiology and the § Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received for publication, February 9, 2000, and in revised form, April 28, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 cytochrome c 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·.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 and 2). Antiapoptotic effects are associated with low levels (10 nM to 1 µM) of exposure from the activation of endogenous NO· synthases and slow release rates from NO· donors (3-12). Specific molecular targets include inhibition of Bcl-2 cleavage (3), inactivation of caspases by S-nitrosation (4-10), and cGMP-mediated effects (11, 12). However, exposure of cells to high NO· concentrations (>1 µM) results in extensive inhibition of mitochondrial ATP synthesis (13-15), and while apoptosis is inhibited, necrotic cell death results (16, 17). This is consistent with the greater ATP dependence of apoptosis compared with necrosis (16, 17). Under some conditions, such as inflammation or neurodegenerative diseases, NO·-dependent apoptosis has been observed (18-24). In this case, the hypothesis that NO· reacts with superoxide to form peroxynitrite (25) has been suggested as a mechanism leading to apoptotic cell death (22-24). 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 cytochrome c from mitochondria. This is important, since it is becoming increasingly apparent that mitochondria are important mediators of apoptosis (26-29), and is possibly related to recent evidence for a mitochondrial NO· synthase that would produce NO· within the organelle (30, 31). The release of cytochrome c and apoptosis-inducing factor from mitochondria leads to activation of caspases 9 and 3 (29), and it was recently reported that caspase 3 can activate caspase 8, which can in turn trigger cytochrome c release (32). 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), 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, 34). 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).

The reported effects of NO· on the mitochondrial PTP appear to be inconsistent. In some studies, mitochondria were exposed to S-nitrosothiols or high concentrations of NO· (0.6-10 µM), and PTP opening was observed (36, 37). It has been suggested that these effects were not mediated by NO· but by other reactive nitrogen species such as peroxynitrite (22-25, 38) or by S-nitrosation of mitochondrial proteins (37). In contrast, it was also suggested that NO· may inhibit PTP in a subpopulation of mitochondria, but the effects on cytochrome c release were not reported (36). 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, 39). 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). In addition, inhibition of the putative mitochondrial NOS facilitates Ca2+ accumulation, consistent with either NO·-dependent prevention of Ca2+ uptake or enhanced release (31). 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).

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 cytochrome c 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 c release were determined.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals and Materials-- Male Harlan Sprague-Dawley rats, 250-300 g in weight, were handled in accordance with recommendations in Ref. 48. 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.

Isolation of Mitochondria-- Liver mitochondria were isolated according to standard procedures (41) 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) against a standard curve constructed using bovine serum albumin.

Mitochondrial PTP Assay-- Mitochondrial PTP opening was assayed essentially as described by Packer and Murphy (38). 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 nM NO·/s, this relationship became nonlinear.

Measurement of Mitochondrial Cytochrome c Release-- 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) and electroblotted to nitrocellulose membranes (44). 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. Cytochrome c was not detectable by spectrophotometric methods (reduced/oxidized A550) 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.

Measurement of Mitochondrial Calcium Uptake-- 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). 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.

Measurement of Mitochondrial Membrane Potential (Delta psi )-- 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). 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). Readings were taken every 1 s, and data were smoothed using a 4-s moving integration window.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fig. 1A 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. 1B shows 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. 1A). 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.


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  		Fig. 1.   The effects of NO· on mitochondrial PTP opening. Mitochondria were incubated as described under "Experimental Procedures." A, Ca2+ was added after 2 min as indicated by the arrow except in the trace marked No Ca2+. NO· donors were present from the start of the incubation (~20 s before mitochondria) as follows: DEA NONOate, 1.3 mM; DETA NONOate, 3.1 mM; spermine NONOate, 2.2 mM. CsA, cyclosporin A, 5 µM. Data are means of at least three experiments. Error bars (S.E.) are omitted from all but control data for clarity. B, dose response of Ca2+-induced mitochondrial PTP to NO·. Mitochondria were incubated as described under "Experimental Procedures." Spermine NONOate (open circle ), DETA NONOate (), or DEA NONOate (black-triangle) was present from the start of incubation (~20 s before mitochondria). Ca2+ was added at 2 min, and the maximum swelling rate achieved within 20 min was recorded. NO· release rates were calculated as detailed under "Experimental Procedures." Data are means ± S.E. from at least five independent experiments.

Fig. 2A shows the effects of the NO· donor spermine NONOate on mitochondrial Ca2+ uptake measured by the Arsenazo III method (38). 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 Delta psi were determined using TPMP+ (45). Development of Delta psi results in accumulation of TPMP+ by mitochondria and a decreased signal from the TPMP+ electrode. Consistent with the literature (40), Fig. 2B shows that the presence of NO· prevented development of Delta psi upon the addition of succinate as substrate. These data support the hypothesis that NO· inhibits the PTP by preventing membrane potential-driven Ca2+ accumulation.


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  		Fig. 2.   The effects of NO· on mitochondrial Ca2+ accumulation and membrane potential. A, mitochondria were incubated at 0.25 mg of protein/ml in the presence of Arsenazo III (100 µM). After 30 s, CaCl2 (10 µM) was added as indicated by the arrow, followed by FCCP (1 µM) and then EGTA (1 mM) as indicated. Traces are annotated as follows. Control, additions as detailed above; Ru Red, in the presence of ruthenium red (10 µM); NO, in the presence of spermine NONOate (1.1 mM) from the start of the incubation (~20 s before mitochondria). Data are representative of a larger experimental population (n = 3). B, mitochondria were incubated at 1 mg of protein/ml in conditions identical to those in Fig. 1, except for the presence of nigericin (100 nM). Membrane potential (Delta psi ) was measured using an electrode sensitive to TPMP+, whereby a decrease in the TPMP+ signal indicates development of a Delta psi . TPMP+ additions (1 µM each) were made as indicated by the arrows (T), followed by succinate (5 mM) and FCCP (1 µM) as indicated. Data are representative of a larger experimental population (n = 3).

To establish that inhibition of PTP opening required the continuous generation of NO·, the effects of oxyhemoglobin (oxyHb) were examined. Fig. 3A 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. 3B shows that the well characterized induction of the PTP by Ca2+ plus tert-butyl hydroperoxide can also be inhibited by NO·. Similar results were obtained when Ca2+ plus phosphate was used to induce PTP (not shown).


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  		Fig. 3.   A, effects of oxyhemoglobin on NO· inhibition of PTP. Mitochondria were incubated as described under "Experimental Procedures." Spermine NONOate (2.2 mM) was present where indicated from the start of incubations (~20 s before mitochondria). Ca2+ was added at 2 min as indicated by the arrow. Oxyhemoglobin (Hb; 10 µM heme) was added as indicated by the arrow. B, effects of NO· on PTP opening induced by Ca2+ plus t-BuOOH. Mitochondria were incubated as in Fig. 1, except that t-BuOOH (100 µM) was added where indicated. Data are means ± S.E. from at least three independent experiments. Error bars (S.E.) in A and B are omitted from all but control data for clarity.

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. 4C shows typical mass spectra from the same postmitochondrial supernatants as in Fig. 4A. 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.


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  		Fig. 4.   The effects of NO· on mitochondrial cytochrome c release. Cytochrome c was detected in mitochondrial supernatants by Western blotting or MALDI-TOF mass spectrometry as detailed under "Experimental Procedures." A, typical developed Western blot. Lane 1, Ca2+ at 2 min, swollen mitochondria; lane 2, as lane 1, with spermine NONOate (2.2 mM) at the start of the experiment; lane 3, as lane 1, with no Ca2+; lane 4, authentic cytochrome c (positive control). B, densitometric analysis of blots of the type shown in A. Results are means ± S.E. of at least three experiments. C, MALDI-TOF mass spectrometric analysis of the effects of NO· on proteins released from mitochondria upon Ca2+ induced PTP. Analysis was performed using 1 µl of concentrated supernatants. Trace designations (1-3) are as for lanes on Western blot (A).

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 cytochrome c release. This was examined in the next series of experiments. Fig. 5A 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.


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  		Fig. 5.   Time course of the effects of NO· on PTP opening and cytochrome c release. A, time course of cytochrome c release during swelling experiments (closed circles). Mitochondria were incubated as in Fig. 1, without NO· present. At set time intervals, EGTA (1 mM) and cyclosporin A (5 µM) were added to halt swelling (see "Experimental Procedures"), and supernatants were analyzed for cytochrome c by Western blotting as in Fig. 4A. Data are means ± S.E. of at least four independent experiments. Swelling data (line, no symbols, with errors) were taken from Fig. 1. B, effects of time of NO· addition on swelling and cytochrome c release. Mitochondria were incubated as in Fig. 1. Ca2+ was added at 2 min as indicated by the arrow. Spermine NONOate (2.1 mM) was added at different times during the incubation. The arrows (A-D) indicate the time of NO· addition, and labels beside traces correspond to these arrows (e.g. the addition at time A resulted in trace A). Data are the means of at least three independent experiments. Error bars are omitted from all but control data for clarity. Inset, effects of the time of the NO· addition on cytochrome c released from mitochondria in B. Data are means ± S.E. of at least four independent experiments.

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. 5B 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. 5A). This is confirmed in the inset to Fig. 5B, which shows the effect of time of the addition of NO· on cytochrome c release.

The relationship between the degree of swelling and cytochrome c 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.


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  		Fig. 6.   The relationship between degree of swelling and cytochrome c release and the effects of NO·. Closed symbols, mitochondrial swelling and cytochrome c release were halted at various stages during incubations, as in Fig. 5. Cytochrome c release and degree of swelling were then calculated and expressed as percentages of the maximum that day. Open symbols, following the addition of NO· at different time points during swelling (Fig. 5B), cytochrome c release and degree of swelling were calculated and expressed as percentages of the maximum that day. All data are means ± S.E. of at least four independent experiments (*, p < 0.1; **, p < 0.05 between corresponding points on control and NO·-inhibited curves).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, 28, 33, 35) and that NO· has specific interactions with mitochondria at the level of both cytochrome c oxidase and complex III (13-15, 39). 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). 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), 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 encountered in vivo, even under pathologic conditions with maximal stimulation of iNOS (46). It is likely that at such high concentrations NO· may form S-nitrosating species or react with superoxide to form ONOO- (25). Both of these reactive nitrogen species have been shown to induce PTP opening when added to Ca2+-loaded mitochondria (37, 38), and ONOO- is capable of inducing apoptosis (22-24).

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. 5B shows 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) 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. 3B 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. 3A). 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) or complex III (39). The binding of NO· to cytochrome c oxidase is competitive with oxygen (13), 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 µM O2).

The release of apoptogenic proteins including cytochrome c from 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, 17). 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 cytochrome c release (Fig. 5, B and inset). In the absence of NO·, the relationship between cytochrome c release and swelling is hyperbolic in character, such that very little swelling is required for almost complete cytochrome c release. This result may partly explain some observations that cytochrome c release can occur in cells without appreciable signs of PTP opening (47). 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 cytochrome c release. This has significant implications for the role of NO· in apoptosis and suggests that acute control of cytochrome c 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 cytochrome c 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.

    ACKNOWLEDGEMENT

We thank Rakesh Patel for providing purified oxyHb.

    FOOTNOTES

* The University of Alabama at Birmingham mass spectrometry core facility is funded in part by a National Institutes of Health (NIH) instrumentation grant, by the Howard Hughes Medical Institute, and by a NCI, NIH, core research grant to the University of Alabama at Birmingham comprehensive cancer center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by American Heart Association Postdoctoral Fellowship 9920144V.

|| Participant in the NASA SHARP plus program in the laboratories of V. M. D. U. and P. G. A.

Dagger Dagger Supported by NIH Grants RO1-HL64937, RO1-HL58115, and P6-HL58418.

§§ Supported by the American Heart Association, American Diabetes Association, and NIH Grant RO1 58031.

¶¶ Supported by NIH Grants RO1-HL058895 and RO1-HL58209. To whom correspondence should be addressed. Tel.: 205-9342414; Fax: 205-9341775; E-mail: pga@uab.edu.

Published, JBC Papers in Press, April 28, 2000, DOI 10.1074/jbc.M001077200

    ABBREVIATIONS

The abbreviations used are: NO·, nitric oxide; DEA NONOate, 2-(N,N-1-diethylamino)-diazenolate-2-oxide; DETA NONOate, (Z)-1-[2-(2-aminoethyl)-N-(ammonio-ethyl)amino]diaze-1-ium-1,2-diolate; FCCP, carbonyl-cyanide-p-(trifluoromethoxy)-phenylhydrazine; oxyHb, oxyhemoglobin; PTP, permeability transition pore; t-BuOOH, tert-butyl hydroperoxide; MALDI-TOF, matrix-assisted laser-desorption time-of-flight; TPMP+, triphenylmethylphosphonium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Brune, B., von Knethen, A., and Sandau, K. (1999) Cell Death Differ. 6, 969-975
2. Kim, Y-M., Bombeck, C. A., and Billiar, T. R. (1999) Circ. Res. 84, 253-2563
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