ΔpH Induced Calcium Fluxes in Rat Liver Mitochondria

Eur. J. Biochem. 102, 555-562 (1979)

ApH Induced Calcium Fluxes in Rat Liver Mitochondria

Paolo BERNARD1 and Giovanni Felice AZZONE

with the technical assistance of Paolo Veronese

Consiglio Nazionale delle Ricerche Unit for the Study of Physiology of Mitochondria, and Institute of General Pathology, University of Padova

(Received June 22, 1979)

1. The paper reports an investigation on dpH-driven Ca" fluxes in rotenone-treated rat liver mitochondria. The H + concentration gradient (acidic inside) required to drive CaL + influx is obtained from the K + concentration gradient through a H + / K + exchange. The rate of H + / K + exchange is very low in native mitochondria and is markedly enhanced by the electroneutral ionophore nigericin. The rate of Ca2+ influx depends on the rate of generation of ApH; it is negligible in native mitochondria and fast after nigericin.

2. The process of ApH-driven Ca2+ influx shows three basic features. First, the rate of Ca2+ influx is markedly increased by the addition of uncouplers. Second, the rate of CaL+ influx is inhibited by ruthenium red with the same sensitivity as the processes driven by the H f pump or K + diffusion. The electroneutral Hf /Ca2+ ionophore A231 87 restores Ca2+ influx in mitochondria inhibited by ruthenium red. Third, both rate and extent of ApH-driven Ca2+ influx are inhibited by the addition of weak bases but not of weak acids.

3. The process of ApH-driven Ca2+ influx is accompanied by H + efflux. A correlation also exists between generation of ApH (acidic inside) due to H + / K + exchange and utilization of ApH due to H+/Ca2+ exchange. The process of ApH-driven, A23187-catalyzed, H+/Ca2+ exchange is not preceeded by rapid H + / K + exchange in the absence of nigericin.

4. After termination of the phase of Ca2+ influx a phase of Ca2+ efflux ensues. Ca2+ efflux is not coupled to H + or K + reentry and is completely inhibited by ruthenium red. Ca2+ efflux is not observed when Ca2+ transport is catalyzed by A23187 in mitochondria inhibited by ruthenium red. Furthermore A23187 abolishes Ca2+ efflux also when Ca2+ influx occurs in the absence of ruthenium red.

5 . The data suggest that the ApH-driven Ca2+ influx and the subsequent Ca2+ efflux take place through the native carrier as electrical uniport.

Rat liver mitochondria translocate divalent cations, such as Ca2+, Mn2+ and Sr2+, at a high rate across the inner membrane [l-41. The process occurs through a component defined as Ca2+ carrier (or divalent cation carrier). The Ca2+ carrier catalyzes an electrophoretic Ca2+ transport with a charge of 2 and is specifically inhibited by ruthenium red and La3+ [5 - 81. Thermodynamic and kinetic evidence however suggests that, during efflux, Ca2 + may be transported also through an alternative and independent pathway.

The existence of an electrophoretic Ca2 + uniport predicts that the steady-state distribution of free Ca2+

Ahbrc,virrtion.s. FCCP, carbonylcyanidep-trifluoromethoxyphen- ylhydrazone; Tris, 2-amino-2-hydroxymethyl-l,3-propan.ediol; Mops, 4-morpholinepropane sulfonic acid; Hepes: 4-(2-hydroxy- ethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylene-bis(oxo- ethylenenitrilo)]tetraacetic acid.

~ _ _ _ ~

across the membrane corresponds to A $ (the membrane potential across the inner membrane determined from the Nernst distribution of other permeant cations such as K + in the presence of valinomycin or triphenyl- methylphosphonium). However this prediction holds only at low A $ . At high A $ the distribution ratio of free Ca2 + is smaller than predicted from electrochemical equilibrium [9 - 131.

Further evidence is provided by the effect of ruthenium red. Would Ca2+ attain electrochemical equilibrium via an electrophoretic uniport, should ruthenium red not affect the CaZ+ distribution ratio. However if ruthenium red is added in steady-state, after Ca" accumulation, calcium begins to leak out at an extent which is proportional to the degree of inhibition of the Ca2+ carrier [S, 10,12,14,15]. These considerations support the concept of steady-state

556

Ht

ApH-Induced Calcium Fluxes in Rat Liver Mitochondria

Leak or FCCP *

calcium cycling where the uptake occurs via the electrophoretic uniport and the efflux via a d$-independent pathway [12,13,3 61.

While there seems to be agreement on the concept that, at high A $ , Ca2+ attains a kinetic rather than a thermodynamic steady state, the problem still open is the nature of the efflux pathway. Fiskum and Cockrell [17] have recently provided new evidence in favour of an alternative pathway describing a ruthenium-red- insensitive Ca2+ influx driven by ApH in respiratory- inhibited mitochondria. The present study is an ex- tension of this work. The dpH-driven Ca2+ influx has been characterized with respect to several features such as sensitivity to ruthenium red, effect of uncouplers and of weak bases, restoration of Ca2+ influx by A23187 after ruthenium red inhibition. From our study it appears that most of the ApH-driven Ca2+ fluxes go through the native carrier and that the rate of ruthenium-insensitive H+/Ca2+ exchange is below 0.1 nmol x mg protein-' x min-'.

MATERIALS AND METHODS

Rat liver mitochondria were prepared by standard centrifugation procedures in 0.25 M sucrose, 10 mM Tris-HCI pH 7.4 and 2 mM EDTA [19]. The last washing was carried out in an EDTA-free medium and the mitochondria1 protein was assayed with the biuret method. The incubation medium is specified in the legends.

Calcium movements were followed as absorbance changes of the dye Antipyrilazo 111 at the wavelength pair 720-790 nm with an Aminco dual-wavelength spectrophotometer with magnetic stirring and thermo- static control. Antipyrilazo 111 has been purified in our laboratory by Dr M. Beltrame according to Scarpa et al. [20]. As photometric calibration with Antipyrilazo 111 is very sensitive to the composition of the incubation medium, the procedure was adopted to add known amounts of CaC12 both at the beginning and at the end of each experiment. The spectrophotometer traces were corrected for the slight absorbance shifts, not corresponding to Ca2+ transport, following addition of ionophores or ruthenium red.

Proton movements were followed either in the same spectrophotometer using phenol red as pH indicator at the wavelength pair 560-623 nm or with a Radiometer pH meter (model 26). The two procedures gave comparable results. All chemicals were of analytical grade. Matrix pH was determined on the distribution of [3H]acetate or of [14C]methylamine [21]. Mitochondria were incubated under the specified conditions in the presence of either 10 pM [3H]acetate or of 14.7 pM ['4C]methylamine and the reaction terminated by rapid centrifugation in the Rotor SE12 of the Sorvall RC2B Supercentrifuge. The

(4) Ca2'

Ca2' (5) Antiporter

(6) H'

Fig.1. Scheme for Cu2+ floit.s. For explanation see the text. I , inside; 0, outside; RR, ruthenium red; Nig, nigericin

pellets were dissolved by incubation at 45 "C for 60 min in 0.3 ml of 0.01 M NaOH, 1 mercaptoethanol and 10 sodium dodecylsulphate. The dissolved pellet was counted in a Packard Tri-Carb 2425 Liquid Scintillation Spectrometer after addition of the emul- sifier Scintillator MI-96. The counts were converted to nmol x mg protein-' and then to concentrations on the basis of a matrix volume of 0.5 pl x mg protein-' and an activity coefficient of 1.

Rutionale of' ApH-Driven Culcium Transport

In Fig. 1 are reported the hypothetical transport reactions in the experimental system used.

Formation (if ApH. A ApH originates through the utilization of the K + concentration gradient through reactions 1 or 2. Reaction 1 is very slow because of the low permeability of the inner membrane to K + and H + . Valinomycin cannot be used in this system because in its presence the diffusion potential created by K f ef- fluxdrivesdirectly Ca" influx without the intermediate formation of ApH. Formation of ApH at the expense of the K + concentration gradient is markedly accelerated by the ionophores of the nigericin type.

Calcium Influx. The influx is indicated in reactions 3 ,4 and 5. In reaction 3 Ca2+ uptake is coupled to H f efflux either via leakage or via uncoupler and occurs via the native ruthenium-red-sensitive carrier. In reaction 4 Ca2+ uptake occurs via the exogenous electroneutral ionophore A23187 [22]. In reaction 5 Ca2+ uptake occurs through an hypothetical ruthenium-red-insensitive electroneutral H+/Ca2+ exchange carrier. Therefore while reaction 3 is sensitive

557 P. Bernardi and G. F. Azzone

Fig. 2. Thr ApH-hivef l Cud + ,/lures. Effrci of u~icouplcvs o i i d ruiIfw- ium red. The incubation medium contained: 0.25 M sucrose, 20 mM Tris-Mops pH 7.0, 3 pM rotenone, 0.5 pg oligomycin x mg protein-', 50 pM Antipyrilazo 111. In (A) 2 pM FCCP. Mitochon- dria 4mg/ml, final volume 2 m l ; 25°C. 40 pM CaC12. When indicated 69 pmol nigericin x mg protein-' and 1 nmol ruthenium red x mg protein -' were added

Fig. 3. The ApH-(lrivm Cu2 + ,f/u.rtJs in the presmce of rurherliunl rc,d. ,5&i I$ A23187. Experimental conditions as in Fig.2. 1 nmol ruthenium red x mg protein-'. When indicated 69 pmol nigericin x mg protein-' and 191 pmol A23187 x mg protein-' were added

to ruthenium red and stimulated by uncoupler, reactions 4 and 5 are insensitive to ruthenium red and not stimulated by uncouplers.

Calcium EjTlux. The efflux can occur either through reactions 6 and 7 or through reversal of reactions 4 and 5. Ca2+ efflux is coupled in reaction 6 to H+ influx either via leakage or uncoupler, and in reaction 7 to efflux of an unknown anionic species.

RESULTS

Fig.2 shows the general properties of the ApH- driven Ca2+ transport. Rotenone-treated and oligomycin-treated rat liver mitochondria were incubated in the presence of 40 pM CaC12. In Fig. 2A 2 pM FCCP was present. In the absence of nigericin there was a negligible rate of CaZ+ influx due to the low permeability of the membrane to H + and K + , i.e. reaction 1 was very slow. Addition of nigericin caused a phase of rapid Ca2+ influx corresponding in amount to about 6 nmol x mg protein-'. The rate was about 40 nmol x m g protein-'xmin-' in the presence and about 8 nmol x mg protein-' x min-' in the absence of FCCP, respectively. The extent of Ca2+ uptake, as expected, was partially enhanced by the presence of uncoupler.

After the phase of Ca2+ uptake a slow, spontaneous efflux ensued. The rate was about 0.4 nmol x mg-' xmin- ' in the presence, and about 0.3 nmol x mg protein-' x min-' in the absence of FCCP, respectively.

Fig.2 also shows that addition of ruthenium red resulted in a complete inhibition of the nigericin- induced Ca2+ influx. The rate of ruthenium-red- insensitive Ca2+ influx was so low that it was possible to calculate only an upper limit for the process, say a rate below 0.1 nmol x mg protein-' x min-'. This

rate was not increased by nigericin. Finally Fig.2 shows that also the phase of Ca2+ efflux following completion of Ca2+ influx was completely abolished by ruthenium red.

Fig.3 shows the process of ApH-driven Ca2+ influx in ruthenium-red-treated mitochondria. In accord with what shown in Fig. 2 addition of nigericin did not result in stimulation of Ca2+ influx when the carrier was blocked by ruthenium red. On the other hand addition of A23187 resulted in a marked ac- celeration of the rate of Ca2+ influx (Fig. 3A). The rate. of the A23187-catalyzed Ca2+ influx was much faster in the presence than in the absence of nigericin (Fig. 3B). This is in accord with the view that in the presence of a rapid H+/Ca2+ exchange via A23187, the rate of Ca2+ influx depends on the rate of generation of dpH. The crucial question however in the experiment of Fig.3B is the mechanism of generation of acidity in the mitochondria1 matrix. In the absence of nigericin the H + / K + exchange is very slow and the Kf electrode provides no indication of K + efflux after addition of A23187. Thus utilization of H + to drive Ca2+ influx requires generation of H + by A23187 through a mechanism which is largely independent of the exchange with K'. A further explanation involves the hypothesis of a A23 187-induced decrease of Ca2+ activity in the matrix (see Dis- cussion).

Fig. 4 and 5 show the H + fluxes accompanying the transformation of the K + concentration gradient into H + concentration gradient and the subsequent utilization of the H + concentration gradient to drive Ca2+ influx. Fig.4 shows the H + fluxes following addition of nigericin in the presence of FCCP under three conditions: absence of Ca2+, presence of Ca2+ and ruthenium red and presence of Ca2+ without ruthenium red. In all cases addition of nigericin caused a fast Hf influx followed by a biphasic H + efflux. A

558

Niqericin Nigericin

ApH-Induced Calcium Fluxes in Rat Liver Mitochondria

Nigericin

A23187 HCI

A23187

HCI 100 5

Fig.4. H' fluxes in the presence of uncouplers. The incubation medium contained: 0.25 M sucrose, 0.5 mM Hepes-NaOH pH 7.0, 3 pM rotenone, 0.5 pg oligomycinx mg prot-I, 2 pM FCCP, 30 pM phenol red. In (B) 1 nmol ruthenium red x mg protein-'. Mitochondria 4 mg/ml, final volume 2 ml, 25 T. When indicated: 40 pM CaC12, 69 pmol nigericin x mg protein-', 191 pmol A23187 x mg protein-', 10 pM HC1

Nigericin Nigericin Nigericin

!1 I

Fig. 5. H + ,fluxes in the absence of' uncouplers. Experimental conditions as in Fig.4 without FCCP in the incubation medium. In (B) 1 nmol ruthenium red x mg protein-'. CaCI2, nigericin, A23187 and HCI were added in the same amounts as in Fig.4

comparison of the traces indicates the following. When Ca2+ was present (Fig.4C) the extent of rapid H + influx was reduced to about 60% with respect to that occurring without Ca2+ (Fig.4A) or with Ca2+ in the presence of ruthenium red (Fig. 4B). Addition of A23187 resulted in a large H + efflux when Ca2+ was present and Ca2+ transport was inhibited by ruthenium red (Fig. 4B).

Fig.5 shows the same experiment carried out in the absence of FCCP. Similarly to Fig. 4, the presence of Ca2+ resulted in a marked depression of the extent of H + influx (Fig.5C), inhibition of the Ca2+ carrier by ruthenium red (Fig. 5 B) restored the extent of H + influx occurring in the absence of Ca2+ (Fig.5A) and A23187 addition resulted in a large H + efflux only when Ca2+ was present and its transport was inhibited by ruthenium red (Fig.5B). The experiments of Fig. 4 and 5 show that Ca2+ efflux, after termination of the influx phase, is not accompanied by H + reentry. Thus charge balance does not occur in this case because of coupling of Ca2+ with H + fluxes (cf. reaction 6 of Fig. 1). Since also the K + electrode provides no indication of K + influx (not shown) the alternative is that Ca2+ efflux is coupled to that of an

I I

- 3 - 2 -1

log [ K'lo. M

Fig. 6. Dependence on K' gradient ojnigericin-induced CaZ+ uptake. Experimental conditions as in Fig.2A. Variations of the K + gradient was achieved by increasing the concentration of external K'. The osmolarity of the medium was kept constant by reducing the sucrose concentration proportionally to the increase of KCI. The reaction was initiated by nigericin. Values on the ordinate refer to the initial rates of Ca2+ influx following nigericin

Table 1. Dimensiun of ApH during ApH-driven CaZ+ influx The incubation medium contained: 0.25 M sucrose, 20 mM Tris- Mops pH 7.0,2 pM rotenone, 2 pM FCCP, 0.5 pg oligomycin x mg protein-', 14.7 pM ['4C]methylamine, 20 pM CaCI2. In the experiments with succinate (2 mM) the medium contained: 0.25 M sucrose, 20 m M Tris-Mops pH 7.0, 2 pM rotenone, 10 pM [3H]- Acetate, 20 pM CaCl2. Final volume 1 ml, mitochondria 2 mg. The experiments were performed as described in Materials and Methods. Additions: ruthenium red, 1 nmol x mg protein-' ; A23187, 191 pmol x mg protein-'; nigericin, 69 pmol x mg protein-'

Addition ~- _ _ _ - -~ ~-

Matrix pH

without with nigericin nigericin

~~- - ~~

~ ..-__ ~~ _____. ~

Succinate 7.2 None 6.43 6.22 Ruthenium red 6.23 5.92 A23187 6.42 6.40 Ruthenium red + A23187 6.7 6.49

- ~

unknown anionic species (reaction 7 of Fig.1). Although the experiments of Fig.4 and 5 clearly indicate a qualitative correlation between H + efflux and CaZf influx, the interpretation of the kinetics of H' efflux is not easy because the reactions leading to pH equilibration are presumably multiple and their nature is unknown.

Fig.6 shows the dependence of the initial rate of nigericin induced Ca2+ influx on the K + gradient. I t is seen that increase of the external K + concentration from 60 pM and 60 mM resulted in a linear decrease of the rate of Ca2+ influx in a semilogarithmic plot. This is in accord with the rate of Ca2+ influx being proportional to the log of the [K+Ii/[K+]o ratio.

In Table 1 are reported the values of matrix pH under the various conditions used to study the ApH- driven Ca2+ influx. The dimension of dpH may be

P. Bernard; and G . F. Azzone

B C A

559

Fig.7. Inhibition of ApH-driven Cu" influx by weak bases. The incubation medium contained: 0.25 M sucrose, 20 mM Tris-Mops pH 7.0, 3 pM rotenone, 0.5 pg oligomycinxmg protein-', 2 p M FCCP. In (B) and (C) 1 nmol ruthenium r e d x m g protein-'. Where indicated, 2, 10 or 20 m M (NH&S04 was present. Antipyrilazo I11 was 50 pM in the absence of (NH&S04, 55.75 pM with 2 mM (NH4)2S04. 67.14 pM with 10 mM (N&)2S04 and 75.71 pM with 20 m M (NH4)2S04 in order to obtain the same sensitivity to C a 2 + movements. Mitochondria 4 mg/ml, final volume 2 ml, 25°C. 40 PM CaCI2. When indicated 69 pmol nigericinx mg protein-' and 191 pmol A23187 x mg protein-' were added

calculated since the pH of the medium was kept constant at the level of 7.0. It is seen that a ApH of 0.57 acidic inside was present in respiratory-inhibited mitochondria presumably due to slow H + / K + exchange. The matrix pH was lowered from 6.43 to 6.23 by ruthenium red and increased to 6.7 when A23187 was present together with ruthenium red. This indicates that the dimension of dpH was maximal when the H+/Ca2+ exchange was abolished and minimal when the H+/Ca2 + exchange was operating. A similar pattern was observed in the presence of nigericin although the values of matrix pH were lower (and the ApH larger) due to the H + / K + exchange catalyzed by nigericin.

Fiskum and Cockrell [I71 reported that the process of ApH-driven Ca2+ influx is inhibited by inorganic phosphate. We have tested the effect of both 2 m M phosphate and of 10 mM acetate and have found no inhibitory effect either on the rate or extent of Ca2+ influx (not shown). On the other hand the rate and extent of ApH-driven Ca2+ influx were found to be sensitive to the addition of weak bases. Fig.7 shows the effect of increasing concentrations of NH: on the rate and extent of Ca2+ influx either induced by nigericin (Fig. 7A) or, in the presence of ruthenium red, by A23187 with nigericin (Fig.7B) or A23187 without nigericin (Fig.7C). In all cases addition of NH: resulted in an inhibition which was proportional to the amount of NH:. At 20 mM (NH4)2S04 the process of Ca2+ influx was completely inhibited in the absence but not in the presence of A23187. This suggests that A23187 gives rise to another driving force, presumably a Ca2+ concentration gradient. also under conditions where ApH is abolished by weak bases.

The experiments of Fig. 8 and 9 are concerned with the problem of the distribution of Ca2+ at the end of the phase of Ca2+ influx. An apparent discrepancy

I A23187

A B

Fig. 8. Ejject c$ A23187 on Cu2+ efflux. Experimental conditions as in Fig.2 (A) and (B), respectively. When indicated: 69 pniol nigericin and 191 pmol A23187 x m g protein-.'

I , nos , Fig.9. EJj i r t ofnigericin on CaZf distribution ufter- A231K7 ciddition. Experimental conditions as in Fig.2 with 2 pM FCCP and 1 nmol ruthenium red x mg protein-' in the incubation medium. When indicated 191 pmol A23187 and 69 pmol nigericin x mg protein-' were added

seems to exist between the traces of Fig.2 and those of Fig.3. In Fig.2 in the absence of A23187 the phase of CaL+ influx was followed by another of Ca2+ efflux. In Fig.3 in the presence of

560 dpH-Induced Calcium Fluxes in Rat Liver Mitochondria

A23187 the phase of Ca2+ influx was not followed by Ca2+ efflux. Fig. 8 shows that addition of A23187 after completion of the phase of Ca2+ influx, completely abolished the phase of Ca2+ efflux. The effect of A23187 was independent of whether FCCP was present. Finally Fig.9 shows that, when Ca2+ influx was catalyzed by A231 87 in mitochondria treated with FCCP + ruthenium red, after the attainment of an apparent equilibrium, addition of nigericin was followed by a short cycle of Ca2+ influx and efflux. It should be reminded that the initial phase of Ca2+ influx was not accompanied by K + efflux while that following addition of nigericin was. The experiments of Fig.8 and 9 thus indicate that the equilibrium distribution of Ca2+ was not identical whether A23187 was absent or present, and it was independent of the order of addition of nigericin and A23187.

DISCUSSION

Coupling of transport to metabolism is common in biological systems. Since the coupling may have a profound effect on the kinetic and thermodynamic properties of the transport reaction, it is generally felt advisable to study the two reactions separately, by splitting transport from metabolism. This concept has been fruitfully applied in the case of Ca2+ transport, by using artificially generated diffusion potentials to study Ca2+ influx in rat liver mitochondria [23 -261. Concentration gradients of K + in the presence of valinomycin [23-2.51 and of permeant anions [26] have been used for this purpose. The approach has led to the identification of Ca2+ transport as an electrophoretic process, to the determination of its stoichiometry, and to the establishment of kinetic and thermodynamic properties of the Ca2 + carrier [18,25]. A recent development in the field of Ca2+ transport is the view that mitochondria possess, in addition to the well established electrical Ca2+ uniport, also an electroneutral H + /Ca2 + exchange carrier. Both Ca2+ and H+ concentration gradients would cooperate to render this exchange carrier operating in the direction of Ca2+ efflux during matrix Ca2+ accumulation either in v i t m or in vivo.

A ApH-driven Ca2+ influx can be studied in fresh rat liver mitochondria after transformation of the K + into H + concentration gradient [17]. Such transformation tends to occur spontaneously in mitochondria incubated in a low K + medium, but at a slow rate because of the low membrane permeability for H + and K + . A rapid transformation of K + in H + concentration gradient occurs after addition of the nigericin type of ionophores. Once ApH is generated, acidic inside, H + will tend to come out from the matrix, generating a driving force for ApH-driven cation influx. It is important to note that as shown in Fig. 1

the dpH-driven process may be either electrical or electroneutral, i.e. Ca2+ influx may proceed either through the native elecrrical carrier, or through exogenous electroneutral H +/Ca2 + ionophores. Thus the fact that the process is ApH-driven does not imply that the underlying molecular mechanism is electroneutral since the ApH may be used also to drive Ca2+ uptake through formation of a H + diffusion potential.

Distinction between a Ca2+ influx proceeding through the uniport Ca2+ carrier or through a H+/Ca2+ exchange carrier can be achieved by two criteria: ruthenium red sensitivity and the effect of uncouplers. As to the first criterion, since there are no examples in mitochondria of ruthenium-red-insensitive Ca2 + influx the inhibition by ruthenium red may be taken as an indication for operation of the CaL+ uniport. The sensitivity to ruthenium red of Ca2+ efflux is not a similar unequivocal test (see below). As to the second criterion, as indicated in reaction 3 of Fig.1, Ca2+ influx through the native carrier requires that H +

efflux proceeds through an independent, electrical pathway. In native mitochondria such pathway does not exist except for the H + leakages. Ca2+ influx through reaction 3 is therefore expected to be slow and to be enhanced by uncouplers. Application of both criteria suggests that the nigericin-induced ApH- driven Ca2+ influx proceeds through reaction 3. First, Ca2+ influx is inhibited by ruthenium red with the same titer as the K+-driven or the H+-pump- driven Ca2+ influx. Second, the rate of Ca2+ influx is markedly increased by FCCP: the rates were 40 and 8 nmol Ca2+ x mg protein-' xmin- ' in the presence and absence of FCCP respectively. We are unable to provide an exact measurement of the ruthenium-red- insensitive ApH-driven Ca2+ influx because of the overlap of real Ca2+ fluxes with spectrophotometric drifts at slow Ca2+ fluxes. However the upper limit for the ruthenium-red-insensitive process is around 0.1 nmol Ca2+ x mg protein-' x min-'. Furthermore a basic feature of this ruthenium-red-insensitive Ca2 +

influx is its insensitivity to the addition of nigericin. After a cycle of Ca2+ influx, a process ofCa2+

efflux ensues in the absence but not in the presence of A23187. Two problems here arise, one, the nature of the charge balancing species, and two, the reason for the A23187 effect. As to the first problem, the pH measurements indicate a negligible H + reentry during Ca2+ efflux. This suggests that Ca2 + efflux is balanced by some other species, presumably anionic. Several attempts were made to identify such species, but without success. As to the second problem the ApH- driven Ca2+ influx levels off when

Aj& = A P C , . 11) Since the electrical component of the H + and Ca2+ electrochemical gradients neutralize each other, Eqn (1) reduces to the equivalence of the H + and Ca2+

P. Bernardi and G. F. Azzone 56 1

activity gradients. Thus when the Ca2+ activity gradient equals the H + activity gradient, no further movement of Ca2+ is expected. The Ca2+ distribution may be altered by the efflux of anionic species es- tablishing a diffusion potential, negative outside. The ability of A23187 to abolish Ca2+ efflux and the constancy of the Ca2+ distribution in the presence of A23187, may be explained in two ways. First, a further H+/Ca2+ exchange catalyzed by A231 87 may com- pensate the hypothetical anion induced Ca2+ efflux. This explanation implies that a ApH, acidic inside, still exists after nigericin-induced Ca2 + influx, as indicated by data of Table 1 and by the traces of Fig. 4C and 5 C. Second, A23187 may lower the matrix Ca2+ activity catalyzing an exchange of Ca2+ with H + donating groups otherwise not accessible in the absence of the ionophore. This is partially supported by the residual Ca2+ influx in presence of A23187 in spite of the col- lapsing effect on ApH by weak bases. The two ex- planations are not in conflict and may be both correct.

In the study of Fiskum and Cockrell [17] data on either extents or rates of Ca2+ influx are not provided. However in that study the rate of the ruthenium-red- insensitive Ca2+ influx is less than 10% the A23187 induced rate. This corresponds to a ruthenium-red- insensitive Ca2+ influx of 2 nmol x mg protein-' x min-' if their rate for the A23187 induced influx is equal to our rate, namely 20 nmol Ca2 + x mg protein-' xmin- ' in the presence of nigericin. A major discrepancy, on the other hand, concerns the reported inhibition of the ApH-driven Ca2+ influx by weak acids. We find that the process is inhibited by weak bases and is insensitive to weak acids. This could be predicted since a ApH, acidic inside, is collapsed by the uptake of weak bases but not by the influx of weak acids. There is no way to explain the discrepancy neither on experimental nor on theoretical grounds.

Tlie Problem qf the A$-Independent Efflux Patliuuy

Extensive evidence has been provided for an independent Na+/Ca2+ exchange carrier in rat heart mitochondria ([27,29] but see also [36]). As to rat liver mitochondria the view of an independent pathway for Ca2+ efflux has been proposed by several authors on the basis of different lines of evidence [8,10, 13, 14, 17, 30, 33, 35, 371.

The heterogeneity of the arguments for an additio- nal Ca2+ transport pathway requires a comment. The present authors feel that an independent Ca2 +

efflux pathway can be rationalized, and distinguished from the uniport pathway, in so far as it possesses two features, namely an electroneutral driving force and a slow rate. The necessity for electroneutrality arises from the discrepancy between the membrane potentials calculated on the Nernst distribution of

permeant cations and of free Ca2+ [9-131; physio- logical considerations suggest that also in vivo Ca2+ is out of electrochemical equilibrium [9,12]. The discrepancy is overcome by assuming that Ca2 +

distribution is regulated by a kinetic steady state where the rate of influx is balanced by the rate of efflux; since the influx via the uniport is under the control of A $ the efflux must be A$-independent and presumably under the control of ApH. The necessity for a slow rate arises from the simultaneous presence of the A$-dependent uniport. Operation of a ApH-dependent Ca2+ efflux in combination with a A$-dependent Ca2+ influx leads to Ca2+ cycling, the rate of which, being the rate of Ca2+ influx via the uniport in large excess, is determined by the rate of Ca2+ efflux. This is what actually happens after addition of the exogenous H+/Ca2 + ionophore A231 87 to steady state mitochondria, i.e. Ca2+ cycling and uncoupling proportionally to the amount of A231 87 [22,34]. The rate of the A$-independent pathway must therefore be compatible with the notion that the respiratory rate of static head mitochondria, which reflects all dissipative processes including H +

leaks and Ca2+ cycling, is very low, say around 10 n m o l 0 x mg protein-' x min-'.

An argument for the A$-independent pathway is that of the ruthenium-red-insensitivity of the FCCP- induced Ca2+ efflux. This argument does not fit with the two features discussed above. First, this Ca2+ efflux is coupled to the H + influx via FCCP. Being H + influx via FCCP electrical, also that of Ca2+ must be so, thus via the uniport. Second, the FCCP- induced Ca2+ efflux is very fast, say even faster that the rate of H + extrusion via the pump. Mitochondria possessing a ApH-dependent pathway operating at this rate, would be permanently uncoupled. We think that also the Ca2+ efflux induced by oxidation of the pyridin nucleotides [33] cannot be explained with this mechanism. If the efflux would be due to activation of an electroneutral H+/Ca2+ exchange, oxidation of pyridine nucleotide should cause an increase of the respiratory rate. Once defined the two features of the A$-independent Ca2+ efflux pathway, namely electroneutrality and slow rate, the question arises as to the nature of this pathway. The present study, where no evidence is found for independent ApH-driven H+/Ca2+ exchange carriers in rat liver mitochondria, supports the view of a specific modulation of the native Ca2+ carrier for Ca2+ efflux [12,32]. This modulation might involve the induction of a latent activity.

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2. Azzone, G. F. & Massari, S. (1973) Biochim. Biophys. Actu, mol. Relut. Areus Mol. Bid . 29, 259- 320.

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562 P. Bernardi and G. F. Azzone: dpH-Induced Calcium Fluxes in Rat Liver Mitochondria

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ΔpH Induced Calcium Fluxes in Rat Liver Mitochondria