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Characteristics of Ca2+ transport by Trypanosoma cruzi mitochondria in situ
ARCHIVES
OF BIOCHEMISTRY
Vol. 272, No. 1, July,
AND
BIOPHYSICS
pp. 122-129,1989
Characteristics ROBERTO
of Ca*+ Transport by Trypanosoma Mitochondria in Situ’ DOCAMPO*r’AND
ANIBAL
cruzi
E. VERCESIt
*Institute de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21961, Rio de Janeiro, Brazil, and -fInstituto de Biologia, Universidade Estadual de Cam&as, Camp&as 13100, Sao Pa&o, Brazil Received
November
l&1988;
and in revised
form
March
13,1989
The use of digitonin to permeabilize Trypanosoma cruxi plasma membrane has allowed the study of Ca2+ transport and oxidative phosphorylation in mitochondria in situ (R. Docampo and A. E. Vercesi (1989) J. BioZ. Chew. 264,108-111). The present results show that these mitochondria are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Cazf uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Addition of Cazf or ethylene glycol bis (/3aminoethyl ether) N-N-tetraacetic acid to these preparations under steady-state conditions was followed by Ca2+ uptake or release, respectively, tending to restore the original Ca2+ “set point” at about 0.9 PM. In addition, large amounts of Ca2+ were retained by T. cruxi mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butyl hydroperoxide, diamide, and the 1,2-naphthoquinone ,&lapachone. However, when ascorbate plus N,N#J’-tetramethyl-p-phenylenediamine in the presence of antimycin A was used as subtrate, P-lapachone caused pyridine nucleotide oxidation, and Cazf accumulation by these mitochondria was considerably lower than in control preparations, this effect being dose-dependent. o 1989Academic press, I,,~.
The difficulties in isolation of coupled mitochondria from most trypanosomatids have hampered the study of energy-linked functions in this group of parasites for years (1). However, we have shown recently that the use of digitonin to permeabilize Trgpanosoma cruxi plasma
membrane allows the study of oxidative phosphorylation and Cazf transport in mitochondria in situ (2). This saponin has previously been used to permeabilize the plasma membrane of a wide variety of cells (3-6), including protozoa (7), without significantly affecting the gross structure and function of intracellular organelles such as mitochondria and endoplasmic reticulum. It is now widely accepted that mitochondria isolated from most tissues of vertebrates (8-13) as well as from some lower life forms (14) possess an active Ca2+ transport system believed to participate in intracellular Ca2+ homeostasis. Uptake of the cation occurs through a uniport mechanism driven electrophoretically by the inside-negative membrane potential (A$) while the efflux pathway appears to promote the electroneutral exchange of matrix Ca2+ by external Na+ or Hf (9-12). The
i This work was supported in part by National Institutes of Health Grant AI-23259, the United Nations Development Project/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, the Conselho National de Desenvolvimento Cientifico e Tecnologico, the Financiadora de Estudos e Projetos, the Fundacao de Amparo a Pesquisa do Estado de SHo Paulo, and the Funda+ de Amparo a Pesquisa do Estado de Rio de Janeiro, Brazil. * Present address: Department of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399. 0003-9861/89 Copyright All rights
$3.00
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
122
Ca2+ TRANSPORT
BY TrZlpanosma
simultaneous operation of these pathways under steady-state conditions establishes a “set point” for extramitochondrial free Ca2+concentration in the range of 0.5 to 1.0 PM depending on the experimental conditions (9-12). There is evidence that alterations in mitochondrial Ca2+fluxes by toxic agents are associated with disruption in intracellular Ca2+ homeostasis and cell damage (15). It has been shown that Ca2+ retention in mitochondria is related to the redox state of intramitochondrial pyridine nucleotides (16) and thiols (17). Both t-butyl hydroperoxide (18) and 1,4-naphthoquinone menadione increase the oxidation of intramitochondrial pyridine nucleotides and thiols (15). These modifications result in a sustained increase in cytosolic Ca2+levels (15) which could cause the formation of numerous small blebs on the surface of the cells (15) and the activation of catabolic enzymes, such as proteases and lipasas (15). These processes may lead to cell death (15). P-Lapachone is a 1,2-naphthoquinone that, like menadione in hepatocytes (15), increases 0, and H202 formation in T. cruxi mitochondrial preparations and causes the formation of blebs on the surface of !l! cruxi at different stages and cell death (19-21). Since the formation of surface blebs by the action of naphthoquinones has been associated with mitochondrial Ca2+ release (15), we investigated the effect of P-lapachone on digitonin-permeabilized T. cruzi epimastigotes. Despite the importance of the knowledge of the mode of action of trypanocidal compounds for the identification of targets for drug action there is no evidence for the involvement of alterations in T. cruzi Ca2+ homeostasis by P-lapachone or other NAD(P)H oxidants. In the present work we have further characterized the mitochondrial Ca2+transport system of T. cruzi epimastigotes and studied the effect of NAD(P)H oxidants on this transport system. MATERIALS
AND METHODS
Rat liver mitochondria were isolated by the method of Schneider (22) from overnight-fasted male Wistar rats weighing approximately 250 g.
cruzi MITOCHONDRIA
123
T. cruzi culture forms (Y strain) were grown at 28°C under constant shaking (120 rpm) in the liquid medium described by Warren (23) supplemented with 5% fetal calf serum. Five days after inoculation, cells were collected by centrifugation and washed twice with 0.154 M NaCl. The protein concentration was determined by the biuret assay in the presence of 0.2% deoxycholate (24). ATP, oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),3 EGTA, N,N,N’, N’-tetramethyl-p-phenylenediamine (TMPD), tetraphenylphosphonium bromide (TPP+), and digitonin were purchased from Sigma. fi-Lapachone (3,4-dihydro - 2,2-dimethyl-W-naphtho[l,2-blpyran-5,6-dione) was a gift from Dr. A. V. Pinto. All other reagents were of analytical grade. Changes in Ca*+ concentration in the suspending medium were followed using a Ca2+ selective electrode (Radiometer, F2112 Calcium Selectrode). Signals from the Ca2+ electrode were fed into an amplifier and then into a dual channel strip chart recorder (Linear Model 1202). The response on the Ca2+ electrode was calibrated in each system by addition of internal standards to the medium and the concentrations were expressed on the ordinate axis in terms of total Ca2+ in the medium, except in the experiments in which the intramitochondrial steady-state free Ca2+ was determined at a final pH of 7.0,25”C. In this case the electrode was calibrated by the addition of the concentrations of EGTA (dissodium salt) calculated to achieve the desired values of [Ca”+] (25-27) adjusting the pH after each EGTA addition. The value 4.7 X lo6 Mm’ was used for the association constant of the Ca-EGTA complex under our experimental conditions. The internal standards were Ca’+EGTA buffers and the pH, temperature, and ionic strength of the medium were taken into account in their preparation. Mitochondrial membrane potential was monitored indirectly in terms of TPP+ activity in the incubation medium using a TPP+-selective electrode in combination with a calomel reference electrode as described by Kamo et al. (28). The electrode signals were amplified and the output was registered with a dual-channel recorder (Linear Model 1202). Changes in NAD(P)H redox state were measured fluorometrically (366 + 450 nm) in an Aminco Bowman spectrofluorometer.
‘Abbreviations used: FCCP, carbonyl cyanide p trifluoromethoxyphenylhydrazone; EGTA, ethylene glycol bis @-aminoethyl ether) N,N-tetraacetie acid, TPP+, tetraphenylphosphonium bromide; TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine; ASC, ascorbate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
124
DOCAMPO RESULTS
In a previous communication (2) we have shown that mitochondria in situ of digitonin-permeabilized T. cruxi epimastigotes were able to accumulate large amounts of Ca2+ parallel to respiratory stimulation. This suggests that as in mitochondria from vertebrate tissues (8-13) this Ca2+ uptake is driven by the membrane potential. In order to clarify this mechanism and to evaluate the phosphorylative capacity of our preparations we have applied the technique of TPP+ distribution across the inner mitochondrial membrane to follow the alterations in membrane potential during Cazf accumulation and ADP phosphorylation in these mitochondria in situ. These experiments were compared with experiments using isolated rat liver mitochondria where these processes have been extensively studied. The experiments in Fig. 1 show the effect of ADP and Ca2+on the membrane potentials developed by liver mitochondria and digitonin-permeabilized epimastigotes incubated in an aerobic medium containing succinate as respiratory substrate. The permeant cation TPP+ was taken up by intact liver mitochondria (Fig. 1A) or when the epimastigotes were permeabilized with digitonin (Figs. 1B and 1C). Addition of EGTA (Fig. 1C) to digitonin-permeabilized epimastigotes increased the potential in part by preventing Ca2+cycling since addition of ruthenium red to the experiment in Fig. 1B decreased this difference by about 30% (not shown). However, considering the complexity of the digitonin-permeabilized cell system the remaining effect of EGTA could be due to other factors, e.g., decreased endogenous ADP-ATP turnover. Addition of ADP or Ca2’ resulted in a small transient loss of TPP+ while addition of FCCP caused immediate loss of most of the incorporated TPP+, followed by the extrusion of the rest of the incorporated TPP+. Similarly, addition of ADP and Ca2+ to rat liver mitochondria caused the expected transient decrease of membrane potential, while addition of FCCP caused its immediate loss (Fig. 1A). Figure 2 shows that digitonin-permeabilized epimastigotes had the ability to
AND VERCESI TPP+QM)
*ImInI-RiM
4
3min +
i
FIG. 1. Mitochondrial membrane potential measured in digitonin-permeabilized T. cruzi epimastigotes and rat liver mitochondria. (A) The reaction mixture (total volume, 2 ml) contained 240 mM mannitol, 15 mM KCl, 2.0 mM Hepes buffer (pH 7.2), 5 mM succinate, 2 mM potassium phosphate buffer (pH 7.2), 5 pM rotenone, 0.1% bovine serum albumin. Additions were TPP+ as indicated, 1 mg/ml rat liver mitochondria (RLM), 100 nmol ADP, 40 nmol Ca’+, and 0.5 pM FCCP. (B) The reaction mixture (total volume, 2 ml) contained 200 ITIM sucrose, 10 mM Tris-HCl buffer (pH 7.4), 1 mM potassium phosphate buffer (pH 7.4), 2 mM succinate, and 0.005% digitonin. Additions were TPP+ as indicated, 20 nmol Ca2+, 0.5 pM FCCP, and 1.7 mg/ml epimastigotes (E). In the dashed line, 0.005% digitonin was added where indicated. (C)The same as in (B) but in the presence of 0.5 mM EGTA. The addition was 400 nmol ADP.
buffer external free Ca2+at concentrations in the range of 0.75 to 1.0 PM (trace a) under the same experimental conditions as those in which rat liver mitochondria maintain external free Ca2+in the range of 0.5 to 0.75 PM (trace b). Trace c of this figure shows the capacity of Ca2+ accumulation insensitive to oligomycin and FCCP. This indicates that in trace a most of the Ca2+present in the medium was taken up by T. cruxi mitochondria. Addition of Ca2+ or EGTA to these preparations under steady-state conditions was followed by Ca2+uptake or
Ca2+ TRANSPORT
0.50
-
+
3min
BY
Trypanmma
+-
FIG. 2. Ca2+ transport by digitonin-permeabilized T. err& epimastigotes and rat liver mitochondria. The test system (total volume, 2 ml) contained 200 mM sucrose, 10 mM Tris-HCl buffer (pH 7.0), 2 mM succinate, 1 mM potassium phosphate buffer (pH 7.0), and in a and c, 0.005% digitonin. Trace c also contained 0.5 NM FCCP and 1 pg/ml oligomycin. Additions were 1.7 mg/ml epimastigotes (E), 1 mg/ml rat liver mitochondria (RLM), 20 nmol Ca2+, and 15 nmol EGTA.
release, respectively, tending to restore the original steady-state level (traces a and b). Figure 3 shows experiments in which Ca2+ influx was studied in digitonized epimastigotes energized by different sub-
cruzi
MITOCHONDRIA
125
skates in a medium containing a high Ca2’ concentration. Under these experimental conditions, both the initial rates of Ca2’ accumulation and the Ca2+ set point were similar in mitochondria energized with either succinate (Fig. 3B) or endogenous substrates (Fig. 3A, trace NONE). Similar results were observed when Ca2+ transport was energized by malate/glutamate (data not shown) thus indicating that the presence of exogenous substrates was not necessary to support Ca2’ transport under our experimental conditions. This could be attributed to the presence of intact glycosomes in our preparations (29) that could provide the endogenous substrates required for Ca2+ transport. When ascorbate plus TMPD in the presence of antimycin A were used (Fig. 3A, trace TMPD/ASC) the initial rate of Ca2+ accumulation was slower although the Ca2+ set point was similar to that obtained with succinate, malate/glutamate, or endogenous substrates. In contrast to rat liver mitochondria, Ca2+ was not released by digitonin-permeabilized epimastigotes after addition of NAD(P)H oxidants, such as t-butyl hydroperoxide (0.3 InM) (14,18), and diamide (1.2 mM) (30), or naphthoquinones such as fl-
FIG. 3. Ca2+ transport by digitonin-permeabilized Z! cruzi epimastigotes in the presence of different substrates. (A) The test system (total volume, 1.5 ml) contained 200 mM sucrose, 10 rnbf TrisHCl buffer (pH 7.4), 2 mM MgClz, 1 mM potassium phosphate buffer (pH 7.4), 100 pM CaClz, and where indicated 2 mM ascorbate, 0.1 mM TMPD, and 1 &g/ml antimycin A (TMPD/ASC). Where indicated no substrates were added (NONE). Additions were 2.05 mg/ml epimastigotes (E) and 0.013% digitonin (DiG). (B) The same as in (A) but in the presence of 5 mM succinate (Sue) as substrate.
126
DOCAMPO
AND
VERCESI
t-butyl hydroperoxide and @-lapachone caused oxidation of pyridine nucleotides in rat liver mitochondria energized with succinate in the presence of rotenone (Fig. 6A). In contrast, in digitonized epimastigotes, oxidation of pyridine nucleotides by P-lapachone could be observed only if the preparation was energized by TMPD/ ascorbate in the presence of antimycin A (Fig. 6B). Only a very slow oxidation of pyridine nucleotides by t-butyl hydroperoxide could be observed in these preparations energized by succinate (Fig. 6C). FIG. 4. Effect of @-lapachone on Ca2’ transport by digitonin-permeabilized !l! cruzi epimastigotes. The test system (total volume, 1.5 ml) contained 200 mM sucrose, 10 mM Tris-HCl buffer (pH 7.4), 2 mM MgCla, 1 mM potassium phosphate buffer (pH ‘7.4), and 100 pM CaC12. Additions were 2.05 mg/ml epimastigotes (E), 0.013% digitonin, and where indicated 25,50,100, and 200 PM ,+lapachone.
lapachone (200 PM) (21) in the presence of either succinate or endogenous substrates (data not shown). However, when ascorbate plus TMPD in the presence of antimycin A was used as substrate for digitonized epimastigotes and the cells were exposed to different concentrations of plapachone (but not in the case of t-butyl hydroperoxide) Ca2+ accumulation was considerably lower than in control preparations, this effect being dose-dependent (Fig. 4). This finding indicates that the T. cruzi mitochondrion is more sensitive to the deleterious effect of Ca2+ in the presence of /3-lapachone when TMPD/ascorbate is used as substrate. In contrast, Fig. 5 shows that rat liver mitochondria energized with succinate are very sensitive to ,f3-lapachone (100 PM). In the presence of this naphthoquinone they accumulated only part of the Ca2+ present in the medium (10 PM) and released it under conditions in which most of the Ca2+ was taken up and retained by control mitochondria. Figure 6 shows the effect of t-butyl hydroperoxide and /3-lapachone on the redox state of pyridine nucleotides in rat liver mitochondria and digitonin-permeabilized epimastigotes. It can be observed that both
DISCUSSION
The present study shows that T. cruzi mitochondria in situ take up Ca2+ from the incubation medium by a mechanism associated with depolarization of the membrane potential as shown by a tetraphenylphosphonium-sensitive electrode. The results obtained with this electrode have shown that these mitochondria in situ are able to build up and retain an electrical membrane potential. This indicates that this method is suitable for studies of mitochondrial energy-linked functions in T. cruxi and possibly other trypanosomatids.
FIG. 5. Effect of fi-lapachone on Ca’+ transport by rat liver mitochondria. The test system (total volume, 1.5 ml) contained 200 mru sucrose, 10 mM Tris-HCI buffer (pH 7.4), 10 pM total calcium, and 1 mM MgCla. Additions were 1 mg/ml rat liver mitochondria in the presence or absence of 30 pM 8-lapachone.
Gas+ TRANSPORT
BY Trypanosma
FIG. 6. Effect of P-lapachone and t-butyl hydroperoxide on the redox state of pyridine nucleotides in rat liver mitochondria and in digitonin-permeabilized Z! cruzi epimastigotes. The test system (total volume, 1.0 ml) contained 200 mM sucrose, 10 mM Tris-HCl buffer (pH 7.4), and 1 mM EGTA. (A) The arrow indicates the addition of 200 pM t-butyl hydroperoxide (line a) or 30 pM P-lapachone (line b) to the reaction medium containing rat liver mitochondria (0.5 mg/ ml). (B) The arrow indicates the addition of 30 pM Blapachone to the reaction medium containing T. cruzi epimastigotes (2.3 mg/ml) and 0.013% digitonin in the presence of 5 pM rotenone and 5 mM succinate (line a) or 5 fiM rotenone, 1 pg/ml antimycin A, 2 mM ascorbate, and 0.1 mM TMPD (line b). (C) The arrow indicates the addition of 200 ~.LM t-butyl hydroperoxide to the reaction medium containing digitonin and T. cruzi epimastigotes as in (B). The dashed lines in (A) and (C) represent no additions.
The present results also suggest that mitochondria of digitonin-treated epimastigotes possess similar Ca2+ transporters as do liver mitochondria; i.e., separate pathways for Ca2+ influx and efflux as judged by the response of both types of mitochondria under steady state to the additions of Ca2+ and EGTA. These mitochondria were able to buffer external free Ca2+ at a concentration of about 0.9 PM under experimental conditions similar to those in which isolated liver mitochondria buffer external free Ca2+ at a concentration of about 0.7 PM.
An interesting observation of this work concerns the high capacity of Ca2+ retention by !I? cruxi mitochondria energized by different respiratory substrates and incubated in the presence of phosphate and Ca2+-releasing agents such as t-butyl hy-
cruzi MITOCHONDRIA
127
droperoxide (18) and diamide (30). This behavior is similar to that found in plant mitochondria (14). On the other hand, @-lapachone decreased Ca2’ accumulation and caused oxidation of pyridine nucleotides by digitonized epimastigotes only when TMPD/ascorbate was used as substrate. These results could suggest that the pyridine nucleotide redox-state-mediated Ca2+ efflux system (16) is present in T. cruzi mitochondria as well as in rat liver mitochondria but that the system is normally inoperative in T. cruxi mitochondria. This would be similar to what occurs with hepatoma mitochondria (31). Hepatoma mitochondria have a malic enzyme which reduces either NAD+ or NADP+ during carboxylation of malate to form pyruvate thus preventing Ca2+ efflux (31). A similar malic enzyme has been described in T. cruzi mitochondria (32, 33). The lack of effect of /3-lapachone on Ca2+ accumulation and retention by digitonin-permeabilized epimastigotes in the presence of either succinate or endogenous substrates could indicate, on the other hand, that mitochondrial Ca2+ release is not responsible for the formation of surface blebs in this parasite. The mechanism which causes Ca2+ loss upon peroxide treatment is still controversial. Certain studies would indicate that the irreversible oxidation and hydrolysis of NAD(P)+ is the primary cause, yet liver mitochondria treated in this manner retained a membrane potential (34-36). Other studies indicated that this treatment to induce Ca2+ release also collapsed the membrane potential (37). Some recent work has shown that NAD(P)+ oxidation can be disassociated from thiol oxidation and prevention of the former process does not block peroxide-induced Ca2+ release (38-40). Concerning the lack of effect of tbutyl hydroperoxide on Ca2 retention by digitonin-permeabilized epimastigotes, it is interesting to note that the system in trypanosomes responsible for the maintenance of intracellular thiols in the reduced state is different from that present in other organisms since it depends on a novel and unique trypanothione system (41). Analogous to glutathione peroxidase and reductase from other organisms, trypano-
128
DOCAMPO
somes possess a trypanothione peroxidase and reductase cycle using trypanothione (a spermidine-glutathione conjugate) as cofactor (41). In agreement with the results in Fig. 6C, it has been reported that the specific activity of the trypanothione peroxidase is quite low when compared with glutathione peroxidase in mammalian organs (42). This could explain the lack of effect of t-butyl hydroperoxide on Ca2+ accumulation and retention by digitoninpermeabilized epimastigotes. In recent years, it has been postulated (13) that the mitochondrial Ca2+ transport system regulates the mitochondrial free Ca2+ concentration in a range that allows the regulation of three intramitochondrial dehydrogenases (pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase, and 2-0xoglutarate dehydrogenase). T. cruxi lacks NAD+-isocitrate dehydrogenase activity and possesses only NADP+-isocitrate dehydrogenase (43). In addition, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities have not been detected in T. cruxi (44). This indicates that in contrast to mitochondria from vertebrate tissues, the Ca2+ transport system present in T. cruxi is apparently not linked to the regulation of these enzymes. In conclusion, these results indicate that T. cruxi mitochondria, despite the difference to most vertebrate mitochondria concerning the property of Ca2+ retention, also possess an efficient system for Ca2+ transport. They behave similarly to rat liver mitochondria regarding the ability to buffer external free Ca2+ under in vitro conditions and may have some role in the regulation of Ca2+ distribution in these cells. REFERENCES 1. HILL, G. C. (1976) &o&m Biophys. Acta 456, 149-193. 2. DOCAMPO, R., AND VERCESI, A. E. (1989) J. Biol. Chem 264.108-111. 3. FISKUM, G. (1985) Cell Calcium 6,25-37. 4. HARRIS, S. I., BALABAN, R. S., BARRETT, L., AND MANDEL, L. J. (1981) J. Biol. Chxm. 256,10,31910,329. 5. GRANGER, D. L., AND LEHNINGER, A. L. (1982) J.
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Trypanoscmza
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OF BIOCHEMISTRY
Vol. 272, No. 1, July,
AND
BIOPHYSICS
pp. 122-129,1989
Characteristics ROBERTO
of Ca*+ Transport by Trypanosoma Mitochondria in Situ’ DOCAMPO*r’AND
ANIBAL
cruzi
E. VERCESIt
*Institute de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21961, Rio de Janeiro, Brazil, and -fInstituto de Biologia, Universidade Estadual de Cam&as, Camp&as 13100, Sao Pa&o, Brazil Received
November
l&1988;
and in revised
form
March
13,1989
The use of digitonin to permeabilize Trypanosoma cruxi plasma membrane has allowed the study of Ca2+ transport and oxidative phosphorylation in mitochondria in situ (R. Docampo and A. E. Vercesi (1989) J. BioZ. Chew. 264,108-111). The present results show that these mitochondria are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Cazf uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Addition of Cazf or ethylene glycol bis (/3aminoethyl ether) N-N-tetraacetic acid to these preparations under steady-state conditions was followed by Ca2+ uptake or release, respectively, tending to restore the original Ca2+ “set point” at about 0.9 PM. In addition, large amounts of Ca2+ were retained by T. cruxi mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butyl hydroperoxide, diamide, and the 1,2-naphthoquinone ,&lapachone. However, when ascorbate plus N,N#J’-tetramethyl-p-phenylenediamine in the presence of antimycin A was used as subtrate, P-lapachone caused pyridine nucleotide oxidation, and Cazf accumulation by these mitochondria was considerably lower than in control preparations, this effect being dose-dependent. o 1989Academic press, I,,~.
The difficulties in isolation of coupled mitochondria from most trypanosomatids have hampered the study of energy-linked functions in this group of parasites for years (1). However, we have shown recently that the use of digitonin to permeabilize Trgpanosoma cruxi plasma
membrane allows the study of oxidative phosphorylation and Cazf transport in mitochondria in situ (2). This saponin has previously been used to permeabilize the plasma membrane of a wide variety of cells (3-6), including protozoa (7), without significantly affecting the gross structure and function of intracellular organelles such as mitochondria and endoplasmic reticulum. It is now widely accepted that mitochondria isolated from most tissues of vertebrates (8-13) as well as from some lower life forms (14) possess an active Ca2+ transport system believed to participate in intracellular Ca2+ homeostasis. Uptake of the cation occurs through a uniport mechanism driven electrophoretically by the inside-negative membrane potential (A$) while the efflux pathway appears to promote the electroneutral exchange of matrix Ca2+ by external Na+ or Hf (9-12). The
i This work was supported in part by National Institutes of Health Grant AI-23259, the United Nations Development Project/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, the Conselho National de Desenvolvimento Cientifico e Tecnologico, the Financiadora de Estudos e Projetos, the Fundacao de Amparo a Pesquisa do Estado de SHo Paulo, and the Funda+ de Amparo a Pesquisa do Estado de Rio de Janeiro, Brazil. * Present address: Department of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399. 0003-9861/89 Copyright All rights
$3.00
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
122
Ca2+ TRANSPORT
BY TrZlpanosma
simultaneous operation of these pathways under steady-state conditions establishes a “set point” for extramitochondrial free Ca2+concentration in the range of 0.5 to 1.0 PM depending on the experimental conditions (9-12). There is evidence that alterations in mitochondrial Ca2+fluxes by toxic agents are associated with disruption in intracellular Ca2+ homeostasis and cell damage (15). It has been shown that Ca2+ retention in mitochondria is related to the redox state of intramitochondrial pyridine nucleotides (16) and thiols (17). Both t-butyl hydroperoxide (18) and 1,4-naphthoquinone menadione increase the oxidation of intramitochondrial pyridine nucleotides and thiols (15). These modifications result in a sustained increase in cytosolic Ca2+levels (15) which could cause the formation of numerous small blebs on the surface of the cells (15) and the activation of catabolic enzymes, such as proteases and lipasas (15). These processes may lead to cell death (15). P-Lapachone is a 1,2-naphthoquinone that, like menadione in hepatocytes (15), increases 0, and H202 formation in T. cruxi mitochondrial preparations and causes the formation of blebs on the surface of !l! cruxi at different stages and cell death (19-21). Since the formation of surface blebs by the action of naphthoquinones has been associated with mitochondrial Ca2+ release (15), we investigated the effect of P-lapachone on digitonin-permeabilized T. cruzi epimastigotes. Despite the importance of the knowledge of the mode of action of trypanocidal compounds for the identification of targets for drug action there is no evidence for the involvement of alterations in T. cruzi Ca2+ homeostasis by P-lapachone or other NAD(P)H oxidants. In the present work we have further characterized the mitochondrial Ca2+transport system of T. cruzi epimastigotes and studied the effect of NAD(P)H oxidants on this transport system. MATERIALS
AND METHODS
Rat liver mitochondria were isolated by the method of Schneider (22) from overnight-fasted male Wistar rats weighing approximately 250 g.
cruzi MITOCHONDRIA
123
T. cruzi culture forms (Y strain) were grown at 28°C under constant shaking (120 rpm) in the liquid medium described by Warren (23) supplemented with 5% fetal calf serum. Five days after inoculation, cells were collected by centrifugation and washed twice with 0.154 M NaCl. The protein concentration was determined by the biuret assay in the presence of 0.2% deoxycholate (24). ATP, oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),3 EGTA, N,N,N’, N’-tetramethyl-p-phenylenediamine (TMPD), tetraphenylphosphonium bromide (TPP+), and digitonin were purchased from Sigma. fi-Lapachone (3,4-dihydro - 2,2-dimethyl-W-naphtho[l,2-blpyran-5,6-dione) was a gift from Dr. A. V. Pinto. All other reagents were of analytical grade. Changes in Ca*+ concentration in the suspending medium were followed using a Ca2+ selective electrode (Radiometer, F2112 Calcium Selectrode). Signals from the Ca2+ electrode were fed into an amplifier and then into a dual channel strip chart recorder (Linear Model 1202). The response on the Ca2+ electrode was calibrated in each system by addition of internal standards to the medium and the concentrations were expressed on the ordinate axis in terms of total Ca2+ in the medium, except in the experiments in which the intramitochondrial steady-state free Ca2+ was determined at a final pH of 7.0,25”C. In this case the electrode was calibrated by the addition of the concentrations of EGTA (dissodium salt) calculated to achieve the desired values of [Ca”+] (25-27) adjusting the pH after each EGTA addition. The value 4.7 X lo6 Mm’ was used for the association constant of the Ca-EGTA complex under our experimental conditions. The internal standards were Ca’+EGTA buffers and the pH, temperature, and ionic strength of the medium were taken into account in their preparation. Mitochondrial membrane potential was monitored indirectly in terms of TPP+ activity in the incubation medium using a TPP+-selective electrode in combination with a calomel reference electrode as described by Kamo et al. (28). The electrode signals were amplified and the output was registered with a dual-channel recorder (Linear Model 1202). Changes in NAD(P)H redox state were measured fluorometrically (366 + 450 nm) in an Aminco Bowman spectrofluorometer.
‘Abbreviations used: FCCP, carbonyl cyanide p trifluoromethoxyphenylhydrazone; EGTA, ethylene glycol bis @-aminoethyl ether) N,N-tetraacetie acid, TPP+, tetraphenylphosphonium bromide; TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine; ASC, ascorbate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
124
DOCAMPO RESULTS
In a previous communication (2) we have shown that mitochondria in situ of digitonin-permeabilized T. cruxi epimastigotes were able to accumulate large amounts of Ca2+ parallel to respiratory stimulation. This suggests that as in mitochondria from vertebrate tissues (8-13) this Ca2+ uptake is driven by the membrane potential. In order to clarify this mechanism and to evaluate the phosphorylative capacity of our preparations we have applied the technique of TPP+ distribution across the inner mitochondrial membrane to follow the alterations in membrane potential during Cazf accumulation and ADP phosphorylation in these mitochondria in situ. These experiments were compared with experiments using isolated rat liver mitochondria where these processes have been extensively studied. The experiments in Fig. 1 show the effect of ADP and Ca2+on the membrane potentials developed by liver mitochondria and digitonin-permeabilized epimastigotes incubated in an aerobic medium containing succinate as respiratory substrate. The permeant cation TPP+ was taken up by intact liver mitochondria (Fig. 1A) or when the epimastigotes were permeabilized with digitonin (Figs. 1B and 1C). Addition of EGTA (Fig. 1C) to digitonin-permeabilized epimastigotes increased the potential in part by preventing Ca2+cycling since addition of ruthenium red to the experiment in Fig. 1B decreased this difference by about 30% (not shown). However, considering the complexity of the digitonin-permeabilized cell system the remaining effect of EGTA could be due to other factors, e.g., decreased endogenous ADP-ATP turnover. Addition of ADP or Ca2’ resulted in a small transient loss of TPP+ while addition of FCCP caused immediate loss of most of the incorporated TPP+, followed by the extrusion of the rest of the incorporated TPP+. Similarly, addition of ADP and Ca2+ to rat liver mitochondria caused the expected transient decrease of membrane potential, while addition of FCCP caused its immediate loss (Fig. 1A). Figure 2 shows that digitonin-permeabilized epimastigotes had the ability to
AND VERCESI TPP+QM)
*ImInI-RiM
4
3min +
i
FIG. 1. Mitochondrial membrane potential measured in digitonin-permeabilized T. cruzi epimastigotes and rat liver mitochondria. (A) The reaction mixture (total volume, 2 ml) contained 240 mM mannitol, 15 mM KCl, 2.0 mM Hepes buffer (pH 7.2), 5 mM succinate, 2 mM potassium phosphate buffer (pH 7.2), 5 pM rotenone, 0.1% bovine serum albumin. Additions were TPP+ as indicated, 1 mg/ml rat liver mitochondria (RLM), 100 nmol ADP, 40 nmol Ca’+, and 0.5 pM FCCP. (B) The reaction mixture (total volume, 2 ml) contained 200 ITIM sucrose, 10 mM Tris-HCl buffer (pH 7.4), 1 mM potassium phosphate buffer (pH 7.4), 2 mM succinate, and 0.005% digitonin. Additions were TPP+ as indicated, 20 nmol Ca2+, 0.5 pM FCCP, and 1.7 mg/ml epimastigotes (E). In the dashed line, 0.005% digitonin was added where indicated. (C)The same as in (B) but in the presence of 0.5 mM EGTA. The addition was 400 nmol ADP.
buffer external free Ca2+at concentrations in the range of 0.75 to 1.0 PM (trace a) under the same experimental conditions as those in which rat liver mitochondria maintain external free Ca2+in the range of 0.5 to 0.75 PM (trace b). Trace c of this figure shows the capacity of Ca2+ accumulation insensitive to oligomycin and FCCP. This indicates that in trace a most of the Ca2+present in the medium was taken up by T. cruxi mitochondria. Addition of Ca2+ or EGTA to these preparations under steady-state conditions was followed by Ca2+uptake or
Ca2+ TRANSPORT
0.50
-
+
3min
BY
Trypanmma
+-
FIG. 2. Ca2+ transport by digitonin-permeabilized T. err& epimastigotes and rat liver mitochondria. The test system (total volume, 2 ml) contained 200 mM sucrose, 10 mM Tris-HCl buffer (pH 7.0), 2 mM succinate, 1 mM potassium phosphate buffer (pH 7.0), and in a and c, 0.005% digitonin. Trace c also contained 0.5 NM FCCP and 1 pg/ml oligomycin. Additions were 1.7 mg/ml epimastigotes (E), 1 mg/ml rat liver mitochondria (RLM), 20 nmol Ca2+, and 15 nmol EGTA.
release, respectively, tending to restore the original steady-state level (traces a and b). Figure 3 shows experiments in which Ca2+ influx was studied in digitonized epimastigotes energized by different sub-
cruzi
MITOCHONDRIA
125
skates in a medium containing a high Ca2’ concentration. Under these experimental conditions, both the initial rates of Ca2’ accumulation and the Ca2+ set point were similar in mitochondria energized with either succinate (Fig. 3B) or endogenous substrates (Fig. 3A, trace NONE). Similar results were observed when Ca2+ transport was energized by malate/glutamate (data not shown) thus indicating that the presence of exogenous substrates was not necessary to support Ca2’ transport under our experimental conditions. This could be attributed to the presence of intact glycosomes in our preparations (29) that could provide the endogenous substrates required for Ca2+ transport. When ascorbate plus TMPD in the presence of antimycin A were used (Fig. 3A, trace TMPD/ASC) the initial rate of Ca2+ accumulation was slower although the Ca2+ set point was similar to that obtained with succinate, malate/glutamate, or endogenous substrates. In contrast to rat liver mitochondria, Ca2+ was not released by digitonin-permeabilized epimastigotes after addition of NAD(P)H oxidants, such as t-butyl hydroperoxide (0.3 InM) (14,18), and diamide (1.2 mM) (30), or naphthoquinones such as fl-
FIG. 3. Ca2+ transport by digitonin-permeabilized Z! cruzi epimastigotes in the presence of different substrates. (A) The test system (total volume, 1.5 ml) contained 200 mM sucrose, 10 rnbf TrisHCl buffer (pH 7.4), 2 mM MgClz, 1 mM potassium phosphate buffer (pH 7.4), 100 pM CaClz, and where indicated 2 mM ascorbate, 0.1 mM TMPD, and 1 &g/ml antimycin A (TMPD/ASC). Where indicated no substrates were added (NONE). Additions were 2.05 mg/ml epimastigotes (E) and 0.013% digitonin (DiG). (B) The same as in (A) but in the presence of 5 mM succinate (Sue) as substrate.
126
DOCAMPO
AND
VERCESI
t-butyl hydroperoxide and @-lapachone caused oxidation of pyridine nucleotides in rat liver mitochondria energized with succinate in the presence of rotenone (Fig. 6A). In contrast, in digitonized epimastigotes, oxidation of pyridine nucleotides by P-lapachone could be observed only if the preparation was energized by TMPD/ ascorbate in the presence of antimycin A (Fig. 6B). Only a very slow oxidation of pyridine nucleotides by t-butyl hydroperoxide could be observed in these preparations energized by succinate (Fig. 6C). FIG. 4. Effect of @-lapachone on Ca2’ transport by digitonin-permeabilized !l! cruzi epimastigotes. The test system (total volume, 1.5 ml) contained 200 mM sucrose, 10 mM Tris-HCl buffer (pH 7.4), 2 mM MgCla, 1 mM potassium phosphate buffer (pH ‘7.4), and 100 pM CaC12. Additions were 2.05 mg/ml epimastigotes (E), 0.013% digitonin, and where indicated 25,50,100, and 200 PM ,+lapachone.
lapachone (200 PM) (21) in the presence of either succinate or endogenous substrates (data not shown). However, when ascorbate plus TMPD in the presence of antimycin A was used as substrate for digitonized epimastigotes and the cells were exposed to different concentrations of plapachone (but not in the case of t-butyl hydroperoxide) Ca2+ accumulation was considerably lower than in control preparations, this effect being dose-dependent (Fig. 4). This finding indicates that the T. cruzi mitochondrion is more sensitive to the deleterious effect of Ca2+ in the presence of /3-lapachone when TMPD/ascorbate is used as substrate. In contrast, Fig. 5 shows that rat liver mitochondria energized with succinate are very sensitive to ,f3-lapachone (100 PM). In the presence of this naphthoquinone they accumulated only part of the Ca2+ present in the medium (10 PM) and released it under conditions in which most of the Ca2+ was taken up and retained by control mitochondria. Figure 6 shows the effect of t-butyl hydroperoxide and /3-lapachone on the redox state of pyridine nucleotides in rat liver mitochondria and digitonin-permeabilized epimastigotes. It can be observed that both
DISCUSSION
The present study shows that T. cruzi mitochondria in situ take up Ca2+ from the incubation medium by a mechanism associated with depolarization of the membrane potential as shown by a tetraphenylphosphonium-sensitive electrode. The results obtained with this electrode have shown that these mitochondria in situ are able to build up and retain an electrical membrane potential. This indicates that this method is suitable for studies of mitochondrial energy-linked functions in T. cruxi and possibly other trypanosomatids.
FIG. 5. Effect of fi-lapachone on Ca’+ transport by rat liver mitochondria. The test system (total volume, 1.5 ml) contained 200 mru sucrose, 10 mM Tris-HCI buffer (pH 7.4), 10 pM total calcium, and 1 mM MgCla. Additions were 1 mg/ml rat liver mitochondria in the presence or absence of 30 pM 8-lapachone.
Gas+ TRANSPORT
BY Trypanosma
FIG. 6. Effect of P-lapachone and t-butyl hydroperoxide on the redox state of pyridine nucleotides in rat liver mitochondria and in digitonin-permeabilized Z! cruzi epimastigotes. The test system (total volume, 1.0 ml) contained 200 mM sucrose, 10 mM Tris-HCl buffer (pH 7.4), and 1 mM EGTA. (A) The arrow indicates the addition of 200 pM t-butyl hydroperoxide (line a) or 30 pM P-lapachone (line b) to the reaction medium containing rat liver mitochondria (0.5 mg/ ml). (B) The arrow indicates the addition of 30 pM Blapachone to the reaction medium containing T. cruzi epimastigotes (2.3 mg/ml) and 0.013% digitonin in the presence of 5 pM rotenone and 5 mM succinate (line a) or 5 fiM rotenone, 1 pg/ml antimycin A, 2 mM ascorbate, and 0.1 mM TMPD (line b). (C) The arrow indicates the addition of 200 ~.LM t-butyl hydroperoxide to the reaction medium containing digitonin and T. cruzi epimastigotes as in (B). The dashed lines in (A) and (C) represent no additions.
The present results also suggest that mitochondria of digitonin-treated epimastigotes possess similar Ca2+ transporters as do liver mitochondria; i.e., separate pathways for Ca2+ influx and efflux as judged by the response of both types of mitochondria under steady state to the additions of Ca2+ and EGTA. These mitochondria were able to buffer external free Ca2+ at a concentration of about 0.9 PM under experimental conditions similar to those in which isolated liver mitochondria buffer external free Ca2+ at a concentration of about 0.7 PM.
An interesting observation of this work concerns the high capacity of Ca2+ retention by !I? cruxi mitochondria energized by different respiratory substrates and incubated in the presence of phosphate and Ca2+-releasing agents such as t-butyl hy-
cruzi MITOCHONDRIA
127
droperoxide (18) and diamide (30). This behavior is similar to that found in plant mitochondria (14). On the other hand, @-lapachone decreased Ca2’ accumulation and caused oxidation of pyridine nucleotides by digitonized epimastigotes only when TMPD/ascorbate was used as substrate. These results could suggest that the pyridine nucleotide redox-state-mediated Ca2+ efflux system (16) is present in T. cruzi mitochondria as well as in rat liver mitochondria but that the system is normally inoperative in T. cruxi mitochondria. This would be similar to what occurs with hepatoma mitochondria (31). Hepatoma mitochondria have a malic enzyme which reduces either NAD+ or NADP+ during carboxylation of malate to form pyruvate thus preventing Ca2+ efflux (31). A similar malic enzyme has been described in T. cruzi mitochondria (32, 33). The lack of effect of /3-lapachone on Ca2+ accumulation and retention by digitonin-permeabilized epimastigotes in the presence of either succinate or endogenous substrates could indicate, on the other hand, that mitochondrial Ca2+ release is not responsible for the formation of surface blebs in this parasite. The mechanism which causes Ca2+ loss upon peroxide treatment is still controversial. Certain studies would indicate that the irreversible oxidation and hydrolysis of NAD(P)+ is the primary cause, yet liver mitochondria treated in this manner retained a membrane potential (34-36). Other studies indicated that this treatment to induce Ca2+ release also collapsed the membrane potential (37). Some recent work has shown that NAD(P)+ oxidation can be disassociated from thiol oxidation and prevention of the former process does not block peroxide-induced Ca2+ release (38-40). Concerning the lack of effect of tbutyl hydroperoxide on Ca2 retention by digitonin-permeabilized epimastigotes, it is interesting to note that the system in trypanosomes responsible for the maintenance of intracellular thiols in the reduced state is different from that present in other organisms since it depends on a novel and unique trypanothione system (41). Analogous to glutathione peroxidase and reductase from other organisms, trypano-
128
DOCAMPO
somes possess a trypanothione peroxidase and reductase cycle using trypanothione (a spermidine-glutathione conjugate) as cofactor (41). In agreement with the results in Fig. 6C, it has been reported that the specific activity of the trypanothione peroxidase is quite low when compared with glutathione peroxidase in mammalian organs (42). This could explain the lack of effect of t-butyl hydroperoxide on Ca2+ accumulation and retention by digitoninpermeabilized epimastigotes. In recent years, it has been postulated (13) that the mitochondrial Ca2+ transport system regulates the mitochondrial free Ca2+ concentration in a range that allows the regulation of three intramitochondrial dehydrogenases (pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase, and 2-0xoglutarate dehydrogenase). T. cruxi lacks NAD+-isocitrate dehydrogenase activity and possesses only NADP+-isocitrate dehydrogenase (43). In addition, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities have not been detected in T. cruxi (44). This indicates that in contrast to mitochondria from vertebrate tissues, the Ca2+ transport system present in T. cruxi is apparently not linked to the regulation of these enzymes. In conclusion, these results indicate that T. cruxi mitochondria, despite the difference to most vertebrate mitochondria concerning the property of Ca2+ retention, also possess an efficient system for Ca2+ transport. They behave similarly to rat liver mitochondria regarding the ability to buffer external free Ca2+ under in vitro conditions and may have some role in the regulation of Ca2+ distribution in these cells. REFERENCES 1. HILL, G. C. (1976) &o&m Biophys. Acta 456, 149-193. 2. DOCAMPO, R., AND VERCESI, A. E. (1989) J. Biol. Chem 264.108-111. 3. FISKUM, G. (1985) Cell Calcium 6,25-37. 4. HARRIS, S. I., BALABAN, R. S., BARRETT, L., AND MANDEL, L. J. (1981) J. Biol. Chxm. 256,10,31910,329. 5. GRANGER, D. L., AND LEHNINGER, A. L. (1982) J.
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Trypanoscmza
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