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

Starting from the obser-vation that isolated mitochondria oxidize NADH present outside the mitochondria only if cytochrome c cyto-c is also added to the incubation medium, and supported

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

1 Department of Biochemistry and Molecular Biology, University of Bari, Italy

2 Institute of Biomembranes and Bioenergetics (IBBE - CNR), University of Bari, Italy

The oxidative and energetic metabolism of glucose

requires cytosolic NADH to be oxidized by the

respi-ratory 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

mitochon-drial 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 flavopro-teins inside the mitochondria Starting from the obser-vation 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

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 5

September 2008, accepted 14 October

2008)

doi:10.1111/j.1742-4658.2008.06741.x

Cytochrome c (cyto-c), added to isolated mitochondria, activates the oxida-tion of extramitochondrial NADH and the generaoxida-tion 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 protec-tive 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 pro-motes 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 exoge-nously added cyto-c We suggest that, in physiological low-amplitude swell-ing, magnesium ions may have the function, together with other factors, of modulating the amount of cyto-c molecules transferred from the mitochon-drial intermembrane space into the cytosol, required for the correct execu-tion of the apoptotic programme and/or the activaexecu-tion of the NADH/ cyto-c electron transport pathway

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; DY m , mitochondrial membrane potential change.

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

com-ponents of this additional electron transport chain are

sited on the ‘respiratory contact sites’ [5,6], but

endogenous cyto-c, present in the mitochondrial

inter-membrane 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

experimen-tal 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

sys-tem 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

pro-ton gradient [3] In apoptotic cells, with the

impair-ment 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

path-way are as follows: NAD-dependent dehydrogenases

present in the cytosol; cytosolic NADH; the

rotenone-insensitive 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-depen-dent anion channel or porin of MOM is juxtaposed to

the cytochrome oxidase molecules spanning MIM All

of these components are required for the correct

exe-cution 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

meth-ods, 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

prerequisite to study the activity of the respiratory chain, but becomes mandatory for the new, additional and independent NADH/cyto-c electron transport sys-tem to counteract the criticism that the results obtained can be ascribed to damaged or broken mito-chondria One test, based on the determination of sul-fite/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 oxido-reductase 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 limit-ing 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 meable to exogenous NADH and with MOM not per-meable 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 incu-bated 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 med-ium, 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

cyto-c 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 oxi-dase of MIM is in contrast with the already men-tioned 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 mito-chondria is not permeable through MOM [7], we car-ried 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

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 1 (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

pres-ence 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

sub-stances, an incubation medium simply consisting of

sucrose and buffer to stabilize the pH was utilized

Figure 1 (trace a) also shows that the oxidation rate

was blocked by cyanide, suggesting the involvement of

cytochrome oxidase The addition of 4 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 subse-quent 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 signifi-cantly 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 1 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 1 Effect of magnesium ions on exogenous NADH oxidation by mitochondria incubated in isotonic and hypotonic media Rat liver mito-chondria (3 mg protein) were incubated in 3.0 mL of isotonic (traces a, b) or hypotonic (traces c–e) medium containing 250 and 25 m M

sucrose, respectively, plus 20 m M Hepes (pH 7.4), 6 l M rotenone and 6 l M myxothiazol After 2 min of incubation, 0.2 m M NADH (N) was added Further additions: 10 l M cytochrome C (C); 4 m M MgCl2(Mg); 1 m M 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 differ-ent 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 5 of trace d versus value 1.2 of trace e); P = 2.5 · 10)4(value 9 of trace

e versus value 5 of trace d); P = 8.8 · 10)5(value 24 of trace d versus value 45 of trace e).

medium, magnesium has a differential stimulatory

effect depending on whether it is added to the

incuba-tion 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

oxy-gen 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 4 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

Magnesium-dependent binding of exogenous cyto-c and release of endogenous cyto-c The effect of Mg2+on the distribution of both endog-enous and exogendog-enous cyto-c of mitochondria incubated

in isotonic and hypotonic media was determined in the experiments summarized in Fig 2 The amounts pres-ent in pellets of 6 mg of mitochondrial protein, incu-bated in 6 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 sam-ples 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 sam-ples with magnesium present in the medium, the con-tent of cyto-c was the same when stopped at either 5

A

B

Fig 2 Effect of magnesium ions on both the content of

endoge-nous cyto-c (A) and the binding of exogeendoge-nous cyto-c (B) to

mito-chondria incubated in isotonic and hypotonic media Mitomito-chondria

(6 mg protein) were incubated in 6 mL of 250 m M (Iso) or 25 m M

(Hypo) sucrose-based medium for a total time of 10 min (A) and

15 min (B), and then centrifuged at 10 000 g for 10 min at 4 C.

Sequence of additions: (A) at zero time, mitochondria and the

reac-tion stopped by centrifugareac-tion either immediately or at 10 min (m);

at 5 min, 4 m M MgCl 2 and the reaction stopped at 10 min (m-Mg);

at zero time, mitochondria added to the medium already containing

4 m M MgCl2 and the reaction stopped at either 5 or 10 min

(Mg-m); (B) at zero time, mitochondria, at 5 min addition of 2 l M

exogenous cyto-c, and reaction stopped at either 10 or 15 min (a);

alternatively, 2 l M cyto-c added at 10 min and reaction stopped at

15 min (a); at 5 min addition of 4 m M MgCl 2 , at 10 min addition of

2 l M cyto-c, and the reaction stopped at 15 min (Mg-c); at 5 min

addition of 2 l M cyto-c, at 10 min addition of 4 m M MgCl2, and the

reaction stopped at 15 min (c-Mg); 4 m M MgCl2 present in the

medium, at 5 min addition of 2 l M cyto-c and reaction stopped at

15 min (Mg-m) In all samples, at 2 min, 6 l M rotenone plus 6 l M

myxothiazol were added to the incubation medium The number of

nanomoles of cyto-c present in the pellets of 6 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

iso-tonic).

or 10 min (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

med-ium, the presence of magnesmed-ium, 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

incuba-tion 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

previ-ously [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

pres-ence of exogenously added 2 lm cyto-c, 3.9 nmol was

found in the pellets of 6 mg of mitochondrial protein

incubated in 6 mL of isotonic medium However, not

all of these molecules are of exogenous cyto-c;

accord-ing 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

mito-chondria, giving a value of 0.42 nmol bound per

milli-gram 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 nmolÆmg)1, corrected for the

1.39 nmol of endogenous cyto-c From Fig 1 (traces

a, b), it can be observed that magnesium added before

or after the mitochondria does not influence the

activ-ity 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

value of 1 nmolÆmg)1, calculated after the correction for the 1.2 nmol of endogenous cyto-c, with an increase of 2.4 times compared with isotonic mitochon-dria It is interesting to note that the extra binding is almost completely prevented in the presence of magne-sium as, in both isotonic and hypotonic medium, 2.1 nmol of cyto-c was found in the pellets More rele-vant 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 exo-genous 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 nmolÆmg)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 6 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 nmolÆmin)1Æmg)1 (Fig 1, trace d) with magnesium present in the med-ium, compared with a value of 45 nmolÆmin)1Æmg)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 mem-brane 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 3 As a

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 3 (trace a) shows that DYm, supported by

the oxidation of endogenous substrates, is abolished by

the addition of the respiratory chain inhibitors

(rote-none 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

con-tinuously produced outside the mitochondria by the

activity of added alcohol dehydrogenase, which

cataly-ses the oxidation of ethanol present in the incubation

medium as the solvent of rotenone and myxothiazol

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 3 (trace a) also shows that the uncoupler carbonyl cyanide p-(trifluoromethoxy)phen-ylhydrazone (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 cyto-chrome oxidase is involved in this process Moreover, the experimental approach in Fig 3 mimics in vitro the generation of cytosolic NADH, as well as the activa-tion of the NADH/cyto-c system when, in physio-path-ological conditions (e.g apoptosis), a catalytic amount

of cyto-c is released from the mitochondria Magne-sium 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 DYmlinked to the oxidation of exogenous NADH Results identical to those illustrated in Fig 3 (trace b) were obtained when MgCl2 was added to the isotonic medium before the mitochondria (not reported)

Figure 3 (trace c) shows that, also with hypotonic mitochondria, the activation of the NADH/cyto-c elec-tron 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 intermedi-ate However, with hypotonic mitochondria, a 1 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 3 (traces c–f), mitochondria were preincubated for 5 min

in the presence of respiratory chain inhibitors (rote-none and myxothiazol) to suppress the membrane potential generated by the oxidation of endogenous substrates (Fig 3, traces a, b) In the presence of mag-nesium (Fig 3, trace d), DYmwas generated even with-out 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 3 also shows that, if magnesium is present in the incubation medium before the addition of mitochon-dria (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 3 (traces e and f) gives a clear view of the

Fig 3 Mitochondrial membrane potential generated by the

oxida-tion of either endogenous respiratory substrates or extra

mitochon-drial NADH in isotonic (A) and hypotonic (B) media (A)

Mitochondria (3 mg protein, ‘mito’) were added to 3 mL of 250 m M

sucrose isotonic medium containing 0.1 mgÆmL)1 BSA, 50 l M

EGTA and 10 l M safranine (B) Mitochondria (3 mg protein) were

incubated for 5 min in 3 mL of 25 m M sucrose hypotonic medium

containing 6 l M rotenone, 6 l M myxothiazol, 0.1 mgÆmL)1 BSA,

50 l M EGTA and 10 l M safranine Further additions: 6 l M rotenone

plus 6 l M myxothiazol (RM); 5 l M and, only in trace c, 1 l M cyto-c

(C); 166 l M NAD + (N); 45 IU alcohol dehydrogenase (A); 4 m M

MgCl 2 (Mg); 1.6 l M FCCP (F); 1 m M potassium cyanide (CN); 30 l M

TMPD (T) In trace e, 4 m M MgCl2was already present in the

incu-bation medium before the addition of mitochondria Experiments

reported are representative of seven performed with five different

mitochondrial preparations.

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-phenylene-diamine (TMPD) must be added to achieve its

com-plete 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 2 and [7]),

may also directly affect the activity of the NADH/

cyto-c system was tested in the experiments reported

in Fig 4 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

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, usu-ally not higher than 15%, was observed in both iso-tonic and hypoiso-tonic media In Fig 4 (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 1 on the oxidation of exo-genous 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 1 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 interpreta-tion 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 mitochon-dria 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

Fig 4 Reduction and oxidation of exogenous cyto-c by

mitochon-dria incubated in isotonic and hypotonic media (A) Activity of

rote-none-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 5 min in

3 mL of 250 m M (Iso) or 25 m M (Hypo) sucrose-based medium

con-taining 6 l M rotenone and 6 l M myxothiazol (A) 15 l M ferricyto-c

plus 1 m M potassium cyanide were also present, and the reaction

was started with the addition of 0.2 m M NADH (N) (B) The reaction

was started with 15 l M ferrocyto-c Incubations were made in the

absence (traces a and c) or presence of 4 m M MgCl 2 , added either

before (traces b, d and f) or 2 min after (traces b and d) the addition

of mitochondria; in trace e, magnesium was either absent or added

2 min 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.

protective envelope not permeable to either exogenous

cyto-c or trypsin [7] The activity of

succinate/exoge-nous cyto-c reductase and the oxidation of exogesuccinate/exoge-nous

ferrocyto-c, long proposed to be the expression of

bro-ken and damaged mitochondria [22–24], are both

mis-leading and inappropriate to extrapolate the

percentage of intact mitochondria Previously

pub-lished 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

activ-ity 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

activ-ity of the cytosolic NADH/cyto-c electron transport

system, our data show a new finding, not elicited

pre-viously, that magnesium presents a dual effect very

clear in hypotonic but not visible in isotonic

mitochon-dria 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

permeabil-ity 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 nmolÆmin)1Æmg)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

signifi-cantly, together with the frequency of contact sites

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 mag-nesium to prevent and remove the binding of exoge-nous 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 conse-quence of MOM stretching with the disclosure of addi-tional nonspecific binding sites However, this increase

is not responsible for the increased rate of NADH oxi-dation, as the extra binding is completely removed by the addition of magnesium, but the oxidation rate is not affected and still remains high (Figs 1 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 mitochon-dria 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 elec-trons between the NADH/cyto-b5 reductase and the respiratory contact sites (see the scheme in [6]) How-ever, it can be speculated that, corrected for the amount of endogenous cyto-c, the 120 pmol of exo-genous cyto-c bound per milligram of protein of iso-tonic 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-b5complex 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 insen-sitive to magnesium added after mitochondria is

200 pmolÆmg)1protein These results correlate with the differential rate of NADH oxidation by isotonic and hypotonic mitochondria reported in Fig 1 As the

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

insensi-tivity to magnesium ions could essentially be the

expression of the increased number of respiratory

con-tact sites Additional and direct experimental data are

required to provide further support for these

specula-tions

The finding that MgCl2 promotes the release of an

additional amount of endogenous cyto-c with

hypo-tonic but not isohypo-tonic 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

exter-nal 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,

consis-tent with the desorption mechanism [8,16], the

possibil-ity 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

pro-moting the release of cyto-c from mitochondria In our

previous publications, we have emphasized that,

acti-vated by the presence of cyto-c outside the

mitochon-dria, 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

mitochon-dria promotes an impairment of the respiratory chain,

followed by a relevant decrease in the energy content

of the cell in relation to the amount of cyto-c trans-ferred 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 mem-brane potential [25–27] which, according to some authors, can be ascribed to hydrolysis, inside the mito-chondria, 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 apop-totic processing before the membrane potential dissipa-tion step is activated Indicadissipa-tions 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 apopto-somes [29] and a smaller amount of cyto-c has been found relative to that of Apaf-1 in mature apopto-somes [30] The availability of energy, either as ATP

or in the form of a membrane potential, is a prerequi-site to make apoptotic cell death a programmed and controlled process distinct from necrosis, which does not require energy as it is characterized by acute dis-ruption of cellular metabolism Therefore, the activa-tion 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 find-ing that magnesium ions regulate the permeability of mitochondria and the binding of cyto-c may have rele-vant implications in the bioenergetics of the cell, as well as possible consequences in therapeutic applica-tions 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 mito-chondria into the cytosol

Materials and methods

Incubation of mitochondria

Rat liver mitochondria were isolated by differential

centrifu-gation in 250 mm sucrose medium, as described previously

[3] Incubations were carried out at 25C 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

intact-ness of mitochondrial membranes was routinely determined

by four different but convergent and already described [7]

integrity tests based on the following activities: (a) the

oxida-tion 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

intermem-brane 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

oxi-do-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

oxida-tion test (a) and sulfite/exogenous cyto-c test (c), were utilized

(see also [7]) NADH oxidation was determined

spectropho-tometrically at 340–374 nm (e = 4.28 mm)1Æcm)1) and the

redox state of cyto-c at 548–540 nm (e = 21 mm)1Æcm)1) to

minimize the interference of cyto-b5sited 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 6 mg protein of mitochondria incubated for

10 min in 6 mL of both isotonic and hypotonic media, and

then centrifuged at 10 000 g for 10 min at 4C Magnesium

ions were added at a concentration of 4 mm according to

the sequence of additions specified in the legend to Fig 2

The capability of magnesium ions to both prevent and

remove the binding of exogenously added cyto-c was

deter-mined in isotonic and hypotonic media with two

experi-mental protocols In the first procedure, 4 mm MgCl2was

added 5 min after the mitochondria, but before 2 lm cyto-c

was added at 10 min, and the reaction was stopped at

15 min In the second procedure, 2 lm of cyto-c was added

at 5 min, 4 mm MgCl2 at 10 min and the reaction was

stopped at 15 min Details of the sequence of additions are

reported in the legend to Fig 2 To increase the reliability

of the results and to better appreciate the changes induced

by magnesium, mitochondria containing 6 mg of protein

were incubated in 6 mL of medium, centrifuged as specified

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 ferricya-nide as oxidant and sodium dithionite as reductant Spectra and kinetic determinations were carried out with Hitachi-Perkin 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)] spec-trophotometers

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 sup-ported by grants from MIUR (Prin 2005–2007 ‘Bioen-ergetica: meccanismi molecolari e aspetti fisiopatologici dei sistemi bioenergetici di membrana’), CNR (Insti-tute of Biomembranes and Bioenergetics, IBBE, Bari, Italy) and University of Bari, Bari, Italy

References

1 Palmieri F (2004) The mitochondrial transporter family (SLC25): physiological and pathological implications Pflu¨gers Arch 447, 689–709

2 Lofrumento NE, Marzulli D, Cafagno L, La Piana G

& Cipriani T (1991) Oxidation and reduction of exogenous cytochrome c by the activity of the respiratory chain Arch Biochem Biophys 288, 293–301

3 Marzulli D, La Piana G, Cafagno L, Fransvea E & Lofrumento NE (1995) Proton translocation linked to the activity of the bi-trans-membrane electron transport chain Arch Biochem Biophys 319, 36–48

4 La Piana G, Fransvea E, Marzulli D & Lofrumento

NE (1998) Mitochondrial membrane potential