Báo cáo khoa học: Effect of magnesium ions on the activity of the cytosolic NADH/cytochrome c electron transport system pptx

Effect of magnesium ions on the activity of the cytosolic NADH/cytochrome c electron transport system Gianluigi La Piana1, Vincenza Gorgoglione1, Daniela Laraspata1, Domenico Marzulli2 and Nicola E Lofrumento1 Department of Biochemistry and Molecular Biology, University of Bari, Italy Institute of Biomembranes and Bioenergetics (IBBE - CNR), University of Bari, Italy Keywords cytosolic NADH oxidation; magnesium ions and mitochondrial membrane permeability; mitochondria, cytochrome c and apoptosis; mitochondrial contact sites and respiration; mitochondrial membrane potential Correspondence N E Lofrumento, Department of Biochemistry and Molecular Biology, University of Bari, via Orabona 4, 70126 Bari, Italy Fax: +39 80 5443317 Tel: +39 80 5443325 E-mail: e.lofrumento@biologia.uniba.it (Received 25 July 2008, revised September 2008, accepted 14 October 2008) Cytochrome c (cyto-c), added to isolated mitochondria, activates the oxidation of extramitochondrial NADH and the generation of a membrane potential, both linked to the activity of the cytosolic NADH/cyto-c electron transport pathway The data presented in this article show that the protective effect of magnesium ions on the permeability of the mitochondrial outer membrane, supported by previously published data, correlates with the finding that, in hypotonic but not isotonic medium, magnesium promotes a differential effect on both the additional release of endogenous cyto-c and on the increased rate of NADH oxidation, depending on whether it is added before or after the mitochondria At the same time, magnesium prevents or almost completely removes the binding of exogenously added cyto-c We suggest that, in physiological low-amplitude swelling, magnesium ions may have the function, together with other factors, of modulating the amount of cyto-c molecules transferred from the mitochondrial intermembrane space into the cytosol, required for the correct execution of the apoptotic programme and/or the activation of the NADH/ cyto-c electron transport pathway doi:10.1111/j.1742-4658.2008.06741.x The oxidative and energetic metabolism of glucose requires cytosolic NADH to be oxidized by the respiratory chain to gain the maximal production of ATP and to regenerate the cytosolic NAD+ necessary for continuous and efficient glycolytic flux In mammalian cells, because of the impermeability of the mitochondrial inner membrane (MIM) to pyridine nucleotides, many translocating pathways are available that may be involved in the transfer of reducing equivalents from the cytosol to the mitochondrial matrix, and vice versa [1] Among them, two shuttle systems are well known: the a-glycerophosphate and malate– aspartate shuttles All the proposed systems, however, promote an indirect transfer of reducing equivalents from cytosolic NADH to either NAD+ or flavoproteins inside the mitochondria Starting from the observation that isolated mitochondria oxidize NADH present outside the mitochondria only if cytochrome c (cyto-c) is also added to the incubation medium, and supported by many converging data, we proposed the existence in liver mitochondria of the cytosolic NADH/cyto-c electron transport pathway in addition to and independent of the electron pathway of the respiratory chain [2–7] In the presence of a catalytic Abbreviations cyto-b5, cytochrome b5; cyto-c, cytochrome c; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; ferrocyto-c, ferrocytochrome c; MIM, mitochondrial inner membrane; MIS, mitochondrial intermembrane space; MOM, mitochondrial outer membrane; TMPD, N,N,N¢,N¢-tetramethyl-p-phenylenediamine; DYm, mitochondrial membrane potential change 6168 FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS G La Piana et al amount of cyto-c outside the mitochondria, the NADH/cyto-c system promotes [2–6,8–10]: (a) the oxidation of externally added NADH molecules; (b) the consumption of molecular oxygen by cytochrome oxidase; and (c) the generation of an electrochemical membrane potential We have shown that some components of this additional electron transport chain are sited on the ‘respiratory contact sites’ [5,6], but endogenous cyto-c, present in the mitochondrial intermembrane space (MIS), is not involved in this process [3] With no knowledge of our series of publications and therefore independent of our data, a report in late 2005 [11], with the support of detailed experimental approaches, considered a direct interaction between the voltage-dependent anion channel and cytochrome oxidase The existence of ‘a novel type of contact site’ as such, but with no specific function, was inferred and outlined in a scheme very similar to that reported in our paper published at the beginning of 2005 [6] The NADH/cyto-c electron transport system may have the function, in the physio-pathological conditions associated with the extra formation of cytosolic NADH, to promote its oxidation by the direct transfer of reducing equivalents to cytochrome oxidase and the generation of an electrochemical proton gradient [3] In apoptotic cells, with the impairment of the respiratory chain because of the transfer of cyto-c from the mitochondria to the cytosol, the activity of the system may represent an additional, but necessary, source of energy required for correct execution of the death programme To date, the components identified and involved in the cytosolic NADH/cyto-c electron transport pathway are as follows: NAD-dependent dehydrogenases present in the cytosol; cytosolic NADH; the rotenoneinsensitive NADH/cytochrome b5 (cyto-b5) complex present on the external leaflet of the mitochondrial outer membrane (MOM); cyto-c molecules present outside the mitochondria but not those present in MIS; the respiratory contact sites between the two mitochondrial membranes in which the voltage-dependent anion channel or porin of MOM is juxtaposed to the cytochrome oxidase molecules spanning MIM All of these components are required for the correct execution of the cytosolic NADH/cyto-c system The activity of this electron transport pathway was studied and characterized essentially in liver mitochondria as, over time, we devised and improved four different tests for these mitochondria (see Materials and methods, and [7]) to evaluate the intactness of the two mitochondrial membranes and to measure their permeability to both exogenous NADH and cyto-c The integrity of isolated mitochondria is a necessary Magnesium ions and cytosolic cytochrome c oxidation prerequisite to study the activity of the respiratory chain, but becomes mandatory for the new, additional and independent NADH/cyto-c electron transport system to counteract the criticism that the results obtained can be ascribed to damaged or broken mitochondria One test, based on the determination of sulfite/cyto-c oxido-reductase activity, is highly specific for measurement of the permeability of MOM to exogenous cyto-c, but requires the presence of sulfite oxidase in MIS This enzyme is highly expressed in liver, barely in heart and absent in skeletal muscle [12] The rotenone-insensitive NADH/cyto-c oxidoreductase activity is 10 times lower in the heart than in rat liver [13] The NADH/cyto-b5 complex of MOM is responsible for the reduction of exogenous cyto-c, and therefore its activity should not be a limiting factor for the oxidation of cytosolic NADH With the support of the four tests in our laboratory, we routinely utilize mitochondrial preparations containing more than 98% of mitochondria with MIM not permeable to exogenous NADH and with MOM not permeable to both endogenous and exogenous cyto-c In support of the intactness of MOM, adenylate kinase and sulfite oxidase, present in MIS, are not released outside the mitochondria Recently, unexpected new findings have shown that exogenous cyto-c does not permeate into MIS even when mitochondria are incubated in a strongly hypotonic medium [7] However, some authors maintain that the NADH/cyto-c system is catalysed by broken and/or damaged mitochondria, on the basis of the observation that magnesium ions, added to mitochondria incubated in a hypotonic medium, promote the oxidation of exogenous NADH even in the absence of externally added cyto-c [8–10] The possibility that the free molecules of endogenous cytoc present in the intermembrane space may have the additional function (proposed in 1969 [14] and invoked in 1981 [15] and 2002 [16]) to shuttle electrons between the cyto-b5 of MOM and the cytochrome oxidase of MIM is in contrast with the already mentioned and well-known finding that intact mammalian mitochondria are unable to oxidize exogenous NADH unless cyto-c molecules are also present outside the mitochondria With the support of the above-mentioned results indicating that, even with mitochondria incubated in hypotonic medium, cyto-c present outside the mitochondria is not permeable through MOM [7], we carried out a series of experiments to ascertain the role and effect of magnesium ions on the activity of the cytosolic NADH/cyto-c electron transport system of mitochondria incubated in isotonic 250 mm sucrose and hypotonic 25 mm sucrose media FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS 6169 Magnesium ions and cytosolic cytochrome c oxidation G La Piana et al Results Magnesium stimulates the NADH/cyto-c system only in mitochondria incubated in hypotonic medium Returning to an experiment similar to that carried out in 1989 and reported in [2], Fig (trace a) shows the property of isolated rat liver mitochondria, incubated in isotonic 250 mm sucrose medium, to promote the oxidation of exogenously added NADH in the presence of respiratory chain inhibitors (rotenone and myxothiazol) and a catalytic amount of exogenous cyto-c To test the effect of magnesium ions and avoid any interference or synergistic activity of other substances, an incubation medium simply consisting of sucrose and buffer to stabilize the pH was utilized Figure (trace a) also shows that the oxidation rate was blocked by cyanide, suggesting the involvement of cytochrome oxidase The addition of mm MgCl2 before the cyto-c (Fig 1, trace b) did not influence the NADH oxidation rate Mitochondria incubated in hypotonic medium (25 mm sucrose; Fig 1, traces c–e) oxidized exogenous NADH before the addition of cyto-c, even if at a very low rate, which was increased about eight times by magnesium ions (Fig 1, trace e) The oxidation rate was further increased by the subsequent addition of cyto-c, but reached a value similar to that obtained in the absence of magnesium (Fig 1, trace c) Moreover, when MgCl2 was already present in the medium, before the addition of mitochondria (Fig 1, trace d), NADH oxidation was higher than in the control, but, on addition of cyto-c, it was significantly lower than that obtained either in the absence (Fig 1, trace c) or presence of magnesium added after the mitochondria (Fig 1, trace e) Magnesium added to the isotonic medium before the mitochondria had no effect on the rate of NADH oxidation (Fig 1, trace a) The results illustrated in Fig are consistent, at least in part, with those already reported in recent years by two research groups [8–10], showing that the exogenous NADH/cyto-c oxidation rate is greatly increased in hypotonic medium As a new finding, not reported previously, we have shown that, in hypotonic Fig Effect of magnesium ions on exogenous NADH oxidation by mitochondria incubated in isotonic and hypotonic media Rat liver mitochondria (3 mg protein) were incubated in 3.0 mL of isotonic (traces a, b) or hypotonic (traces c–e) medium containing 250 and 25 mM sucrose, respectively, plus 20 mM Hepes (pH 7.4), lM rotenone and lM myxothiazol After of incubation, 0.2 mM NADH (N) was added Further additions: 10 lM cytochrome C (C); mM MgCl2 (Mg); mM potassium cyanide (CN) In trace a, when present, and in trace d, magnesium ions were added to the medium before mitochondria The traces reported are representative of 12 obtained with nine different mitochondrial preparations, and the values reported are the number of nanomoles of NADH oxidized per minute per milligram of protein Statistical significance in hypotonic medium: P = 3.7 · 10)8 (value of trace d versus value 1.2 of trace e); P = 2.5 · 10)4 (value of trace e versus value of trace d); P = 8.8 · 10)5 (value 24 of trace d versus value 45 of trace e) 6170 FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS G La Piana et al medium, magnesium has a differential stimulatory effect depending on whether it is added to the incubation medium before or after the mitochondria The plurality and differential effects of magnesium ions on the activity of the respiratory chain, as well as on many enzymatic reactions and biological processes, have been studied extensively [17–19] Experiments, not reported here, on the effect of Mg2+ on the oxygen uptake supported by succinate oxidation confirmed the results reported by Panov and Scarpa [17], and showed that magnesium, in both isotonic and hypotonic mitochondria, improves the ratio of state 3/state respiration; moreover, no appreciable difference was observed when added either before or after the mitochondria In the succinate oxidation experiments, it appears that the prevailing effect of MgCl2 consists of the stabilization of ADP and ATP molecules, the substrate and product of ATP synthase activity, respectively We have obtained indications that the effect of magnesium ions on the oxidation of substrates present in the matrix space (such as succinate) and catalysed by the respiratory chain is completely different from their effect on the oxidation of exogenous NADH Fig Effect of magnesium ions on both the content of endogenous cyto-c (A) and the binding of exogenous cyto-c (B) to mitochondria incubated in isotonic and hypotonic media Mitochondria (6 mg protein) were incubated in mL of 250 mM (Iso) or 25 mM (Hypo) sucrose-based medium for a total time of 10 (A) and 15 (B), and then centrifuged at 10 000 g for 10 at °C Sequence of additions: (A) at zero time, mitochondria and the reaction stopped by centrifugation either immediately or at 10 (m); at min, mM MgCl2 and the reaction stopped at 10 (m-Mg); at zero time, mitochondria added to the medium already containing mM MgCl2 and the reaction stopped at either or 10 (Mg-m); (B) at zero time, mitochondria, at addition of lM exogenous cyto-c, and reaction stopped at either 10 or 15 (a); alternatively, lM cyto-c added at 10 and reaction stopped at 15 (a); at addition of mM MgCl2, at 10 addition of lM cyto-c, and the reaction stopped at 15 (Mg-c); at addition of lM cyto-c, at 10 addition of mM MgCl2, and the reaction stopped at 15 (c-Mg); mM MgCl2 present in the medium, at addition of lM cyto-c and reaction stopped at 15 (Mg-m) In all samples, at min, lM rotenone plus lM myxothiazol were added to the incubation medium The number of nanomoles of cyto-c present in the pellets of mg protein, expressed as the mean value (± standard deviation) of duplicate samples of seven different mitochondrial preparations, are reported Statistical significance: (A) **P £ 0.003 (control hypotonic versus control isotonic); *P £ 0.03 (m-Mg hypotonic versus control hypotonic); *P £ 0.02 (Mg-m hypotonic versus control hypotonic); (B) ***P £ 0.0002 (control a hypotonic versus control a isotonic); **P £ 0.002 (Mg samples in isotonic medium versus control a isotonic) Magnesium ions and cytosolic cytochrome c oxidation Magnesium-dependent binding of exogenous cyto-c and release of endogenous cyto-c The effect of Mg2+ on the distribution of both endogenous and exogenous cyto-c of mitochondria incubated in isotonic and hypotonic media was determined in the experiments summarized in Fig The amounts present in pellets of mg of mitochondrial protein, incubated in mL of medium and then centrifuged, were determined to better appreciate the difference between each sample As reported in Fig 2A, we found that the content of endogenous cyto-c in the pellets of samples of isotonic mitochondria stopped at zero time (i.e immediately after the addition of mitochondria) was the same as that of samples stopped at the end of a 10-min incubation (samples, m) In addition, in samples with magnesium present in the medium, the content of cyto-c was the same when stopped at either A B FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS 6171 Magnesium ions and cytosolic cytochrome c oxidation G La Piana et al or 10 (samples, Mg-m) The same was observed with hypotonic mitochondria Therefore, these results indicate that endogenous cyto-c is released in a rapid and complete process, and not slowly during the course of incubation Moreover, with an isotonic medium, the presence of magnesium, added either before (Mg-m) or after (m-Mg) the mitochondria, did not influence the cyto-c content of the pellets Hypotonic medium per se promotes the release of no more than 14% of cyto-c from mitochondria (m) and, in this case, the addition of magnesium after a 5-min incubation of mitochondria increased to 35% the release of cyto-c compared with isotonic mitochondria (the additional release was 21%) The latter result can be considered in some aspects to be in line with the concept of the desorption mechanism proposed by Bodrova et al [8] and supported by Lemeshko [9,10] and Scorrano et al [16] However, when magnesium was already present in the hypotonic medium before the addition of mitochondria (Mg-m), the release of cyto-c was significantly lower and amounted to 21% (the additional release was only 7%) The decrease in the release of cyto-c when magnesium is present in the medium recalls its protective effect (reported previously [7]) on the release induced by hypotonic medium of sulfite oxidase and adenylate kinase, which, similar to cyto-c, are both present in MIS Two experimental protocols have been designed (see Materials and methods) to analyse the effect of Mg2+ in preventing or removing the binding of exogenous cyto-c added to isolated mitochondria The results of these experiments are reported in Fig 2B In the presence of exogenously added lm cyto-c, 3.9 nmol was found in the pellets of mg of mitochondrial protein incubated in mL of isotonic medium However, not all of these molecules are of exogenous cyto-c; according to the data reported in Fig 2A, 1.39 nmol derives from endogenous cyto-c More precisely of the total of 12 nmol added, 2.51 nmol remains bound to mitochondria, giving a value of 0.42 nmol bound per milligram of protein The values of the supernatants (not shown) were complementary to those of the pellets in both the absence and presence of Mg2+ Magnesium added before cyto-c (Mg-c) greatly limited its binding to a value of 0.12 nmolỈmg)1, corrected for the 1.39 nmol of endogenous cyto-c From Fig (traces a, b), it can be observed that magnesium added before or after the mitochondria does not influence the activity of the NADH/cyto-c system This may suggest that cyto-c molecules not bound in the presence of magnesium may not be involved directly in the oxidation of exogenous NADH In hypotonic mitochondria, the binding capability is increased to a 6172 value of nmolỈmg)1, calculated after the correction for the 1.2 nmol of endogenous cyto-c, with an increase of 2.4 times compared with isotonic mitochondria It is interesting to note that the extra binding is almost completely prevented in the presence of magnesium as, in both isotonic and hypotonic medium, 2.1 nmol of cyto-c was found in the pellets More relevant is the finding that the total binding of 2.1 nmol of cyto-c (endogenous + exogenous) in both isotonic and hypotonic medium remains the same whether Mg2+ is added before (Mg-c; preventing effect) or after (c-Mg; removal effect) cyto-c The same value of 2.1 nmol was obtained with magnesium present in the medium before the addition of mitochondria (Mg-m) However, in hypotonic medium, the binding of exogenous cyto-c in the presence of magnesium, added either before or after cyto-c, was still higher than that in isotonic medium, with a value of 0.2 nmolỈmg)1 obtained by subtracting, from the 2.1 nmol, the value of 0.91 nmol of endogenous cyto-c reported in Fig 2A (and divided by mg of protein) The finding that the oxidation rate of exogenous NADH after the addition of cyto-c is essentially the same with the hypotonic medium, in the absence or presence of magnesium (Fig 1, traces c, e), may suggest that only the nanomoles of cyto-c still bound in the presence of magnesium are directly involved in the activity of the NADH/cyto-c system Notwithstanding that, in the presence of magnesium and independent of the sequence of additions, the total binding of cyto-c remains the same (Fig 2B, Mg present), the rate of NADH oxidation is decreased to 24 nmolỈmin)1Ỉmg)1 (Fig 1, trace d) with magnesium present in the medium, compared with a value of 45 nmolỈmin)1Ỉmg)1 with magnesium added after the mitochondria (Fig 1, trace e) This may indicate that remodelling of the mitochondrial structure is involved [16,20] Magnesium-dependent cyto-c release activates, in hypotonic medium, the NADH/cyto-c system with the generation of a membrane potential As reported for the first time in 1995 [3], the activity of the cytosolic NADH/cyto-c electron transport pathway is coupled, similar to the activity of the respiratory chain, to the generation of an electrochemical proton gradient determined also as the mitochondrial membrane potential change (DYm) [4,21] The change in DYm generated by the NADH/cyto-c system is similar to that supported either by succinate oxidation or ATP hydrolysis [21] The fluorimetric determination of DYm of mitochondria incubated in isotonic medium with 10 lm safranine as probe is reported in Fig As a FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS G La Piana et al Fig Mitochondrial membrane potential generated by the oxidation of either endogenous respiratory substrates or extra mitochondrial NADH in isotonic (A) and hypotonic (B) media (A) Mitochondria (3 mg protein, ‘mito’) were added to mL of 250 mM sucrose isotonic medium containing 0.1 mgỈmL)1 BSA, 50 lM EGTA and 10 lM safranine (B) Mitochondria (3 mg protein) were incubated for in mL of 25 mM sucrose hypotonic medium containing lM rotenone, lM myxothiazol, 0.1 mgỈmL)1 BSA, 50 lM EGTA and 10 lM safranine Further additions: lM rotenone plus lM myxothiazol (RM); lM and, only in trace c, lM cyto-c (C); 166 lM NAD+(N); 45 IU alcohol dehydrogenase (A); mM MgCl2 (Mg); 1.6 lM FCCP (F); mM potassium cyanide (CN); 30 lM TMPD (T) In trace e, mM MgCl2 was already present in the incubation medium before the addition of mitochondria Experiments reported are representative of seven performed with five different mitochondrial preparations result of the very low electron pressure provided by the oxidation of endogenous substrates and the activity of the NADH/cyto-c system, small amounts of both BSA and EGTA were added to stabilize the membrane potential Consistent with the previously reported data [4,21], Fig (trace a) shows that DYm, supported by the oxidation of endogenous substrates, is abolished by the addition of the respiratory chain inhibitors (rotenone and myxothiazol) The subsequent activation of the NADH/cyto-c system with the sequential addition of cyto-c, NAD+ and alcohol dehydrogenase restores DYm With this experimental approach, NADH is continuously produced outside the mitochondria by the activity of added alcohol dehydrogenase, which catalyses the oxidation of ethanol present in the incubation medium as the solvent of rotenone and myxothiazol Magnesium ions and cytosolic cytochrome c oxidation solutions It can be observed that the addition of cyto-c promotes a nonspecific change in fluorescence, which is proportional to the amount of cyto-c added and is not abolished by uncouplers, and therefore was electrically reset down Figure (trace a) also shows that the uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) dissipates DYm, supported by the oxidation of NADH present outside the mitochondria Dissipation of the membrane potential can also be obtained with the addition of cyanide (Fig 3, trace b) These results confirm that the fluorescence signal is the expression of an electrical charge gradient between the outside and inside of the mitochondria, and that cytochrome oxidase is involved in this process Moreover, the experimental approach in Fig mimics in vitro the generation of cytosolic NADH, as well as the activation of the NADH/cyto-c system when, in physio-pathological conditions (e.g apoptosis), a catalytic amount of cyto-c is released from the mitochondria Magnesium ions added after the mitochondria, but before the activation of the NADH/cyto-c system (Fig 3, trace b), promote a slight and sometimes not appreciable decrease in DYm linked to the oxidation of exogenous NADH Results identical to those illustrated in Fig (trace b) were obtained when MgCl2 was added to the isotonic medium before the mitochondria (not reported) Figure (trace c) shows that, also with hypotonic mitochondria, the activation of the NADH/cyto-c electron transport system generates a membrane potential According to the sequence of additions, it can be seen that the generation of DYm is strictly linked to the presence of exogenous cyto-c as the electron intermediate However, with hypotonic mitochondria, a lm or lower cyto-c concentration is sufficient to obtain the full expression of the membrane potential (see Fig 3, traces a and c) It should be noted that, in Fig (traces c–f), mitochondria were preincubated for in the presence of respiratory chain inhibitors (rotenone and myxothiazol) to suppress the membrane potential generated by the oxidation of endogenous substrates (Fig 3, traces a, b) In the presence of magnesium (Fig 3, trace d), DYm was generated even without the addition of exogenous cyto-c, but was still sensitive to the uncoupler dissipation effect These results are substantially consistent with those reported in [8,9] Furthermore, as a new and original finding, Fig also shows that, if magnesium is present in the incubation medium before the addition of mitochondria (Fig 3, trace e), cyto-c is required to generate DYm, similar to the results obtained in the absence of Mg2+ (Fig 3, trace c) The comparison of the results in Fig (traces e and f) gives a clear view of the FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS 6173 Magnesium ions and cytosolic cytochrome c oxidation G La Piana et al differential effect of magnesium ions, whether already present in the hypotonic medium or added after the mitochondria It also provides direct evidence that, in these latter conditions, the addition of magnesium mimics the results obtained with the addition of cyto-c, the only difference being that the potential is lower and N,N,N¢,N¢-tetramethyl-p-phenylenediamine (TMPD) must be added to achieve its complete expression Effect of magnesium on the two semi-reactions of the NADH/cyto-c system The possibility that magnesium, other than having a protective effect on the permeability of mitochondria incubated in hypotonic medium (see Fig and [7]), may also directly affect the activity of the NADH/ cyto-c system was tested in the experiments reported in Fig The activity of the system was split into two main steps: (a) the reduction of exogenous cyto-c induced by the oxidation of NADH in cyanide-inhibited respiration (Fig 4A); and (b) the oxidation of exogenous ferrocytochrome c (ferrocyto-c) (Fig 4B) It was found (Fig 4A) that the NADH/ cyto-c reaction rate was similar in both isotonic and Fig Reduction and oxidation of exogenous cyto-c by mitochondria incubated in isotonic and hypotonic media (A) Activity of rotenone-insensitive NADH-cytochrome c reductase (B) Oxidation of ferrocyto-c present outside the mitochondria Mitochondria (90 lg protein in A and 1.0 mg protein in B) were incubated for in mL of 250 mM (Iso) or 25 mM (Hypo) sucrose-based medium containing lM rotenone and lM myxothiazol (A) 15 lM ferricyto-c plus mM potassium cyanide were also present, and the reaction was started with the addition of 0.2 mM NADH (N) (B) The reaction was started with 15 lM ferrocyto-c Incubations were made in the absence (traces a and c) or presence of mM MgCl2, added either before (traces b, d and f) or after (traces b and d) the addition of mitochondria; in trace e, magnesium was either absent or added after the mitochondria Values on the traces represent the nanomoles of cyto-c reduced (A) or oxidized (B) per minute per milligram of protein, and are representative of eight experiments performed with five different mitochondrial preparations 6174 hypotonic mitochondria This is an expected result if it is considered that the reaction being catalysed by the NADH/cyto-b5 complex sited on the external side of MOM occurs outside the mitochondria, and therefore should be independent of the osmolarity of the medium In the presence of magnesium (Fig 4, trace b), an increased rate of cyto-c reduction, usually not higher than 15%, was observed in both isotonic and hypotonic media In Fig (traces c and d), it is shown that the oxidation rate of exogenous cyto-c by isotonic mitochondria is not affected by the presence of MgCl2, added either before or after the mitochondria With hypotonic mitochondria, the oxidation rate is greatly increased (Fig 4, trace e) and magnesium decreases this rate only when present in the medium (Fig 4, trace f), but has no effect when added after the mitochondria (Fig 4, trace e) The data reported in Fig 4B are consistent with and similar to those in Fig on the oxidation of exogenous NADH as the expression of the activity of the complete system Discussion The data presented provide new insights into the role of magnesium ions in the permeability of MOM to both endogenous and exogenous cyto-c, and provide further support for the functional activity, in liver mitochondria, of the cytosolic NADH/cyto-c electron transport system The oxidation of exogenous NADH occurs exclusively if cyto-c is also present outside the mitochondria It is not relevant if cyto-c is added externally, as in the case of isotonic mitochondria, or is released outside from MIS when mitochondria are incubated in hypotonic medium (Figs and 2) In both cases, the activity of the system generates DYm (Fig 3), which contradicts the interpretation that the system could represent the expression of completely broken mitochondria [8–10] and/or of mitochondria with MOM broken at leopard’s spots This interpretation is also in contrast with the finding that exogenous cyto-c is unable to react with sulfite oxidase present in MIS [7], and that dextran sulfate inhibits exogenous NADH oxidation in intact mitochondria, but not in mitoplast preparations [6] Comparing the data reported in [7] with those in Fig 2, it is clear that, in hypotonic mitochondria, endogenous cyto-c is released outside, although in a limited amount (14%), but cyto-c present in the medium is unable to move from outside into MIS [7] Therefore, MOM of mitochondria incubated in either isotonic sucrose or very low osmotic medium (25 mm sucrose) is not broken, as generally believed, but still maintains the function of a FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS G La Piana et al protective envelope not permeable to either exogenous cyto-c or trypsin [7] The activity of succinate/exogenous cyto-c reductase and the oxidation of exogenous ferrocyto-c, long proposed to be the expression of broken and damaged mitochondria [22–24], are both misleading and inappropriate to extrapolate the percentage of intact mitochondria Previously published results [5–7], and those presented in this report on the effect of magnesium ions, are consistent with the view that both the succinate/cyto-c reductase activity and the oxidation rate of exogenous ferrocyto-c are correlated with the frequency of specific contact sites With regard to the effect of magnesium on the activity of the cytosolic NADH/cyto-c electron transport system, our data show a new finding, not elicited previously, that magnesium presents a dual effect very clear in hypotonic but not visible in isotonic mitochondria The first effect involves protection against the increase in permeability of MOM when magnesium is present in the hypotonic medium before the addition of mitochondria Experimental data have shown that contact sites [5,6], indicated as respiratory contact sites, are the mitochondrial structures in which the main components of the NADH/cyto-c system are localized In 1995, we defined the respiratory contact sites as dynamic but not fixed structures, which could be visualized as frequently forming and breaking bridges between different points of the inner and outer membranes; over time, the two membranes could be involved in the contact sites, forming these structures in all their parts [3] Therefore, as a result of the much greater area of MIM than MOM, the increase in the matrix volume by hypotonic medium pushes MIM against MOM, which becomes stretched; its permeability is increased and the contact points between the two mitochondrial membranes are also expected to increase In these conditions, the oxidation rate of exogenous NADH is greatly increased (Fig 1) If Mg2+ is added before the mitochondria, the release linked to the hypotonic medium of adenylate kinase, sulfite oxidase and endogenous cyto-c is, to a large extent, prevented relative to the findings obtained when Mg2+ is added after the mitochondria ([7] and Fig 2), and the oxidation rate of NADH is also decreased from 45–49 to 24 nmolỈmin)1Ỉmg)1 (Fig 1) Tentatively, it could be hypothesized that magnesium, with its two positive charges, may function as a linker between the negative charges of phospholipids and/or proteins MOM and MIM become more compact, offering more resistance to the stretching caused by the pressure induced by the increased volume of the matrix The permeability of MOM is decreased significantly, together with the frequency of contact sites Magnesium ions and cytosolic cytochrome c oxidation Therefore, the rates of NADH oxidation (Fig 1) and of the succinate/exogenous cyto-c reductase [7] are both decreased greatly The second effect is linked to the property of magnesium to prevent and remove the binding of exogenous cyto-c depending on whether it is added before or after cyto-c (Fig 2B) In hypotonic medium, cyto-c binding increases, which tentatively could be the consequence of MOM stretching with the disclosure of additional nonspecific binding sites However, this increase is not responsible for the increased rate of NADH oxidation, as the extra binding is completely removed by the addition of magnesium, but the oxidation rate is not affected and still remains high (Figs and 2B) Inhibition of the rate is observed when magnesium is already present in the medium to prevent the binding of cyto-c, which, however, is identical to the binding observed with magnesium added after the mitochondria or when Mg2+ is already present in the medium Therefore, the decreased rate must be ascribed to the above-mentioned protective effect of magnesium on the structural remodelling of the two mitochondrial membranes, elicited when present in the medium (but not when added after the mitochondria), rather than to the binding of cyto-c All of these considerations indicate that cyto-c present outside the mitochondria is essentially in the free form, available to shuttle electrons between the NADH/cyto-b5 reductase and the respiratory contact sites (see the scheme in [6]) However, it can be speculated that, corrected for the amount of endogenous cyto-c, the 120 pmol of exogenous cyto-c bound per milligram of protein of isotonic mitochondria, and insensitive to the presence of magnesium, may be the expression of the molecules tightly bound essentially to both the cytosolic side of the respiratory contact sites and the NADH/cyto-b5 reductase complex, rather than to nonspecific sites Therefore, one possible mechanism may be that, of all the cyto-c molecules added to isolated mitochondria or present in the cytosol, only a few, in relation to the binding sites available, remain firmly bound, some to the NADH/cyto-b5 complex and some to contact sites The majority of cyto-c molecules are free to move and, in the oxidized state (with an intermolecular process), accept electrons from reduced molecules bound to the NADH/cyto-b5 system; in the reduced state, they transfer their electrons to oxidized cyto-c bound to contact sites With hypotonic mitochondria, it can be calculated that the amount of cyto-c bound and insensitive to magnesium added after mitochondria is 200 pmolỈmg)1 protein These results correlate with the differential rate of NADH oxidation by isotonic and hypotonic mitochondria reported in Fig As the FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS 6175 Magnesium ions and cytosolic cytochrome c oxidation G La Piana et al number of units per milligram of protein of the NADH/cyto-b5 complex should be independent of the osmolarity of the medium, the increase in the binding of cyto-c with hypotonic mitochondria and the insensitivity to magnesium ions could essentially be the expression of the increased number of respiratory contact sites Additional and direct experimental data are required to provide further support for these speculations The finding that MgCl2 promotes the release of an additional amount of endogenous cyto-c with hypotonic but not isotonic mitochondria is consistent with the view that, in physiological low-amplitude swelling, similar to the experimental large-amplitude swelling induced by hypotonic medium, the contact area between the two mitochondrial membranes is increased extensively Cyto-c molecules still bound to the external leaflet of MIM turn to face the medium and are more accessible to displacement by magnesium This interpretation correlates with the observation that the large increase in binding of externally added cyto-c, observed in hypotonic mitochondria, is completely removed or prevented by magnesium However, consistent with the desorption mechanism [8,16], the possibility that cyto-c bound to MIM is removed by magnesium, and then released outside because of the increased permeability of MOM, cannot be excluded The data presented here, together with those reported in [7], show that magnesium may have a dual role in the permeability of mitochondrial membranes: (a) it counteracts the remodelling of membrane structures induced by low-amplitude physiological swelling or, in general, by cell injury; and (b) it contributes to the correct execution of the cell death programme by promoting the release of cyto-c from mitochondria In our previous publications, we have emphasized that, activated by the presence of cyto-c outside the mitochondria, the NADH/cyto-c system may have at least two functions The first involves the promotion, in healthy cells, of the oxidation of cytosolic NADH utilizing the mitochondrial machinery to generate ATP with the energy preserved in the membrane potential (Fig 3) This activity becomes essential for cell survival in the presence of an impairment of the respiratory chain at the level of one of the first three respiratory complexes The second function concerns its role in the apoptotic programme It is well known that, in the early stages of this process, cyto-c is released into the cytosol where it participates in the formation of apoptosomes, responsible for the activation of caspases, leading to nuclear condensation and the formation of apoptotic bodies However, the release of cyto-c from mitochondria promotes an impairment of the respiratory chain, 6176 followed by a relevant decrease in the energy content of the cell in relation to the amount of cyto-c transferred into the cytosol This raises the problem of the energy source required for the correct execution of the apoptotic programme Indeed, in the early stages of apoptosis, mitochondria continue to generate a membrane potential [25–27] which, according to some authors, can be ascribed to hydrolysis, inside the mitochondria, of ATP generated by glycolytic activity [28] We maintain that, in these conditions, the cytosolic cyto-c activates the NADH/cyto-c electron transport pathway, and more energy is made available for apoptotic processing before the membrane potential dissipation step is activated Indications have been obtained which support the transient participation of cyto-c in the formation of apoptosomes, as cyto-c has not been found in preparations of precipitated native apoptosomes [29] and a smaller amount of cyto-c has been found relative to that of Apaf-1 in mature apoptosomes [30] The availability of energy, either as ATP or in the form of a membrane potential, is a prerequisite to make apoptotic cell death a programmed and controlled process distinct from necrosis, which does not require energy as it is characterized by acute disruption of cellular metabolism Therefore, the activation of the NADH/cyto-c system may represent an additional source of energy for the correct execution of the apoptotic programme Recently, it has been reported that, in homogenates of apoptotic HeLa cells, the reduction rate of added cyto-c is lower than that in homogenates of control cells [31] In the presence of azide, the reduction rate is increased and the values obtained are identical in both types of homogenate The decreased rate in the reduction of cyto-c has been ascribed to an increased involvement of mitochondria present in homogenates of apoptotic cells, responsible for the oxidation of reduced cyto-c Data have also been reported showing that, in mice liver mitochondria, magnesium ions are involved in Bax- and Bid-induced cyto-c release [32,33] The finding that magnesium ions regulate the permeability of mitochondria and the binding of cyto-c may have relevant implications in the bioenergetics of the cell, as well as possible consequences in therapeutic applications In tumour cells, an increase in the concentration of cytosolic cyto-c may contribute to activate the apoptotic process Experiments are in progress in our laboratory to measure and characterize, in healthy and apoptotic HeLa cells, the activity of the cytosolic NADH/cyto-c electron transport system, and the role of magnesium ions in modulating the transfer of cyto-c from mitochondria into the cytosol FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS G La Piana et al Materials and methods Incubation of mitochondria Rat liver mitochondria were isolated by differential centrifugation in 250 mm sucrose medium, as described previously [3] Incubations were carried out at 25 °C at pH 7.4 in media consisting of 20 mm Hepes/Tris and either 250 mm (isotonic medium) or 25 mm (hypotonic medium) sucrose The intactness of mitochondrial membranes was routinely determined by four different but convergent and already described [7] integrity tests based on the following activities: (a) the oxidation of exogenously added NADH in the absence of both rotenone and exogenous cyto-c to assess the impermeability of NADH through MIM; (b) the insensitivity of intermembrane adenylate kinase to proteolytic attack by added trypsin to reveal the increased permeability, if any, of MOM during the course of incubation; (c) the sulfite/exogenous cyto-c oxido-reductase activity coupled to its sensitivity to trypsin to assess the impermeability of exogenous cyto-c through MOM; (d) the succinate/exogenous cyto-c oxido-reductase activity to reveal the presence of damaged mitochondria with MIM intact but with MOM permeable to exogenous cyto-c Mitochondrial suspensions containing no more than 2% of damaged mitochondria, according to both the NADH oxidation test (a) and sulfite/exogenous cyto-c test (c), were utilized (see also [7]) NADH oxidation was determined spectrophotometrically at 340–374 nm (e = 4.28 mm)1Ỉcm)1) and the redox state of cyto-c at 548–540 nm (e = 21 mm)1Ỉcm)1) to minimize the interference of cyto-b5 sited on MOM, which has an absorbance peak at 556 nm The protein content was determined by the biuret method Cyto-c content of mitochondria incubated in the absence and presence of exogenous cyto-c The determination of endogenous cyto-c was performed in pellets of mg protein of mitochondria incubated for 10 in mL of both isotonic and hypotonic media, and then centrifuged at 10 000 g for 10 at °C Magnesium ions were added at a concentration of mm according to the sequence of additions specified in the legend to Fig The capability of magnesium ions to both prevent and remove the binding of exogenously added cyto-c was determined in isotonic and hypotonic media with two experimental protocols In the first procedure, mm MgCl2 was added after the mitochondria, but before lm cyto-c was added at 10 min, and the reaction was stopped at 15 In the second procedure, lm of cyto-c was added at min, mm MgCl2 at 10 and the reaction was stopped at 15 Details of the sequence of additions are reported in the legend to Fig To increase the reliability of the results and to better appreciate the changes induced by magnesium, mitochondria containing mg of protein were incubated in mL of medium, centrifuged as specified Magnesium ions and cytosolic cytochrome c oxidation above and the pellets resuspended in 1.0 mL of 50 mm Pi (pH 7.4) supplemented with 0.5% Triton X100 The cyto-c content of the pellets and supernatants was determined from a reduced minus oxidized differential spectrum in the wavelength range 500–650 nm, utilizing potassium ferricyanide as oxidant and sodium dithionite as reductant Spectra and kinetic determinations were carried out with HitachiPerkin Elmer model 557 (Hitachi, Ltd., Tokyo, Japan), Varian Cary model 50 (Varian Inc., Melbourne, Australia) and Aminco DW2A double-wavelength [modernized by OLIS (On Line Instruments Inc., Bogart, GA, USA)] spectrophotometers Determination of mitochondrial membrane potential Time-dependent mitochondrial membrane potential changes (DYm) were followed fluorimetrically with a Perkin-Elmer LS-5B fluorescence luminometer, with 10 lm safranine O at wavelengths of 520 nm (excitation) and 580 nm (emission) [34] Materials All reagents were of analytical grade and mainly obtained from Sigma-Aldrich Chemical Co (St Louis, MO, USA) and Roche Spa (Milan, Italy) Ferrocyto-c was prepared daily as reported in [6] Acknowledgements The authors are grateful to Mr Francesco Felice for his skilled technical assistance This work was supported by grants from MIUR (Prin 2005–2007 ‘Bioenergetica: meccanismi molecolari e aspetti fisiopatologici 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well as possible consequences in therapeutic applications In tumour cells, an increase in the concentration of cytosolic cyto -c may contribute... succinate/cyto -c reductase activity and the oxidation rate of exogenous ferrocyto -c are correlated with the frequency of speci? ?c contact sites With regard to the effect of magnesium on the activity of