spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 15 May 2007
doi: 10.1242/jcs.003228

Journal of Cell Science 120, 1908-1914 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jcs.003228v1
120/11/1908    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guidarelli, A.
Right arrow Articles by Cantoni, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guidarelli, A.
Right arrow Articles by Cantoni, O.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Research Article

Inhibition of complex III promotes loss of Ca2+ dependence for mitochondrial superoxide formation and permeability transition evoked by peroxynitrite

Andrea Guidarelli, Liana Cerioni and Orazio Cantoni*

Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino "Carlo Bo", Via S. Chiara, 27-61029 Urbino (PU), Italy

* Author for correspondence (e-mail: cantoni{at}uniurb.it)

Accepted 14 April 2007


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In intact U937 cells, peroxynitrite promotes the mitochondrial formation of superoxide via a Ca2+-dependent mechanism involving inhibition of complex III. Superoxide then readily dismutates to H2O2 causing lesions on different biomolecules, including DNA. Here we show that formation of H2O2 and DNA damage are suppressed by inhibition of complex I (by rotenone) or ubisemiquinone formation (by myxothiazol), as well as by a variety of manipulations preventing either the mobilization of Ca2+ or its mitochondrial accumulation. In addition, complex III inhibitors promoted rotenone- or myxothiazol-sensitive formation of H2O2 and DNA strand scission in cells exposed to otherwise inactive concentrations of peroxynitrite. However, under these conditions, the intra-mitochondrial concentration of Ca2+ remained unchanged and the effects of peroxynitrite therefore take place via Ca2+-independent mechanisms. H2O2 formation was paralleled by, and causally linked to, the loss of mitochondrial membrane potential associated with the mitochondrial release of cytochrome c and AIF, and with the mitochondrial accumulation of Bax. These events, although Ca2+ independent, were rapidly followed by death mediated by mitochondrial permeability transition, generally considered a typical Ca2+-dependent event. Thus, enforced inhibition of complex III promotes the loss of Ca2+ dependence of those mitochondrial mechanisms regulating superoxide formation and mitochondrial permeability transition evoked by peroxynitrite.

Key words: Peroxynitrite, DNA damage, Cell death, Mitochondrial permeability transition, Calcium ions


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Peroxynitrite, the product of nitric oxide and superoxide, promotes extensive damage on diverse biological molecules including lipids (Radi et al., 1991aGo), proteins (Patel et al., 1999Go) and DNA (Szabó and Ohshima, 1997Go; Guidarelli et al., 2000Go; Guidarelli et al., 2006Go). Although these effects can be directly mediated by peroxynitrite, which is indeed a highly reactive species, it now appears that the oxidant may also promote secondary damage by triggering events resulting in the formation of different damaging species. A good example is provided by our studies showing that most of the DNA single-strand breakage generated by peroxynitrite is in fact mediated by superoxide-derived H2O2 (Guidarelli et al., 2000Go; Guidarelli et al., 2006Go). In particular, peroxynitrite-dependent inhibition of complex III causes the formation of superoxide, in a reaction in which ubisemiquinone serves as an electron donor, promptly dismutated to H2O2 that can now exit the mitochondria, reach the nucleus and promote site-specific hydroxyl-radical-mediated DNA cleavage (Guidarelli et al., 2000Go). As a consequence, the DNA damage induced by peroxynitrite was suppressed by various manipulations preventing ubisemiquinone formation (e.g. inhibitors of electron transport through complex I, or the respiration-deficient phenotype), as well as by catalase or iron chelators, and was remarkably enhanced by complex III inhibitors (Guidarelli et al., 2000Go).

We recently extended our work to show that the process leading to the mitochondrial formation of superoxide in response to peroxynitrite is critically regulated by the availability of mitochondrial Ca2+ (mtCa2+) (Guidarelli et al., 2006Go). In particular, we showed that peroxynitrite mobilizes Ca2+ from the ryanodine (Ry) receptor and that the cation promptly accumulates in the mitochondria thereby enhancing the rate of superoxide and H2O2 formation.

Collectively, the above findings, obtained from studies performed in U937 cells expressing the three Ry receptor isotypes (Hosoi et al., 2001Go), lead to the conclusion that peroxynitrite promotes, in synergy with Ca2+, time-dependent formation of superoxide and H2O2 at the level of complex III of the respiratory chain.

However, the observation that complex III inhibitors promote extensive formation of H2O2 and DNA single-strand breakage in cells exposed to otherwise ineffective concentrations of peroxynitrite, raise some questions as to the Ca2+ requirements under those specific conditions. Maximal formation of superoxide and H2O2 is indeed observed using very low concentrations of peroxynitrite, with hardly any effect on Ca2+ homeostasis, and inhibition of electron transport probably causes loss rather than accumulation of Ca2+ in the mitochondria.

The present study therefore addressed the intriguing possibility that enforced inhibition of complex III might switch the machinery leading to peroxynitrite-dependent superoxide and H2O2 formation to a Ca2+-independent mechanism.

The question we ask has an additional important implication because complex III inhibitors promote rapid necrotic U937 cell death after exposure to otherwise non-toxic concentrations of peroxynitrite (Tommasini et al., 2002aGo; Tommasini et al., 2004aGo), thus suggesting that cell demise might also take place via a Ca2+-independent mechanism. This would be an interesting observation especially if necrosis takes place via a mitochondrial permeability transition (MPT)-dependent mechanism, as previously noticed using intrinsically toxic levels of peroxynitrite (Sestili et al., 2001Go). Ca2+ is indeed generally considered to be an important determinant for MPT (Bernardi, 1999Go; Kowaltowski et al., 2001Go; Brookes et al., 2004Go), an event associated with the release of intramitochondrial material followed by either apoptosis or necrosis.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Exposure of cultured U937 cells to peroxynitrite promotes delayed formation of reactive oxygen species (ROS) and DNA strand scission in a concentration- and time-dependent fashion. For an exposure period of 30 minutes, maximal effects were observed at 200 µM peroxynitrite (Fig. 1A,B) and no further increase was observed at higher concentrations (data not shown). Antimycin A (1 µM, a concentration causing 90±4% inhibition of oxygen consumption) caused a leftward shift in both of the dose-response curves and, under these conditions, maximal effects were observed using 40 µM peroxynitrite (Fig. 1C,D). When the cells were treated for increasing time intervals with 200 µM peroxynitrite, the DNA strand scission was significant at 10 minutes and linearly increased for up to 30 minutes (Fig. 1F). A mixture of peroxynitrite (40 µM) and antimycin A caused a much faster accumulation of DNA lesions, which reached a plateau after only 10 minutes (Fig. 1F). The kinetics of ROS formation was similar to those observed in experiments measuring DNA strand scission (Fig. 1E). Replacing antimycin A with 2-hepthyl-4-hydroxyquinoline (10 µM, HQNO), a structurally unrelated complex III inhibitor (van Ark and Berden, 1977Go), produced identical effects in all of the above experiments (not shown). However, antimycin A, or HQNO, failed to produce detectable effects in the absence of additional treatments (not shown). These results indicate that inhibition of complex III remarkably lowers the concentration and time necessary for the maximal formation of ROS and DNA cleavage mediated by peroxynitrite.


Figure 1
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 1. Antimycin A significantly lowers the threshold concentration and time necessary for maximal formation of ROS and DNA single-strand breaks in cells exposed to peroxynitrite. (A-D) The cells were first treated for 5 minutes with 0.5 µM rotenone, 5 µM myxothiazol or 20 µM ryanodine (Ry), as detailed in the figure, subsequently exposed for 3 minutes to increasing concentrations of peroxynitrite and finally incubated for a further 27 minutes in the absence (A,B) or presence (C,D) of 1 µM antimycin A. (E,F) Cells were exposed for 3 minutes to 0, 40 or 200 µM peroxynitrite and incubated for increasing time intervals in the absence or presence of 1 µM antimycin A. After treatment, cells were analyzed for DNA damage and dihydrorhodamine 123 (DHR)-fluorescence as detailed in the Materials and Methods. Results represent the means ± s.e.m. calculated from three to five experiments. *P<0.01; **P<0.001 compared with levels in untreated cells; (*)P<0.01; (**)P<0.001 compared with levels in cells treated with peroxynitrite alone (A,B) or associated with antimycin A (C,D) (ANOVA followed by Dunnett's test).

 

We recently showed (Guidarelli et at., 2006Go) that in U937 cells peroxynitrite mobilizes Ca2+ from Ry-sensitive stores, an event associated with the mitochondrial accumulation of the cation, critically involved in the formation of ROS, mediating cleavage of genomic DNA. The fluorescent images shown in Fig. 2A indicate that indeed peroxynitrite (200 µM) promotes a Rhod-2-derived fluorescence response superimposable on that obtained with a mitochondrial probe. This response (Fig. 2B), as well as ROS formation (Fig. 1A) and DNA strand scission (Fig. 1B), is sensitive to Ry (20 µM). Interestingly, a mixture of peroxynitrite (40 µM) and antimycin A (or HQNO) failed to enhance mtCa2+ content (Fig. 2B) and triggered ROS formation (Fig. 1C) and DNA damage (Fig. 1D) via Ry-insensitive mechanisms.


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 2. The enhancing effects of antimycin A or 2-hepthyl-4-hydroxyquinoline (HQNO) are mediated by inhibition of electron transport through complex III and are independent on the accumulation of mtCa2+. (A) Representative microscope images (magnification 400x) of cells pre-loaded with Rhod-2 AM and MitoTracker Green and then treated for 10 minutes with 0 or 200 µM peroxynitrite. In the merged image (overlay) regions containing both Rhod-2 and MitoTracker Green fluorescence appear yellow. (B) Rhod 2-AM pre-loaded cells were first treated for 5 minutes with 20 µM ryanodine (Ry), subsequently exposed for 3 minutes to peroxynitrite (40 or 200 µM) and finally incubated for a further 7 minutes in the absence or presence of 1 µM antimycin A or 10 µM HQNO. Fluorescence was then quantified as detailed in the Materials and Methods. Results represent the means ± s.e.m. calculated from three to five experiments. *P<0.001 compared with untreated cells (unpaired Student's t-test). (C) Permeabilized cells were exposed for 10 minutes to 200 µM peroxynitrite (200), or 40 µM peroxynitrite/1 µM antimycin A (40 + Antimycin A), in the absence or presence of 10 µM ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 200 nM ruthenium red (RR), 100 µM LaCl3, 20 µM Ry, 0.5 µM rotenone, 5 µM myxothiazol or 10 U/ml catalase (enzymatically active or heat-inactivated). The DNA cleavage induced by peroxynitrite in the absence or presence of antimycin A was also assessed in respiration-deficient (RD) cells. The level of DNA single-strand breaks was measured immediately after the treatments. Results represent the means ± s.e.m. calculated from three to five experiments. *P<0.001 compared with levels in cells exposed to peroxynitrite alone or associated with antimycin A (unpaired Student's t-test).

 
These results suggest that the mechanism(s) leading to ROS formation in the presence of complex III inhibitors is Ca2+ independent. This notion is in contrast to the previously established Ca2+ dependence of the effects mediated by peroxynitrite alone (Guidarelli et al., 2006Go). However, as we previously showed (Guidarelli et al., 2000Go), ROS formation and DNA damage are in both circumstances dependent on the inhibition of electron transport. Both events (Fig. 1A-D) were indeed sensitive to the complex I inhibitor rotenone (0.5 µM) under conditions in which inhibition of oxygen consumption was also observed (not shown). In addition, the effects of rotenone were mimicked by myxothiazol (5 µM), an agent binding to a site of complex III different from that of antimycin A or HQNO (Rich et al., 1990Go) thereby preventing the electron flow from the reduced coenzyme Q to cytochrome c1, and the ensuing formation of ubisemiquinone (Brand and Hermfisse, 1997Go). Thus, as previously shown (Guidarelli et al., 2000Go; Guidarelli et al., 2006Go), ubisemiquinone serves as an electron donor promoting the time-dependent formation of superoxide and H2O2 in cells exposed to either high concentrations (e.g. 200 µM) of peroxynitrite or low (e.g. 40 µM), otherwise inactive, concentrations of peroxynitrite associated with antimycin A (or HQNO).

We next performed experiments in digitonin-permeabilized cells. The results illustrated in Fig. 2C indicate that the DNA damage induced by 200 µM peroxynitrite is also sensitive to rotenone, myxothiazol and Ry under these conditions. In addition, peroxynitrite failed to promote DNA strand scission in respiration-deficient cells, which do not generate secondary ROS under these conditions (Guidarelli et al., 2000Go; Tommasini et al., 2002aGo). Consistently with the notion that H2O2 represents the final DNA-damaging species, the DNA cleavage was prevented by catalase (10 Sigma units/ml), whereas the boiled enzyme was ineffective. Finally, the observed DNA-damaging response was sensitive to 10 µM ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) – a calcium chelator – and to 200 nM ruthenium red (RR), which, under these conditions, specifically prevents mtCa2+ uptake (Carafoli, 1987Go), as well as to lanthanium ions (100 µM), which are known to competitively inhibit mtCa2+ uptake (Thomas and Reed, 1988Go). Interestingly, the DNA damage induced by 40 µM peroxynitrite with antimycin A was sensitive to rotenone, myxothiazol, the respiration-deficient phenotype or enzymatically active catalase but insensitive to EGTA, RR, lanthanium ions and Ry. Similar results were obtained in studies in which HQNO was used in the place of antimycin A (not shown). These results confirm the notion that maximal formation of ROS and DNA single-strand breaks mediated by a low concentration of peroxynitrite in complex-III-inhibited cells takes place via a Ca2+-independent mechanism.


Figure 3
View larger version (64K):
[in this window]
[in a new window]

  		Fig. 3. mtCa2+-dependent and mtCa2+-independent mechanisms promote toxicity mediated by mitochondrial permeability transition. (A) The cells were first treated for 5 minutes with 0.5 µM rotenone, 20 µM ryanodine (Ry), 10 U/ml catalase, 0.5 µM cyclosporin A (CsA) or 1 µM FK506, subsequently exposed for 3 minutes to peroxynitrite (40 or 200 µM) and finally incubated for a further 57 minutes in the absence or presence of 1 µM antimycin A or 10 µM or 2-hepthyl-4-hydroxyquinoline (HQNO). Toxicity induced by peroxynitrite in the absence or presence of antimycin A, or HQNO, was also tested in respiration-deficient (RD) cells. Cytotoxicity was then determined. Results represent the means ± s.e.m. calculated from three to five experiments. *P<0.001 compared with levels in untreated cells (unpaired Student's t-test). (B) Representative photomicrographs (magnification 400x) of cells treated for 3 minutes with 0 or 40 µM peroxynitrite and finally incubated for a further 7 minutes with MitoTracker Red CMXRos in the absence or presence of the indicated additions. (C) The cells were treated for 5 minutes with 0.5 µM CsA or 1 µM FK506, as detailed in the figure, subsequently exposed for 3 minutes to 40 µM peroxynitrite and finally incubated for a further 7 minutes with MitoTracker Red CMXRos in the absence or presence of 1 µM antimycin A or 10 µM HQNO. Fluorescence was then quantified as detailed in the Materials and Methods. Results represent the means ± s.e.m. calculated from three to five experiments. *P<0.001 compared with levels in untreated cells (unpaired Student's t-test). (D) Cells were exposed for 3 minutes to 40 µM peroxynitrite and finally incubated for a further 7 minutes in the absence or presence of various additions. Cells were then processed to obtain the mitochondrial and cytosolic fractions for western blot analysis using antibodies against cytochrome c, AIF or Bax. The blots were then washed and re-probed for actin or HSP-60.

 
We next measured toxicity mediated by the above conditions over an exposure time of 60 minutes. Treatment with 200 µM peroxynitrite was not toxic for respiration-deficient cells and caused ~20% loss of viability in respiration-proficient cells, which was sensitive to Ry, rotenone and enzymatically active catalase (Fig. 3A). A significantly greater lethal response was observed after treatment with 40 µM peroxynitrite with antimycin A (or HQNO) (Fig. 3A), which generates levels of ROS and DNA cleavage identical to those observed in the above conditions, albeit at a remarkably faster rate (Fig. 1A-F). Toxicity was insensitive to Ry and prevented by catalase, rotenone or by the respiration-deficient phenotype (Fig. 3A). Antimycin A (or HQNO), Ry or a combination of the two agents did not produce DNA cleavage, ROS formation or toxicity in the absence of additional treatments (not shown).

These results indicate that H2O2 generated at the mitochondrial level in response to a low concentration of peroxynitrite and an inhibitor of complex III causes extensive cytotoxicity. The insensitivity to Ry suggests the involvement of Ca2+-independent mechanisms because peroxynitrite would eventually mobilize Ca2+ from the Ry receptor (Guidarelli et al., 2006Go).

As indicated in Fig. 3A, U937 cell death mediated by each of the two different toxicity paradigms was inhibited by cyclosporin A (CsA) (0.5 µM), an inhibitor of MPT (Halestrap et al., 1997Go), thereby implying that toxicity takes place via a MPT-dependent mechanism. Cells were not rescued by FK506 (1 µM), which shares with CsA the ability to inhibit calcineurin, but fails to affect the formation of MPT pores (Henke and Jung, 1993Go). In addition, toxicity was preceded by a CsA-sensitive decline in mitochondrial membrane potential, as measured by MitoTracker Red CMXRos uptake (Fig. 3B,C), and opening of MPT pores, as measured by monitoring the changes in mitochondrial calcein fluorescence after quenching of the cytosolic signal with Co2+. The images obtained in the calcein studies were identical to those published in our previous studies (Tommasini et al., 2004bGo; Guidarelli et al., 2005aGo; Guidarelli et al., 2005bGo) and are not shown here for the sake of brevity. Finally, a mixture of peroxynitrite and antimycin A induced CsA-sensitive early mitochondrial release of cytochrome c and AIF, as well as mitochondrial translocation of Bax (Fig. 3D). These effects were not observed in untreated cells or in cells exposed to peroxynitrite or antimycin A alone (Fig. 3D).

These results indicate that cell death evoked by otherwise non-toxic concentrations of peroxynitrite in complex-III-inhibited cells is mediated by MPT. These results should be further analyzed in parallel with those indicating that there was no increase in mtCa2+ 10 minutes (Fig. 2B) or 15 minutes (not shown) after addition of peroxynitrite to antimycin-A-supplemented cells. The experiments investigating mitochondrial integrity and function (mitochondrial membrane potential, mitochondrial loss of cytochrome c and AIF as well as mitochondrial accumulation of Bax) were also performed 10 or 15 minutes (calcein assay) after addition of peroxynitrite.

Thus, there was no increase in mtCa2+ before or during the onset of MPT. We therefore conclude that MPT, an event in which Ca2+ plays a critical role (Bernardi, 1999Go; Kowaltowski et al., 2001Go; Brookes et al., 2004Go), might also take place via Ca2+-independent mechanisms.


   Discussion
Top
 Summary
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Peroxynitrite causes DNA strand scission via an indirect mechanism involving the mitochondrial formation of superoxide, which, upon dismutation to H2O2, reaches the nucleus to generate DNA cleavage via a reaction of the Fenton type (Guidarelli et al., 2000Go). Superoxide formation takes place via a saturable mechanism involving inhibition of complex III (Guidarelli et al., 2000Go) and mobilization of Ca2+ from Ry-sensitive stores associated with the mitochondrial clearance of the cation (Guidarelli et al., 2006Go). Hence, as we previously showed (Guidarelli et al., 2000Go; Guidarelli et al., 2006Go), ROS formation and DNA damage induced by peroxynitrite (Fig. 1) are sensitive to treatments preventing the electron flow through complex I (i.e. rotenone), or ubisemiquinone formation (i.e. myxothiazol), as well as to a Ry concentration preventing Ca2+ mobilization induced by peroxynitrite (Guidarelli et al., 2006Go). Consistently, the same treatments, along with the respiration-deficient phenotype, catalase and various manipulations preventing the mitochondrial accumulation of Ca2+ were also effective in permeabilized cells (Fig. 2C).

Thus, maximal ROS formation and DNA damage mediated by peroxynitrite require inhibition of electron transport at the level of complex III and the accumulation of Ca2+ in the mitochondrial compartment. In addition, as we previously showed (Guidarelli et al., 2000Go), H2O2 is responsible for DNA strand scission and represents the major species detected by our fluorescent assay after exposure to peroxynitrite.

In the present study, we focused our attention on the effects mediated by complex III inhibitors, because antimycin A or HQNO significantly lowered the threshold concentration necessary for maximal formation of H2O2 and DNA single-strand breaks. In complex-III-inhibited cells, an otherwise inactive concentration of peroxynitrite (40 µM) was as effective as a five times greater concentration of the oxidant in the absence of additional treatments (Fig. 1A-D). Most importantly, however, enforced inhibition of complex III was found to promote maximal responses via a mechanism still dependent on electron transport (i.e. sensitive to rotenone or myxothiazol, Fig. 1C,D) but independent of the accumulation of mtCa2+. The following lines of evidence support the notion of the loss of Ca2+ dependence under these conditions: (1) there was no increase in mtCa2+ (Fig. 2B); (2) Ry had hardly any effect on H2O2 formation and DNA cleavage (Fig. 1C,D); (3) the DNA strand scission mediated in permeabilized cells was insensitive to both EGTA and inhibitors of mtCa2+ uptake (Fig. 2C). These results unequivocally demonstrate that peroxynitrite promotes maximal H2O2 formation and DNA strand scission in complex-III-inhibited cells under conditions in which Ca2+ is not mobilized from Ry-sensitive stores and its concentration is not elevated in the mitochondrial compartment.

Two additional features of the Ca2+-independent mechanism(s) triggered by a low concentration of peroxynitrite (e.g. 40 µM) in complex III-inhibited cells were: (1) the very short time necessary for the onset of the maximal mitochondrial production of H2O2 (Fig. 1E) and strand scission of genomic DNA (Fig. 1F) and (2) a lethal response (Fig. 3A) significantly greater than that mediated by the concentration of peroxynitrite (i.e. 200 µM) resulting in the equivalent formation of H2O2 and DNA cleavage (Fig. 1A-D).

Toxicity, regardless of whether induced by 200 µM peroxynitrite or by 40 µM peroxynitrite with antimycin A (or HQNO), was sensitive to rotenone, catalase or to the respiration-deficient phenotype (Fig. 3A). These findings on the one hand confirm the critical role played by H2O2 in peroxynitrite toxicity (Tommasini et al., 2002aGo; Tommasini et al., 2004aGo) and on the other hand suggest that the rate of H2O2 formation is more important that the net amount of the oxidant that is being generated over time.

This is an additional (see below) important indication that the effects of H2O2 take place soon after peroxynitrite exposure. We previously reported that non-toxic concentrations of peroxynitrite nevertheless commit cells belonging to the monocyte/macrophage lineage – such as the U937 cells used in this study – to MPT-dependent toxicity, which was prevented by a parallel survival signaling driven by cPLA2-released arachidonic acid (Tommasini et al., 2002bGo; Tommasini et al., 2004bGo; Tommasini et al., 2004cGo). Downstream events were subsequently identified in the activation of 5-lipoxygenase (Tommasini et al., 2006Go), activation (Guidarelli et al., 2005aGo) and mitochondrial translocation (Cerioni et al., 2006Go) of protein kinase C{alpha} and the phosphorylation and cytosolic accumulation of Bad (Guidarelli et al., 2005bGo; Cerioni et al., 2006Go). Furthermore, inhibition of each of these critical steps was invariably associated with the mitochondrial translocation of Bad and Bax, which was soon followed by the onset of MPT-dependent necrosis (Guidarelli et al., 2005bGo; Cerioni et al., 2006Go). Interestingly, parallel events resulting in the mitochondrial formation of H2O2 also appear to promote toxicity via upstream inhibition of the survival signaling, in particular at the level of activation of cPLA2 (Tommasini et al., 2004aGo). Recently, we found (L.C. and O.C., unpublished results) that the inhibitory effects of H2O2 are mediated by activation of a tyrosine phosphatase, an event that is followed by de-phosphorylation of ERK1/2 and thus by reduced enzymatic activity of cPLA2. This pathway is critically regulated by ERK1/2-dependent phosphorylation (Tommasini et al., 2005Go; Tommasini et al., 2006Go). As a consequence, the results presented in this study imply that the rapid accumulation of H2O2 observed in complex-III-inhibited cells after addition of peroxynitrite creates optimal conditions for the above response leading to inhibition of cPLA2 activity.

We also determined that, as previously observed in cells exposed to intrinsically toxic levels of the oxidant (Sestili et al., 2001Go), low concentrations of peroxynitrite (e.g. 40 µM) promote MPT-dependent toxicity in complex-III-inhibited cells. This notion was clearly established by showing that cell death was sensitive to CsA (Fig. 3A). In addition, toxicity was preceded by CsA-sensitive loss of mitochondrial membrane potential (Fig. 3B,C) and mitochondrial cytochrome c, or AIF (Fig. 3D), as well as by CsA-sensitive accumulation of Bax in the mitochondria (Fig. 3D). Interestingly, all these events were readily apparent 10 minutes after the addition of peroxynitrite, the same length of time it took for maximal H2O2 formation (Fig. 1E) in the absence of detectable changes in mtCa2+ (Fig. 2B). Not surprisingly, the lethal response observed under these conditions, unlike that triggered by 200 µM peroxynitrite, was insensitive to Ry (Fig. 3A). Hence, as previously noted for the processes of H2O2 formation and DNA cleavage (Fig. 1A-D), it appears that MPT-dependent toxicity elicited by low levels of peroxynitrite in complex III-inhibited cells is also independent of Ca2+.

In conclusion, our results demonstrate that, in cells exposed to otherwise inactive concentrations of peroxynitrite, enforced inhibition of complex III promotes maximal formation of superoxide and H2O2 via a Ca2+-independent mechanism. In addition, under the same experimental conditions, a MPT-dependent lethal response is readily observed. Thus, although the Ca2+ dependence of MPT in a variety of toxicity paradigms is clearly established (Bernardi, 1999Go; Kowaltowski et al., 2001Go; Brookes et al., 2004Go), our results demonstrate that, at least under the specific conditions used, MPT can also take place via a Ca2+-independent mechanism. These findings are potentially important for different reasons, all of which are experimentally testable, relevant for peroxynitrite-dependent DNA strand scission and cytotoxicity. We may predict that cells devoid of Ry receptors, unlike those expressing this Ca2+ pool, will be resistant to the deleterious effects mediated by low levels of peroxynitrite; high levels of peroxynitrite, however, should cause extensive inhibition of complex III thereby causing DNA cleavage and cytotoxicity via Ca2+-independent mechanisms. The same switch to Ca2+ independence may arise in cells in which the glutathione pool is depleted by ROS and/or reduced energy supply after exposure to otherwise non-toxic levels of peroxynitrite (Guidarelli et al., 2005bGo). Finally, our previous studies showed that acute supplementation of ascorbic acid (Guidarelli et al., 2004Go), promotes effects identical to those mediated by antimycin A in U937 cells exposed to low levels of peroxynitrite. Hence, we may also expect the involvement of Ca2+-independent mechanisms under these conditions.


   Materials and Methods
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell culture and treatment conditions
U937 human myeloid leukemia cells were cultured in suspension in RPMI 1640 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (Euroclone, Celbio Biotecnologie, Milan, Italy), penicillin (50 U/ml) and streptomycin (50 µg/ml) (Euroclone), at 37°C in T-75 tissue culture flasks (Corning, Corning, NY) in an atmosphere of 95% air, 5% CO2. Respiration-deficient U937 cells were isolated as described (Tommasini et al., 2002aGo). Peroxynitrite was synthesized by the reaction of nitrite with acidified H2O2, as described previously (Radi et al., 1991bGo), with minor modification (Tommasini et al., 2002aGo). Treatments were performed in pre-warmed saline A (8.182 g NaCl, 0.372 g KCl, 0.336 g NaHCO3 and 0.9 g glucose in 1 l H2O) containing 2.5x105 cells/ml. Permeabilization was achieved by adding digitonin (10 µM, 12.5 µg/105 cells) to a medium consisting of 0.25 M sucrose, 0.1% bovine serum albumin, 10 mM MgCl2, 10 mM K+-HEPES, 5 mM KH2PO4, pH 7.2 at 37°C. Under these conditions, digitonin permeabilizes the plasma membrane but leaves mitochondrial membranes intact (Fiskum et al., 1980Go). All experiments on permeabilized cells were performed in the permeabilization buffer.

Cytotoxicity assay
Cytotoxicity was determined with the trypan blue exclusion assay. Briefly, an aliquot of the cell suspension was diluted 1:1 (v/v) with 0.4% trypan blue and the viable cells (i.e. those excluding trypan blue) were counted with a hemocytometer.

Measurement of DNA single-strand breakage by the alkaline halo assay
DNA single-strand breakage was determined using the alkaline halo assay developed in our laboratory (Sestili and Cantoni, 1999Go), with the modifications described (Guidarelli et al., 2006Go). Processing of fluorescence images and calculation of results were done as described (Guidarelli et al., 2006Go). Results are expressed as relative nuclear spreading factor values calculated by subtracting the nuclear spreading factor values of control cells from those of treated cells.

Measurement of mitochondrial membrane potential
Cells were treated for 3 minutes with peroxynitrite in 35-mm tissue culture dishes containing an uncoated coverslip and post-incubated for a further 7 minutes with various additions and 50 nM MitoTracker Red CMXRos (Molecular Probes, Europe, Leiden, The Netherlands). Under these conditions, U937 cells rapidly attach to the coverslip. Fluorescence images were captured with a BX-51 microscope (Olympus, Japan), equipped with a SPOT-RT camera unit (Diagnostic Instruments, Sterling Heights, MI). The excitation and emission wavelengths were 545 and 610 nm, respectively, with a 5-nm slit width for both emission and excitation. Images were collected with exposure times of 100-400 mseconds, digitally acquired and processed for fluorescence determination at the single cell level on a personal computer using Scion Image software (Scion, Frederick, MD). Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment condition/experiment.

Measurement of ROS
Cells were treated as indicated above, except that 10 µM dihydrorhodamine 123 (DHR) (Molecular Probes) was used in the place of MitoTracker Red CMXRos. After treatment, the cells were washed three times and analyzed with a fluorescence microscope. The excitation and emission wavelengths were 488 and 515 nm, respectively, with a 5-nm slit width for both emission and excitation. Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment condition/experiment.

Measurement of mitochondrial Ca2+
The cells were exposed for 30 minutes (4°C) to 0 or 10 µM Rhod 2-acetoxymethyl ester (AM) (Molecular Probes), washed three times with saline A and finally incubated for 5 hours in RPMI 1640 medium (37°C). This two-step cold loading/warm incubation protocol achieves selective loading of Rhod 2 into the mitochondria (Trollinger et al., 2000Go). Cells were then treated for 3 minutes with peroxynitrite, post-incubated for a further 7 minutes with or without 10 nM MitoTracker Green (Molecular Probes) and finally visualized with a fluorescence microscope. The excitation and emission wavelengths were 540 and 590 nm (Rhod 2), and 488 and 515 nm (MitoTracker Green) with a 55-nm slit width for both emission and excitation. Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment condition/experiment.

Sub-cellular fractionation and western blot analysis
After treatment, the cells were processed to obtain the cytosolic and mitochondrial fractions, as described (Yu et al., 2003Go). Western blot analyses were performed using anti-AIF (BD Transduction Laboratories, Lexington, KY), anti-cytochrome c, anti-Bax, anti-actin and anti-HSP-60 (Santa Cruz, Santa Cruz, CA) antibodies. Details of western blotting apparatus and conditions are reported elsewhere (Guidarelli et al., 2005aGo). Anti-actin and anti-HSP-60 antibodies were used to assess equal loading of the lanes.

Measurement of oxygen consumption
Cells were washed once in saline A and then resuspended in the same medium at a density of 1x107 cells/ml. Oxygen consumption was measured using a YSI oxygraph equipped with a Clark electrode (model 5300, Yellow Springs Instruments, Yellow Springs, OH). The cell suspension (3 ml) was transferred to the polarographic cell and the rate oxygen utilization was monitored under constant stirring for 3 minutes (basal respiration). The rate of oxygen utilization was calculated as described previously (Robinson and Cooper, 1970Go).

Statistical analysis
Statistical analysis of the data for multiple comparisons was performed by ANOVA followed by a Dunnett's test. For a single comparison, the significance of differences between means was determined using the Student's t-test.


   Acknowledgments
 
This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro and from Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Progetti di Interesse Nazionale (O.C.).


   References
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

Bernardi, P. (1999). Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol. Rev. 79, 1127-1155.[Abstract/Free Full Text]

Brand, K. A. and Hermfisse, U. (1997). Aerobic glycolysis by proliferating cells: a protective strategy against reactive oxygen species. FASEB J. 11, 388-395.[Abstract]

Brookes, P. S., Yoon, Y., Robotham, J. L., Anders, M. W. and Sheu, S.-S. (2004). Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am. J. Physiol. Cell. Physiol. 287, C817-C833.[Abstract/Free Full Text]

Carafoli, E. (1987). Intracellular calcium homeostasis. Annu. Rev. Biochem. 56, 395-433.[CrossRef][Medline]

Cerioni, L., Palomba, L., Brüne, B. and Cantoni, O. (2006). Peroxynitrite-induced mitochondrial translocation of PKC{alpha} causes U937 cell survival. Biochem. Biophys. Res. Commun. 339, 126-131.[CrossRef][Medline]

Fiskum, G., Craig, S. W., Decker, G. L. and Lenhinger, A. L. (1980). The cytoskeleton of digitonin-treated rat hepatocytes. Proc. Natl. Acad. Sci. USA 77, 3430-3434.[Abstract/Free Full Text]

Guidarelli, A., Tommasini, I., Fiorani, M. and Cantoni, O. (2000). Essential role of the mitochondrial respiratory chain in peroxynitrite-induced strand scission of genomic DNA. IUBMB Life 50, 195-201.[Medline]

Guidarelli, A., Fiorani, M. and Cantoni, O. (2004). Enhancing effects of intracellular ascorbic acid on peroxynitrite-induced U937 cell death are mediated by mitochondrial events resulting in enhanced sensitivity to peroxynitrite-dependent inhibition of complex III and formation of hydrogen peroxide. Biochem. J. 378, 959-966.[CrossRef][Medline]

Guidarelli, A., Cerioni, L., Tommasini, I., Brüne, B. and Cantoni, O. (2005a). A downstream role for protein kinase C{alpha} in the cytosolic phospholipase A2-dependent protective signaling mediated by peroxynitrite in U937 cells. Biochem. Pharmacol. 69, 1275-1286.[CrossRef][Medline]

Guidarelli, A., Cerioni, L., Tommasini, I., Fiorani, M., Brüne, B. and Cantoni, O. (2005b). Role of Bcl-2 in the arachidonate-mediated survival signaling preventing mitohondrial permeability transition-dependent U937 cell necrosis induced by peroxynitrite. Free Radic. Biol. Med. 39, 1838-1649.

Guidarelli, A., Sciorati, C., Clementi, E. and Cantoni, O. (2006). Peroxynitrite mobilizes calcium ions from ryanodine-sensitive stores, a process associated with the mitochondrial accumulation of the cation and the enforced formation of species mediating cleavage of genomic DNA. Free Radic. Biol. Med. 41, 154-164.[CrossRef][Medline]

Halestrap, A. P., Connern, C. P., Griffiths, E. J. and Kerr, P. M. (1997). Cyclosporin A binding to mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury. Mol. Cell. Biochem. 174, 167-172.[CrossRef][Medline]

Henke, W. and Jung, K. (1993). Comparison of the effects of the immunosuppressive agents FK 506 and cyclosporin A on rat kidney mitochondria. Biochem. Pharmacol. 45, 829-832.

Hosoi, E., Nishizaki, C., Gallagher, K. L., Wyre, H. W., Matsuo, Y. and Sei, Y. (2001). Expression of the ryanodine receptor isoforms in immune cells. J. Immunol. 167, 4887-4894.[Abstract/Free Full Text]

Kowaltowski, A. J., Castilho, R. F. and Vercesi, A. E. (2001). Mitochondrial permeability transition and oxidative stress. FEBS Lett. 495, 12-15.[CrossRef][Medline]

Patel, R. P., McAndrew, J., Sellak, H., White, C. R., Jo, H., Freeman, B. A. and Darley-Usmar, V. M. (1999). Biological aspects of reactive nitrogen species. Biochim. Biophys. Acta 1411, 385-400.[Medline]

Radi, R., Beckman, J. S., Bush, K. M. and Freeman, B. A. (1991a). Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys. 288, 481-487.[CrossRef][Medline]

Radi, R., Beckman, J. S., Bush, K. M. and Freeman, B. A. (1991b). Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266, 4244-4250.[Abstract/Free Full Text]

Rich, P. R., Jeal, A. E., Madgwick, S. A. and Moody, A. J. (1990). Inhibitor effects on redox-linked protonations of the b haems of the mitochondrial bc1 complex. Biochim. Biophys. Acta 1018, 29-40.[Medline]

Robinson, J. and Cooper, J. M. (1970). Method of determining oxygen concentrations in biological media, suitable for calibration of the oxygen electrode. Anal. Biochem. 33, 390-399.[CrossRef][Medline]

Sestili, P. and Cantoni, O. (1999). Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission. Free Radic. Biol. Med. 26, 1019-1026.[CrossRef][Medline]

Sestili, P., Tommasini, I. and Cantoni, O. (2001). Peroxynitrite promotes mitochondrial permeability transition-dependent rapid U937 cell necrosis: survivors proliferate with kinetics superimposable on those of untreated cells. Free Radic. Res. 34, 513-527.[CrossRef][Medline]

Szabó, C. and Ohshima, H. (1997). DNA damage induced by peroxynitrite: subsequent biological effects. Nitric Oxide 1, 373-385.[CrossRef][Medline]

Thomas, C. E. and Reed, D. J. (1988). Effect of extracellular Ca++ omission on isolated hepatocytes. II. Loss of mitochondrial membrane potential and protection by inhibitors of uniport Ca++ transduction. J. Pharmacol. Exp. Ther. 245, 501-507.[Abstract/Free Full Text]

Tommasini, I., Sestili, P. and Cantoni, O. (2002a). Delayed formation of hydrogen peroxide mediates the lethal response evoked by peroxynitrite in U937 cells. Mol. Pharmacol. 61, 870-878.[Abstract/Free Full Text]

Tommasini, I., Sestili, P., Guidarelli, A. and Cantoni, O. (2002b). Peroxynitrite stimulates the activity of cytosolic phospholipase A2 in U937 cells: the extent of arachidonic acid formation regulates the balance between cell survival or death. Cell. Death Differ. 9, 1368-1376.[CrossRef][Medline]

Tommasini, I., Sestili, P., Guidarelli, A. and Cantoni, O. (2004a). Hydrogen peroxide generated at the level of mitochondria in response to peroxynitrite promotes U937 cell death via inhibition of the cytoprotective signaling mediated by cytosolic phospholipase A2. Cell Death Differ. 11, 974-984.[CrossRef][Medline]

Tommasini, I., Guidarelli, A. and Cantoni, O. (2004b). Non-toxic concentrations of peroxynitrite commit U937 cells to mitochondrial permeability transition-dependent necrosis that is however prevented by endogenous arachidonic acid. Biochem. Pharmacol. 67, 1077-1087.[CrossRef][Medline]

Tommasini, I., Guidarelli, A., Cerioni, L. and Cantoni, O. (2004c). The arachidonate-dependent cytoprotective signaling evoked by peroxynitrite is a general response of the monocyte/macrophage lineage. Biochem. Biophys. Res. Commun. 316, 1191-1195.[CrossRef][Medline]

Tommasini, I., Cerioni, L., Guidarelli, A. and Cantoni, O. (2005). ERK1/2-dependent regulation of U937 cell survival after exposure to peroxynitrite. Biochem. Biophys. Res. Commun. 329, 1282-1287.[CrossRef][Medline]

Tommasini, I., Guidarelli, A., Palomba, L., Cerioni, L. and Cantoni, O. (2006). 5-Hydroxyeicosatetraenoic acid is a key intermediate of the arachidonate-dependent protective signaling in monocytes/macrophages exposed to peroxynitrite. J. Leukoc. Biol. 80, 929-938.[Abstract/Free Full Text]

Trollinger, D. R., Cascio, W. E. and Lemasters, J. J. (2000). Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca2+-indicating fluorophores. Biophys. J. 79, 39-50.[Medline]

van Ark, G. and Berden, J. A. (1977). Binding of HQNO to beef-heart sub-mitochondrial particles. Biochim. Biophys. Acta 459, 119-137.[Medline]

Yu, W., Sanders, B. G. and Kline, K. (2003). RRR-{alpha}-tocopheryl succinate-induced apoptosis of human breast cancer cells involves Bax translocation to mitochondria. Cancer Res. 63, 2483-2491.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Guidarelli, L. Cerioni, M. Fiorani, and O. Cantoni
Differentiation-Associated Loss of Ryanodine Receptors: A Strategy Adopted by Monocytes/Macrophages to Prevent the DNA Single-Strand Breakage Induced by Peroxynitrite
J. Immunol., October 1, 2009; 183(7): 4449 - 4457.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Summary Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jcs.003228v1
120/11/1908    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guidarelli, A.
Right arrow Articles by Cantoni, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guidarelli, A.
Right arrow Articles by Cantoni, O.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?