Calcium-dependent mitochondrial oxidative damage promoted by 5-aminolevulinic acid

Biochimica et Biophysica A cta, 1180 (1992) 201-206 © 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00

201

BBADIS 61207

Calcium-dependent mitochondrial oxidative damage promoted by 5-aminolevulinic acid Marcelo H e r m e s - L i m a a, R o g e r F. Castilho a, Valderez G.R. Valle a, Etelvino J.H. Bechara b and Anibal E. Vercesi a " Departamento de Bioquimica, Instituto de Biologia, UniL,ersidade Estadual de Campinas, Campinas (Brazil) and h Departamento de Bioqu[mica, lnstituto de Qufmica, Universidade de Sdo Paulo, Sfto Paulo (Brazil)

(Received 21 April 1992) (Revised manuscript received 20 July 1992)

Key words: 5-Aminolevulinic acid; Porphyria; Reactive oxygen species; Calcium ion homeostasis; Calcium ion; Magnesium ion; Mitochondrial damage; (Rat liver mitochondrion)

Swelling of isolated rat liver mitochondria is shown to be induced by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation, a putative endogenous source of reactive oxygen species (ROS), at concentrations as low as 50-100 ~M. In this concentration range, ALA is estimated to occur in the liver of acute intermittent porphyria patients. Removal of Ca 2+ (10/~M) from the suspension of isolated rat liver mitochondria by added EGTA abolishes both the ALA-induced transmembrane-potential collapse and mitochondrial swelling. Prevention of the ALA-induced swelling by addition of ruthenium red prior to mitochondrial energization by succinate demonstrates the deleterious involvement of internal Ca 2+. Addition of MgCI2 at concentrations higher than 2.5 mM, prevents the ALA-induced mitochondrial swelling, transmembrane potential collapse and Ca 2+ efflux. This indicates that Mg 2+ protects against the mitoehondrial damage promoted by ALA-generated ROS. The ALA-induced mitochondrial damage might be a key event in the liver mitochondrial damage of acute intermittent porphyria patients reported elsewhere.

Introduction

Several authors have connected the generation of reactive oxygen species (ROS) in model systems and their deleterious effects on biomolecules and supramolecular structures with the pathophysiology of various disorders (for a review, see Ref. 1). Bechara and colleagues [2,3] proposed that ROS formed by the metal-catalyzed aerobic oxidation of 5-aminolevulinic acid (ALA), a heme biosynthetic precursor [4], at abnormally high levels, are involved in the etiology of acute intermittent porphyria and lead-poisoning. It is also reported that acute intermittent porphyria patients and lead-exposed workers have high levels of the antioxidant enzymes superoxide dismutase and gluta-

Correspondence to: E.J.H. Bechara, Departamento de Bioquimica, Instituto de Quimica, Universidade de $5o Paulo, CP 20780, 01498, Silo Paulo, SP, Brazil. Abbreviations: ROS, reactive oxygen species; ALA, 5-aminolevulinic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; TPP +, tetraphenylphosphonium bromide; EGTA, ethyleneglycolbis(13-aminoethylether)-N,N,N',N'-tetraacetic acid; RLM, rat liver mitochondria.

tione peroxidase in red blood cells [5,6]. With the aim of screening potential biological targets for ALA-generated ROS, we have tested their effects in mitochondria [7]. In 1974, Biempica et al. [8] reported the accumulation of ferritin and large lipofuscin bodies and the appearance of bizarre forms of mitochondria in liver biopsy samples of acute intermittent porphyria patients. Recently, we observed that ALA-generated ROS promote disfunctions in isolated rat liver mitochondria [7]. Since ALA accumulates in liver of acute intermittent porphyria patients, the in vivo observations of mitochondrial lesions [8] could be due to ROS formation from the aerobic oxidation of ALA. Using the classical mitochondrial swelling technique we demonstrate here that pathological concentrations of ALA (micromolar range [9]).are able to induce ultrastructural changes in isolated mitochondria. The swelling technique follows the net influx of the osmotic support (KCI plus sucrose), indicating a nonspecific increase in membrane permeability [10]. In addition, we report that isolated mitochondria, in media containing 10/zM C a 2+, a r e damaged by ALA-generated ROS just when mitochondria take up Ca 2+. This is in agree-

202 ment with the view that mitochondrial oxidative stress is connected to Ca 2+ metabolism [11-13]. A role of Ca 2+ and Mg 2+ ions in the free radical rationale of the acute intermittent porphyria pathophysiology is discussed here. Materials and Methods

5-Aminolevulinic acid, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid (Hepes), tetraphenylphosphonium bromide (TPP +), ethyleneglycol-bis(/3-aminoethyl ether)-N,N,N',N'-tetraacetic acid ( E G T A ) and ruthenium red were obtained from Sigma. Ruthenium red was purified according to Luft [15]. Stock ALA.HCI solutions (250 mM) were stored at - 2 0 ° C . All other reagents were of analytical grade. The ALA-containing media (in the absence of mitochondria) were pre-incubated at room t e m p e r a t u r e for 15 rain (pH 7.2) before use (see Ref. 7). Rat liver mitochondria (RLM) were isolated as described by Fagian et al. [16] from overnight-fasted male Wistar rats. These mitochondria contain 5 - 1 0 n m o l / m g endogenous calcium as determined by atomic absorbtion spectroscopy. T r a n s m e m b r a n e electrical potential was measured by T P P + activity and Ca 2+ fluxes were followed by measuring the changes in absorbance spectrum of arsenazo III [17], using the SLM Aminco DW2000 spectrophotometer at the wavelenght pair 675-685 nm. Total Ca 2+ present in reaction media was measured by atomic absorption spectroscopy. Free medium Ca 2 + was calculated by titration with different concentrations of E G T A , by employing an iterative computer program [18] modified from that described by Fabiato and Fabiato [19], taking into account the dissociation constants reported by Schwarzenbach et al. [201. The experiments were carried out at 25°C in a basic medium containing 125 mM sucrose, 65 mM KCI, 10 mM Hepes buffer (pH 7.2) and 4.0 p.M rotenone. The swelling experiments were carried out in glass cuvettes with magnetic stirring in a final volume of 2.5 ml and a protein concentration of 0.5 m g / m l . The experiments were run in triplicates and the data reproduced within 15%. Absorbance changes were monitored on an S L M - A M I N C O DW-2000 spectrophotometer at 520 nm [10]. Results

Ca 2 +-dependent ALA-induced mitochondrial damage Mitochondrial disfunctions promoted by the metalcatalyzed aerobic oxidation of A L A were previously demonstrated [7] by monitoring alterations in transm e m b r a n e potentials, Ca 2+ fluxes and state-4 respiratory rates, which were attributed to deleterious ROS. The mitochondrial swelling is now used to verify

Fig. 1. Ca2*-dependent ALA-induced mitochondrial swelling. Rat liver mitochondria (0.5 m g / m l ) were pre-incubated in the basic medium containing: (a), 0.5 mM EGTA; (b), no additions: (c-f) 0.05, 0.1, 0.25, 0.5 and 1.0 mM ALA, respectively. E G T A ({I.5 raM) was added, where shown, to the experiment containing 1.0 mM A L A (dashed line) or was present, under these conditions, from thc begining of the experiment (dotted line). Succinate (2.5 mM) was added after 8 min of mitochondrial preincubation.

whether A L A also promotes ultrastructural changes in the organelles. In fact, Fig. 1 shows that 0.05-1.0 mM A L A (lines c - g ) induces swelling in succinate-energized mitochondria, as compared to control experiments performed in the absence of ALA (lines a and b). In the experiment of line a, the concentration of free Ca 2+ in the incubation medium was lower than 10 - s M due to the presence of 0.5 mM E G T A , whereas in the experiment of line b 10/~M Ca z ~ was present. It can also be observed in this figure that (I.5 mM E G T A completely prevents (dotted line) or interrupts (dashed line) the mitocondrial swelling caused by 1.0 mM ALA. Therefore, the mitochondrial swelling occurs only in media containing Ca 2+. This figure also indicates that no swelling occurs during the 10 rain of mitochondrial pre-incubation with A L A which precedes the addition of succinate. The Ca 2+ requirement for the lesive action of ALA is also shown in Fig. 2. Removal of Ca 2 ~ by pre-addition of 0.5 mM E G T A (dotted line) to the mitochondrial suspension prevents the 1.0 mM ALA-induced transmembrane-potential collapse. This figure indicates that rat liver mitocondria in the succinate-conraining medium take up and retain most of the TPP ~ present (potential close to 150 mV), when A L A was absent (lines a and b). However, when 0.05-1.0 mM A L A (lines c-g) was present, both the extent of TPP ~ accumulation and the ability of its retention by mitochondria were diminished. This again illustrates that A L A (in the presence of Ca 2+) damages mitochondria in a dose-related manner. We have previously observed that o-phenanthroline (a chelator which prevents the iron-catalyzed decomposition of H 2 0 2 [21]) or the antioxidant enzymes catalase or superoxide dismutasc

203 ~

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Fig. 2. The Ca2+-dependent ALA-induced membrane-potential collapse. The experiments were initiated by addition of 3 #M TPP + followed by RLM (1 mg/ml) to the basic medium containing 2.5 mM succinate and: (a), 0.5 mM EGTA; (b), no additions; (c-g), 0.05, 0.1, 0.25, 0.5 and 1.0 mM ALA. The dotted line represents an experiment in which 1.0 mM ALA and 0.5 mM EGTA were present simultaneously. Values of electrical potential were corrected as described in Ref. 45 due to the binding of TPP + to mitochondrial membranes. prevent the A L A - i n d u c e d disruption o f the transmembrane electrical potential [7]. T h e s e results, taken as a whole, indicate that Ca 2+ is an essential factor for the oxygen free-radical-driven mitochondrial damage. Fig. 3 shows the effect of A L A on mitochondrial swelling w h e n the free m e d i u m Ca 2+ varies from 0 - 1 0 tzM (lines a - e ) . It can be observed that a significant swelling occurs even at 0.17 IzM m e d i u m Ca 2+ (line b), a c o n c e n t r a t i o n at which no Ca 2 + uptake occurs due to the low affinity o f the Ca 2+ uniporter [22]. U n d e r these conditions, mitochondrial d a m a g e is certainly d e p e n dent on the p r e s e n c e of e n d o g e n o u s mitochondrial Ca 2+ ( 5 - 1 0 n m o l / m g ) that is redistributed accross the

Fig. 3. Effect of medium Ca 2+ concentrations on ALA-induced mitochondrial swelling. The experiments were initiated by the addition of RLM (0.5 mg/ml) to the basic medium containing EGTA concentrations of 500, 15, 10, 5 and 0 /~M to bring the free Caz+ concentrations close to: (a), 0; (b), 0.17; (c), 0.9; (d), 5.0 and (e), 10 ~M, respectively.

Fig. 4. Effect of ruthenium red on the ALA-induced mitochondrial swelling. The experimental conditions were identical to those of Fig. 1. (a), no additions; (b), ruthenium red (2 ~M) was added 3 min before mitochondrial energization; (c), 1.0 mM ALA was present; (d), in addition to 1.0 mM ALA, 2 /zM ruthenium red was added 3 min before mitochondrial energization. inner m e m b r a n e after mitochondria are a d d e d to the reaction medium. O n e question that arises from the above results is which Ca 2+ pool is involved in the d a m a g e process: intramitochondrial or extramitochondrial? This is app r o a c h e d in the following experiment. Fig. 4 depicts that deenergized mitochondria u n d e r g o no ultrastructural changes, m e a s u r e d by the swelling technique, in the presence of 1.0 m M A L A and 10 IzM Ca 2+. N o A L A - i n d u c e d swelling occurs even after 25 min incubation in these conditions (data not shown). Subsequent addition of succinate p r o m o t e s a fast swelling of mitochondria suspended in 1.0 m M A L A - c o n t a i n i n g m e d i u m (line c). Probably, A L A induces swelling mediated by the succinate-dependent Ca e+ influx. T h e addition of 2 IxM ruthenium red, a c o m p o u n d that blocks the Ca 2+ influx [22], prior to succinate-energization of mitochondria (after mitochondria had released most of the internal Ca2+), indeed prevents the 1.0 m M A L A induced swelling (Fig. 4). In media containing 1.0 m M A L A , addition of ruthenium red 5 rain after mitochondrial energization and Ca 2+ accumulation had no protective effect against mitochondrial swelling (data not shown). In this way, it can be concluded that intramitochondrial Ca 2+ plays a crucial role in the A L A - g e n e r ated R O S - i n d u c e d mitochondrial alterations. This is in accordance with the view that Ca 2+ binding to internal sites of the intramitochondrial m e m b r a n e triggers the process of nonspecific m e m b r a n e permeabilization mediated by pro-oxidants [13,22,23].

Protectiue effect of Mg e+ against the mitochondrial damage process T h e experiment of Fig. 5 shows that the swelling p r o m o t e d by 1.0 m M A L A to succinate-energized

204 important factor involved in the preservation of thc native permeability properties of mitochondrial membranes [23-30]. Discussion

Fig. 5. Magnesium ions antagonize the Ca2+-mediated ALA-induced swelling. The"experiments were initiated by addition of RLM (0.5 mg/ml) to the basic medium containing: (a), no additions; (b), 1.0 mM ALA; (c-f) in addition to 1.0 mM ALA, 0.8, 1.5.2.5 and 5.0 mM MgCI 2 was present, respectively. MgCI 2 (5 mM) was added, under the experimental conditions of line b, where indicated (dotted line).

mitochondria is progressively inhibited by increasing concentrations (up to 5.0 mM) of MgCI 2 (lines c-f). A complete inhibition was caused by 5.0 m M MgCI2, even when the 1.0 m M ALA-induced swelling process had already been initiated. However, within the physiological range of intracellular free Mg 2+ (0.8-1.5 mM) a significant swelling occurs. In addition, 5 m M MgCI 2 totally prevents the deleterious action of 1.0 m M A L A on the mitochondrial capacity of taking up external Ca 2+ and retaining it (Fig. 6). Moreover, 5 mM MgC12 also prevents the t r a n s m e m b r a n e mitochondrial potential collapse induced by 1.0 mM A L A (data not shown). These results are in line with the view that Mg 2+ is an

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Fig. 6. Magnesium ions antagonize the Ca2+-mediated ALA-induced alterations of mitochondrial Ca2+ fluxes. The experiments were started by addition of RLM (1 mg/ml) to the basic medium in the presence of 1.0 mM ALA (line a), 1.0 mM A L A plus 2.5 mM MgCI2 (line b) or in the absence of ALA and MgC12(line c).

Acute intermittent porphyria is an inherited disease with well-known neurological manifestations [4,31]. As proposed by several authors, the clinical syndrome is triggered by accumulation of ALA in liver and bone marrow [4,31] and its distribution to the central nervous system and other organs [32,33]. Studies on hepatic damage may constitute an approach for the investigation of the possible involvement of oxygen free radical involvement in acute intermittent porphyria (see Refs. 3, 7, 8, 34 and 35). We have demonstrated in this work that Ca -'+ and Mg 2* play an important role in the process of in vitro mitochondrial m e m b r a n e permeabilization induced by ALA-generated ROS: intramitochondrial C a ,-+ activates this process, but exogenous Mg 2+ antagonizes it. We suggest that Ca2*-mediated mitochondrial oxidative stress might be related to the liver pathophysiology of acute intermittent porphyria. The chelation of medium Ca 2+ (10 /~M) by 0.5 mM E G T A completely prevents both the A L A - p r o m o t e d swelling (Fig. 1) and the transmembrane-potential collapse (Fig. 2). Calcium ions are not involved in the rate of A L A aerobic oxidation measured by the oxygen uptake technique [36]. As reported here, intramitochondrial Ca ~ can mediate the in vitro deleterious action of oxyradicals. This was concluded from the data obtained with ruthenium red (Fig. 4). On the other hand, we have reported elsewhere [7] that iron ions play a crucial role in the mitochondrial damage process, catalyzing the formation of hydroxyl radicals from ALA-generated 0 2 and H 2 0 2 species. For this reason, the results obtained with ruthenium red also rule out a possible protective E G T A effect merely due to iron-ion chelation and. therefore, prevention of the iron catalyzed oxidativc damage. The fact that intramitochondrial Ca 2+ allows the ROS-induced m e m b r a n e permeabilization could be interpreted as a Ca2+-mediated modification in the targets of ROS oxidation, probably membrane protein targets. This idea comes from previous observations [36] that the mitochondrial damage process promoted by ALA-generated ROS is prevented by the disulfide reducing agent dithiothreitol. This suggested, as reported for the effects of diamide and t-butyl-hydroperoxide [10,13,22,23], the involvement of cross-linking between sulfydryl groups of membrane proteins in the m e m b r a n e permeabilization process promoted by ROS (see Ref. 13). So, intramitochondrial Ca 2. could trigger the opening of 'pores' in the mitochondrial membrane [22] mediated by the oxidized forms of the sul-

205 fydryl proteins, being responsible for the membrane permeabilization. It has been proposed that the increase in mitochondrial membrane permeability promoted by internal Ca 2+ under some experimental conditions is caused by the loss of membrane-bound Mg 2+ and that this is prevented by exogenous Mg 2+ [23-30]. Our results (Figs. 5 and 6) may indicate that the release of membrane-bound Mg 2+ could be involved in the molecular mechanism of ROS-promoted mitochondrial injury. The protective effect of added MgCI 2 cannot be explained by an exchange of Ca 2+ ions bound to intramitochondrial 'trigger sites' for Mg 2+ ions, because Mg 2+ uptake by isolated RLM is extremelly low [37]. Therefore, this protective role of exogenous Mg 2+ against mitochondrial damage promoted by ALA-generated ROS may be due to the restoration of membrane bound-Mg 2+ pool. The pathological changes described by Biempica et al. [8] in liver mitochondria of acute intermittent porphyria patients was previously suggested to be due to ALA-induced oxidative stress [7]. The concentrations of ALA that promote ultrastructural changes in vitro (see Fig. 1) cover the range assumed to occur in the liver of acute intermittent porphyria patients (approx. 100 ~M), as follows: in two cases of acute intermittent porphyria with overwhelming neuropathy, the maximum plasma level of ALA was found to be 9 and 12 p.M, which is approx. 100-times higher than in normal level [9]. However, the liver ALA concentration could be higher: according to McGillion et al. [32], when studying ALA-injected rats (40 m g / k g body weight), the ALA concentration is approx. 10-times higher in the liver as compared to plasma. The actual concentration of ALA in the liver of AIP patients is not known. Recent findings from Pereira [38] (see also Refs. 34 and 35) have shown that ALA plays a pro-oxidative role in vivo. It was verified that ALA-injected rats (40 m g / k g body weight, every two days during 15 days), as compared to a control group, presented: (i), 30 and 40% decrease in hepatic and red fibers (soleus muscle) glycogen content, respectively; (ii), increase in serum concentration of lactate and free fatty acids; (iii), reduction of mitochondrial enzymes activities (citrate synthase and Mn-superoxide dismutase); (iv), reduction of the period for reaching fatigue during swimming exercise (from 92 to 40 min) and (v), a 2.5-fold increase in the level of the antioxidant enzyme CuZn-superoxide dismutase in liver. Interestingly, the white (mitochondria-poor) fibers of the gastrocnemius muscle showed no enzymatic or glycogen alterations under treatment with ALA. These results, taken as a whole, indicate that ALA induced an increase in glycolytic metabolism as a result of mitochondrial damage. The observed enhancement in the CuZn-superoxide dismutase activity could be interpreted as a protective re-

sponse of the organism to ALA-induced oxidative stress (see Ref. 39). The idea that liver mitochondria could be a potential target for ROS generated from the metal-catalyzed ALA aerobic oxidation is also supported by the data from Biempica et al. [8]: the large lipofuscin bodies found in the hepatocites of acute intermittent porphyria patients may result from oxidative damage (see Ref. 40). Moreover, Fe 2+ ions may be crucial for the ALA-induced mitochondrial damage in vivo. The large accumulation of Fe3+-ferritin in the hepatocyte cytoplasm of the acute intermittent porphyria patients is a potential source for Fe z+ ions (Fe 2+ is released from Fe3+-ferritin by the reaction with reductants, such as 0 2 [41]). In addition, it was shown [42] that intramitocondrial non-heine Fe z+ (from isolated rat liver mitochondria) is released when mitochondria take up Ca 2+. This could be another potential source of iron for in vivo hepatic free radical damage in acute intermittent porphyria. Potentially harmful concentrations of iron chelates (such as Fe z +-ATP and Fe 2 +-citrate [43]) could be formed from the above sources of iron. Additionally, Biempica et al. [8] observed lesions to the endoplasmic reticulum of hepatocytes of acute intermittent porphyria and porphyria cutanea tarda patients. These alterations might be responsible for Ca 2+ release to the cytoplasm. If such an increase in Ca 2+ concentration reaches the CaZ+-uniporter K m range (10-6-10 -5 M [22]), mitochondria could take up Ca 2+. This event might trigger the process of ROS-induced Ca2+-dependent mitochondrial membrane permeabilization studied in this paper. On the other hand, the availability of free Mg 2+ could modulate the extension of the oxidative mitochondrial damage in vivo (as part of the cellular Mg 2+ pool is complexed to soluble compounds, such as phosphate- and carboxylate-containing substances [44]). Finally, it is possible to imagine that ROS-induced Ca2+-dependent lesions in mitochondria occur in other organs (where ALA accumulates), such as brain, muscle and kidney, of acute intermittent porphyria patients.

Acknowledgements The authors are grateful for the technical assistance of R.C. Rosseto and the helpful comments of Drs. Ione S. Martins (IB-UNICAMP, Brazil), Sergio T. Ferreira and Adalberto Vieyra (ICB-Universidade Federal do Rio de Janeiro). This work was supported by grants from FAPESP, CNPq and F I N E P (Brazil) to Drs. A.E.V. and E.J.H.B. Dr. M.H.-L. is supported by a CAPES fellowship and a Ph.D. from the Instituto de Bioffsica Carlos Chagas Filho (Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil). V.G.R.V. is a doctoral student at the Escola Paulista de Medicina (Silo Paulo, Brazil) supported by a CNPq felllowship

206 and R.F. Castilho a medical student FAEP-UNICAMP scholarship.

supported

by a

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