Characterization of Ca2+ transport in Euglena gracilis mitochondria

BB, ELSEVIER

Characterization of

Biochimica et Biophysica Acta 1186 (1994) 107-116

Ca 2+

transport in

Biochi!ic~a et BiophysicaA~ta

Euglena gracilis mitochondria

Aida Uribe, Edmundo Chfivez, Mois6s Jim6nez, Cecilia Zazueta, Rafael Moreno-Sfinchez * Departamento de Bioqufmiea, Instituto Nacional de Cardiolog[a, Juan Badiano # 1, Col. Secei6n XVI, M&ico D.F. 014080, Mexico (Received 24 January 1994)

Abstract

The present study was designed to establish the characteristics of the Ca 2+ fluxes in isolated mitochondria of the protist

Euglena gracilis. Uptake of Ca 2+ and Sr 2÷ was supported by succinate and lactate oxidation. Ca z+ influx was slightly inhibited by 5 tzM Ruthenium red and completely blocked by La 3÷ with a half-maximal inhibition attained at 50 /zM. The addition of inorganic phosphate induced a 3-fold stimulation of Ca 2÷ uptake. Ca 2÷ uptake was inhibited by Mg 2÷ only in the absence of phosphate. Ca 2÷ efflux was induced by Na +, Li + and K + through a diltiazem-insensitive reaction. Ca 2+ release, collapse of membrane potential and swelling were induced by I-Ig2+ and Cd 2÷ but not by carboxyatractyloside; cyclosporin A did not prevent the Ca 2+ release induced by the heavy metal ions. Ca 2+ uptake was achieved in the presence of 3/xM antimycin or 0.1 mM cyanide; this finding indicates that the alternative respiratory chain present in Euglena mitochondria can support this energy-dependent reaction. The data obtained suggest similar pathways, but different regulatory mechanisms, for Ca 2÷ transport between protist and mammalian mitochondria.

Key words: Mitochondrion; Calcium ion transport; Heavy metal ion; Alternative respiratory chain; (Euglena)

1. Introduction

It is now well established that mitochondria from virtually all animal tissues are able to transport Ca a+ through an energy-dependent reaction (see for reviews [1,2]). The Ca 2÷ uptake reaction is catalyzed by an electrophoretic uniporter, driven by an internal negative m e m b r a n e potential, and inhibited by Ruthenium red in m a m m a l i a n [1,2] and plant mitochondria [3-5]. In contrast, in protist mitochondria from Euglena [6,7] and trypanosomatids [8-10], the Ca 2+ uptake reaction is only partially sensitive to R u t h e n i u m red, although it is completely abolished by uncouplers and respiratory inhibitors. The Ca 2+ efflux reaction is catalyzed by either a N a + / C a 2+ or H + / C a 2+ exchange in m a m malian mitochondria at low matrix Ca 2+ contents [1,2]. However, in plant and protist mitochondria the Ca 2+ effiux pathway has not as yet been characterized. A n o t h e r pathway for Ca 2+ effiux is apparent in mitochondria loaded with relatively high amounts of

* Corresponding author. Fax: + 52 5 5730926. 0005-2728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 2 8 ( 9 4 ) 0 0 0 0 2 8 - 4

Ca 2+ [1,2]. The activation and opening of this pathway of Ca z+ effiux depends on several factors such as the intramitochondrial N A D H / N A D ÷ ratio [11,12], the redox state of the m e m b r a n e thiol groups [13,14], and the m e m b r a n e orientation of the adenine nucleotide translocase [15]. In particular, the interaction of heavy metal ions with m e m b r a n e thiol groups [14,16,17], and the fixation of the adenine nucleotide translocase in the so-called cytosolic orientation by carboxyatractyloside [15,18], induce the release of mitochondrial Ca 2÷. A potent, specific inhibitor of this Ca z÷ release pathway is cyclosporin A [19]. The presence and functioning of such via for Ca 2÷ effiux in protist mitochondria is unknown. The present study was undertaken with the aim to further characterize the mechanisms involved in Ca 2÷ transport in Euglena gracilis mitochondria. The results indicated that the Ca 2+ transport system in Euglena mitochondria possesses characteristics different to those found in mammalian mitochondria regarding inhibitor sensitivity, cation selectivity, and support of this energy-dependent reaction by the alternative respiratory chain.

108

A. Uribe et al. / B i o c h i m i c a et Biophysica A c t a 1186 (1994) 107 l lO

2. Materials and m e t h o d s

3.1. Ca: + influx

Cell culture and growth conditions of wild-type Euglena gracilis Klebs (a Z-like strain) and isolation of

In agreement with preliminary data [7], Euglena mitochondria were able to take up Ca 2÷ and S r 2+ in an energy-dependent manner as indicated by (i) the cation release induced by the uncoupler CCCP (Fig. 1A,B), (ii) the transient deflection in the magnitude of membrane potential during cation uptake (Fig. 1C,D), and (iii) negligible cation uptake in the absence of

mitochondria were described previously [6,7,20]. The final mitochondrial pellet was resuspended in 250 mM sucrose, 10 mM Tris-Hepes, 0.2% (w/v) fatty acid free albumin (pH 7.2) and kept in ice until use. Ca 2÷ uptake was followed by measuring the changes in absorbance of 50/xM Arsenazo III at 675-685 nm [21] in a dual wavelength DW-2c Aminco spectrophotometer, under stirring, 0 2 gassing and maintenance at 25°C. The incubation media were: (a) the sucrose medium containing 100 mM sucrose, 10 mM Tris-Hepes, 10 mM Tris-phosphate, 10 Tris-succinate, of final pH 7.2, and (b) the K ÷ medium where sucrose was replaced by 50 mM KCI. Ca 2÷ accumulation was also measured with 4 5 C a 2 + (spec. act. 1000 cpm/nmol) by filtration. Mitochondrial transmembrane potential was determined by measuring the changes in absorbance of 5 /zM Safranin O at 511-533 nm [22]. Mitochondrial swelling was followed by changes in optical absorbance at 540 nm. 0 2 consumption was determined with a Clark-type electrode. Mitochondrial protein was measured by the biuret method as described previously [7]. Cyclophilin activity was analyzed photometrically at 390 nm by incubating matrix extracts in media containing 25 /xM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and 0.78/zg chymotrypsin [23]. Arsenazo III and Ruthenium red were further purified. Fresh solutions of Safranin O were used throughout this work. Except for 4 5 C a 2 + (Amersham), all other chemicals were from Sigma.

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3. Results

The respiratory control values and A D P / O ratios obtained for the present Euglena mitochondrial preparation in an EGTA-containing medium were 1.5-2.1 and 1.4-1.9 with succinate and 1.5-2.3 and 1.3-2 with L-lactate as oxidizable substrate, respectively. The appropriate A D P / O ratios and respiratory control values attained with these substrates assure the membrane integrity of these Euglena mitochondria. The addition of 20/~ M carboxyatractyloside (CAT) completely inhibited the rate of ADP-stimulated respiration; this inhibition was released by 1 ~tM of the uncoupler CCCP (not shown). The inhibition exerted by CAT on the ADP-stimulated respiration and ATP hydrolysis [6] indicates that the adenine nucleotide translocation in Euglena mitochondria is carried out by an A T P / A D P translocase system similar to that present in mammalian mitochondria [24].

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Fig. 1. Ca 2+ and Sr 2+ uptake and m e m b r a n e potential in Euglena mitochondria. (A,B) Mitochondria (2 mg protein) were added to 3 ml of the sucrose m e d i u m described in the Methods section along with 33 y.M CaCI 2 (A) or 3 3 / z M SrCI 2 (B). Ca 2÷ uptake was measured by following the changes in the absorbance difference of 50 ~ M Arsenazo III at 675-685 nm. (C,D) Mitochondria (4 mg protein) were incubated in 3 ml of the K ÷ m e d i u m described u n d e r Methods. M e m b r a n e potential was measured by following the changes in the absorbance difference of 5 /zM Safranin O at 511-533 nm. O t h e r additions were 33 p.M CaCI 2 (C), 33 ~ M SrCI 2 (D), 0.33/zM CCCP.

A. Uribe et al. / Biochimica et Biophysica A cta 1186 (1994) 107-116 Table 1 Ca 2+ uptake in Euglena mitochondria Addition

Ca 2+ U p t a k e ( n m o l / m g protein) - Pi

+ 10 m M Pi

+ 5 m M Mg 2÷ + 1 m M spermine

18+3 6+la 13 + 1 b

67+3 605:4 50_+ 7 c

a p < 0.001 versus control. Student t-test for paired samples. b p < 0.01 versus control. c p < 0.05 versus control. Mitochondria (1 mg protein) were incubated in 1.5 ml of the standard m e d i u m additionally containing 5 0 / x M 45Ca2+ for 5 min. T h e n an aliquot of 0.2-0.5 ml was filtered through a 0.45 /.~m diameter Millipore filter and washed with 10 ml cold 0.15 M KC1. T h e radioactivity of the filter was counted in a scintillation counter. O t h e r additions were as indicated. T h e n u m b e r s shown represent the mean_+ standard error of four different mitochondrial preparations.

oxidizable substrate [7]. Substitution of L-lactate by succinate gave an identical Ca 2+ (and Sr 2+) uptake as reported previously [7]. Increase of the sucrose concentration from 100 (Fig. 1) to 250 or 500 mM slightly (10-20%) reduced both the rate and the amount of Ca 2+ accumulated (data not shown); this observation confirms results obtained with mammalian mitochondria [25] that a hypotonic medium favors Ca z+ uptake. Ca 2+ uptake obtained in a K + medium was markedly lower than in the sucrose medium (see Fig. 3B); this result is very likely due to an increased Ca 2+ efflux in the K + medium (see below). Similarly to plant mitochondria [5], Ca 2÷ uptake in Euglena mitochondria was decreased by 70-80% by removal of Pi (Table 1). Acetate (20 mM) did not promote Ca 2+ uptake. The addition of 5 mM Mg 2+ inhibited uptake by 60%, whereas the polyamine sper-

109

mine (1 mM) inhibited the uptake of Ca 2+ by 10-20%, either in the presence or in the absence of phosphate; in the presence of 10 mM phosphate, Mg z+ was not inhibitory (Table 1). A D P (0.2 mM) did not enhance the uptake of Ca 2+ (not shown). The rate of Ca 2+ uptake was dependent on the extramitochondrial Ca 2+ concentration with no apparent saturation up to 300 /zM Ca 2+ (Fig. 2A). Ruthenium red inhibited Ca 2+ uptake in Euglena mitochondria only slightly (Fig. 2B), as previously reported [6,7]; an inhibitory effect was only apparent at Ruthenium red concentrations higher than 1 /zM with a maximal inhibition of 26% at 5 /xM. Conversely, 0.1 txM Ruthenium red completely (90% inhibition) blocked Ca 2+ transport in rat kidney mitochondria. La 3 + competitively inhibits Ca 2 + uptake in mammalian mitochondria [1,2] and also effectively blocked Ca 2+ uptake in Euglena mitochondria (Fig. 2C); the halfmaximal inhibition was attained at 10 and 5 0 / x M La 3+ for rat kidney and Euglena mitochondria, respectively. Thus, the results with Ruthenium red and La 3+ suggest that the Ca 2+ uniporter in Euglena mitochondria seems to lack the glycoprotein moiety required to bind Ruthenium red [1,2] but it preserves the lanthanide reaction site.

3.2. Ca 2+ efflux To measure N a + / C a 2+ exchange in mammalian mitochondria a protocol where Ruthenium red is added to CaZ+-loaded mitochondria prior to the addition of Na + is generally used [1,2]. As Ruthenium red was not an effective inhibitor of Ca 2+ uptake in Euglena mitochondria (see Fig. 2B), La 3+ was used for blocking

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Fig. 2. Effect of R u t h e n i u m red and La 3+ on Ca 2+ uptake in Euglena mitochondria. Mitochondria (1 mg protein) were incubated in 1.5 ml of the sucrose m e d i u m containing the indicated 45Ca2+ concentrations in (A) or 3 3 / z M in (B) and (C). After 5 min an aliquot was withdrawn and filtered, and the filter washed and counted for radioactivity. T h e n u m b e r s shown in the figures represent the a m o u n t of accumulated Ca 2+ after subtraction of 4SCa~+ retained in the filter in the presence of 0.5/xM CCCP at the different Ca 2÷ concentrations used. Euglena (upper traces) or rat kidney (lower traces) mitochondria were incubated with the indicated R u t h e n i u m red (B) or LaCI 3 (C) concentrations for 5 min.

110

A. Uribe et al. / Biochimica et Biophysica Acta 1186 (1994) 107-116

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3.3. CarboxyatractylosideCa e+ release

and heavy metal-induced

Ca 2+ overloading of mitochondria damages the membrane integrity, an effect which is amplified by the addition of several inducing agents such as carboxyatractyloside [15,18] and heavy metal ions [14,16,17]. To follow this membrane transition, properties that depend on the permeability of the mitochondrial membrane are generally examined [1,2]. The addition of CAT to Ca2+-loaded Euglena mitochondria failed to induce Ca 2÷ release in both the sucrose- (Fig. 4A) and K ÷ media (not shown); conversely, CAT induced a fast and complete Ca 2+ release in rat kidney mitochondria (Fig. 4B). The CAT effect was also assessed on the membrane potential (Fig. 4C,D): CAT was ineffective in Euglena mitochondria but it induced a collapse of

Mit

Fig. 3. Efflux of Ca 2+ induced by monovalent cations in Euglena mitochondria. Mitochondria were incubated in the sucrose (A,B) or K ÷ m e d i u m (B, trace 2) as described in Fig. 1A. W h e r e indicated (Me + ) 20 (traces 1,2,4) or 50 m M (traces 3,5,6) Na + , K+, and Li ÷ were added, respectively. CCCP was 0.33 ~ M .

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Ca 2+ uptake; however, a drawback in this protocol may be that La 3÷ also inhibits the Ca 2÷ efflux pathways [1,2]. Interestingly, addition of 0.2 mM La 3+ to mitochondria loaded with 45Ca2+ induced a Ca 2÷ efflux of 10.6 _+ 3 n m o l / m g protein per 6 min (n = 4) in the sucrose medium at 25°C. Therefore, although La 3+ may be inhibiting both Ca 2÷ influx and efflux, the inhibitory effect was stronger on the influx pathway. Li ÷, Na ÷ and K ÷ also induced a significant Ca 2+ efflux (Fig. 3A), with Li ÷ being the most active cation. This monovalent cation-induced Ca 2÷ effiux was not blocked by 1 mM diltiazem (not shown). A Ca 2 ÷ effiux of 7 and 16 nmol Ca2+/mg protein per 6 min (n = 2) as induced by 50 mM K ÷ and Li ÷, respectively, was also determined in mitochondria previously loaded with 45Ca2+. Thus, the diminished level of Ca 2÷ taken up in the K+-medium (Fig. 3B) can be explained by an enhanced K+-induced Ca 2÷ effiux. Therefore, the resuits obtained with La 3÷ and the monovalent cations strongly suggest the presence of active Ca 2+ efflux pathways in Euglena mitochondria. Determination of the pH gradient by laC-DMO and 3 H 2 0 distribution showed a value near to zero for mitochondria incubated in both the sucrose or K medium + 5 0 / z M Ca 2÷, as expected from the high (10 mM) Pi concentration used. This result seems to discard that the K ÷ inducedCa 2÷ efflux is derived from a stimulation of a C a 2 + / H ÷ exchange brought up by matrix alkalinisation caused by a K + / H + exchange.

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Fig. 4. Effect of carboxyatractyloside on Ca 2+ uptake and m e m b r a n e potential in Euglena and kidney mitochondria. Mitochondria from Euglena gracilis (A,B) or rat kidney (A,C) were incubated as indicated in Fig. 1A to m e a s u r e Ca 2+ fluxes (A) or as described in Fig. 1C to m e a s u r e m e m b r a n e potential (B,C). Other additions were 33 /xM Ca 2+, 1 0 / x M carboxyatractyloside, 0.33 p.M CCCP.

A. Uribe et al. /Biochimica et Biophysica Acta 1186 (1994) 107-116

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.vM Ca 2. or 1.192* Fig. 5. Effect of Cd 2+ and Hg 2÷ on Ca 2+ fluxes, membrane potential, and respiration in Euglena mitochondria. Ca ~+ fluxes (A,B) were measured as described in Fig. 1A. (A): 1, 2, 2.5, 3, 5 or 10/zM Cd 2+ was added in traces 1 to 6, respectively. (B): 1, 2, 3, 4, 5 or 10/xM Hg 2+ was added in traces 1 to 6, respectively. The small deflection in the Arsenazo IIl signal obtained after the addition of 10/zM Cd 2+ or Hg 2+ (traces 6, Figs. 5A,B) indicated the low degree of interaction between dye and heavy metals, and which did not interfere with the interaction between dye and Ca 2÷ or Sr 2+ (not shown). Membrane potential (C,D) was measured as described in Fig. 1C. (C): 1 and 3 (trace a) or 2 / z M (trace b) Cd 2÷. (D): 1 (trace a), 2 (trace b) or 3 and 2 / ~ M Hg 2+. Inset: the rate of 0 2 consumption of 2 mg protein incubated in 1.9 ml of sucrose medium was measured in the presence of 33/.~M CaCI 2 at the indicated Cd 2+ and Hg 2+ concentrations. The rate of respiration was 145 ng atoms oxygen/mg protein per min in the absence of heavy metals.

A. Uribeet al. /Biochimica et Biophysica Acta 1186 (1994) 107-116

112

the m e m b r a n e potential in the presence of Ca 2+ in kidney mitochondria. As expected from an effect on the m e m b r a n e integrity, the addition of increasing Cd 2+ and Hg 2+ concentrations to Ca2+-loaded Euglena mitochondria induced a gradual Ca 2+ release (Fig. 5A,13), collapse of m e m b r a n e potential (Fig. 5C,D), and swelling with Hg 2+ but not with Cd 2+ (not shown). Mersalyl (10 /xM), another thiol groups reagent, was also able to induce Ca z+ release (not shown). The Ca 2+ release induced by heavy metals was further confirmed by following the remaining 45Ca2+ after the addition of Cd 2+ and Hg 2+ to mitochondria loaded with 50 /xM 45Ca2+: the release of 3 and 10 nmol C a Z + / m g prot e i n / 6 min (n = 2) was induced by 2 tzM Cd 2+ and Hg 2+, and 24 _+ 5 and 78 _+ 2 nmol C a Z + / m g protein per 6 min (n = 4) by 1 0 / x M Cd 2+ and Hg 2+, respectively. The rate of 0 2 uptake was inhibited by Cd 2+ and Hg 2+ (Fig. 5, inset) at concentrations higher than those required to attain collapse of m e m b r a n e potential or release of Ca 2+. Cyclosporin A up to a concentration of 6.25 ~ g / m g protein only slightly prevented the Ca 2+ release induced by a low heavy metal concentration ( < 2 ~ M ) but it was completely ineffective at higher heavy metal concentrations (not shown). Surprisingly, the lack of cyclosporin A effect was not due to the absence of cyclophilin, a cyclosporin-A-sensitive peptidyl-prolyl cis-trans isomerase present in the

mitochondrial matrix [23]. Assay of the isomerase in Euglena mitochondria showed an activity of 35 + 10 and 7 + 2 nmol of peptide h y d r o l y z e d / m g protein per 30 s (n = 6), in the absence and in the presence of I /zM cyclosporin A, respectively; the cyclophilin activity in kidney mitochondria was 124 _+ 17 and 35 + 14 nmol of peptide h y d r o l y z e d / m g protein per 30 s (n = 6), in the absence and in the presence of 1 /xM cyclosporin A, respectively. In contrast to mammalian mitochondria, Cd 2+ and Hg 2+ also induced release of Sr 2+ (Fig. 6A) and collapse of m e m b r a n e potential in the presence of Sr 2+ (Fig. 6B) in Euglena mitochondria. As Sr 2+ is not recognized by the cyclosporin-A-sensitive Ca 2+ release pathway in mammalian mitochondria (1,2), and the heavy-metal-induced Ca 2+ release in Euglena mitochondria was not sensitive to cyclosporin A, it would a p p e a r that Ca 2+ and Sr 2+ are released by heavy metal ions through different pathways in mammalian and Euglena mitochondria.

3.4. Ca 2+ uptake dricen by the alternative respiratory chain Euglena mitochondria have an antimycin- and cyanide-resistant respiration [7,20,26-28] which is blocked by diphenylamine [27], is able to generate a small, uncoupler sensitive m e m b r a n e potential [6,7],

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! 2" M Fig. 6. Effect of C d 2+ and Hg 2+ o n S r 2+ fluxes and membrane potential in Euglena mitochondria. The same experimental conditions described in Fig. 1B for Ca2÷ fluxes (A) and Fig. 1D for membrane potential (B) were used. (A) 10 p,M Cd2+ or Hg 2+ were added. (B) 3 and 2 #M Cd 2÷ or Hg 2÷ were added as indicated. Sr 2+ was 33 /~M and CCCP was 0.66/zM.

113

A. Uribe et al. /Biochimica et Biophysica Acta 1186 (1994) 107-116 RKM

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Fig. 7. Ca 2+ uptake driven by the alternative respiratory chain in Euglena mitochondria. Euglena (A,C-E) or kidney (B) mitochondria were incubated as described in Fig. 1A to measure Ca 2÷ uptake (A,B) or as described in Fig. 1C to measure membrane potential (C-E). Where indicated, 3.3 (A,D,E) or 0.1 (B) tzM antimycin (Anti), 0.1 mM KCN, 0.2 mM diphenylamine (DPA), 33/zM Ca 2÷, 0.66 ~M CCCP were added. In (C) and (D), 10 mM L-lactate was used instead of succinate as oxidizable substrate.

oxidizes preferentially lactate [7,26,27], and involves the functioning of an alternative oxidase presumably cytochrome o [20,26,27]. Interestingly, in the presence of 3 /zM antimycin or 0.1 m M cyanide, inhibitor concentrations that totally block the cyanide-sensitive

pathway (cytochrome c oxidase) but only slightly affects the alternative pathway (cytochrome o) [7,26,27], both L-lactate (Fig. 7A) or succinate (not shown) were still able to support Ca 2÷ uptake. This result indicated the ability of the alternative respiratory chain to gener-

114

A. Uribe et al. jBiochimica et Biophysica Acta 1186 (1994) 107-110

ate a H + gradient of a sufficient magnitude (Fig. 7C-E) to drive Ca 2+ uptake in Euglena mitochondria. In contrast, 0.1 /zM antimycin or 0.1 mM cyanide induced a complete Ca 2+ release (Fig. 7B) and collapse of membrane potential (not shown) in rat kidney mitochondria. The higher sensitivity of membrane potential to antimycin with succinate (Fig. 7E) than with lactate (Fig. 7D) supports the notion that lactate is preferentially oxidized by an antimycin-resistant alternative respiratory chain [7,26-28]. Ca 2+ uptake driven by L-lactate was abolished by 1 mM cyanide (not shown). A similar Ca 2+ uptake resistant to antimycin and cyanide was obtained in Euglena mitochondria from cells grown with ethanol as carbon source (data not shown): presumably these mitochondria have developed a predominant cyanide-resistant respiratory chain [27].

4. Discussion

The results of this study indicated that the Ca 2÷ influx reaction in Euglena mitochondria was similar to that present in mammalian mitochondria except for the low sensitivity to Ruthenium red and a slight inhibition by spermine. A lack of effect or only a partial inhibition by high Ruthenium red concentrations on Ca 2÷ uptake has also been found in some trypanosomatids mitochondria [8,9]. The presence of an energy-dependent Ca 2+ uptake together with a La 3÷- and monovalent cation-induced Ca 2+ efflux reaction suggests the existence of a complete Ca 2+ transport system of influx and efflux pathways in Euglena mitochondria. As it is unlikely that Euglena has a significant cytosolic Li ÷ concentration, it would appear that the main pathways for Ca 2+ efflux in vivo are the La3+-induced, presumably a H + / C a 2÷ exchange reaction, and the Na ÷ and K+-induced Ca 2÷ effiux reactions. The role of the mitochondrial Ca 2÷ transport system in mammalian mitochondria appears to be that of regulating the matrix free Ca 2÷ concentration [29,30], in the range where the Ca2+-sensitive dehydrogenases - for example, the pyruvate, isocitrate, and 2-oxoglutarate dehydrogenases - can be modulated by Ca 2÷ [29,30]. However, Euglena mitochondria lack the pyruvate dehydrogenase complex [31] and the 2-oxoglutarate dehydrogenase [32], and only contain the isocitrate dehydrogenase [33]. Therefore, regulation of the matrix Ca 2+ concentration by the Ca 2+ transport system would appear to be of little use in Euglena mitochondria, unless other Ca2+-sensitive enzymes such as pyrophosphatase [30] or pyruvate carboxylase [34] were present in the mitochondrial matrix of this protist. The addition of respiratory inhibitors induced an increase in the cytosolic free Ca 2÷ concentration in Leishmania dono-

t;ani [35], suggesting that mitochondria in this trypanosomatid regulate the cytosolic Ca -'+ rather than the matrix Ca 2+ concentration. As Euglenoids and trypanosomatids are the most primitive eukariotes containing mitochondria [36,37], the elucidation of thc physiological role of the mitochondrial Ca 2÷ uptake in these microorganisms might be of relevance for the elaboration of phylogenetic trees in addition to providing with some insight on the Ca 2+ homeostasis in protists. The release of Ca 2÷ through the cyclosporin-A-sensitive pathway has been actively studied in mammalian mitochondria [1,2]. Several models have been postulated for the functioning of this pathway of Ca 2÷ release [12-15,23,38-40], although convincing evidence has yet to be provided. The cyclosporin-A-sensitive Ca 2÷ release has been implied in the mechanism of cell injury during ischemia and reperfusion in heart [41] and liver [42]. However, no attempt has been made to identify this pathway of Ca 2÷ release in mitochondria of invertebrates. In some trypanosomatids mitochondria [8,9] the Ca2÷-releasing agents peroxides and diamide were ineffective. There is a large body of evidence indicating that modification of membrane thiol groups is involved in the underlying mechanism that leads to the membrane permeability transition [1,2]. Heavy metals bind thiol groups with high affinity and it was shown in a previous work [14] that the binding of approximately 1 nmol/mg protein suffices to induce Ca 2÷ release in mammalian mitochondria. Although there are other frequently used Ca 2+ releasing agents such as inorganic phosphate and t-butyl hydroperoxide, they are required in millimolar concentrations. Moreover, the amplitude of membrane damage induced by heavy metals is quite similar to that induced by phosphate or t-butyl hydroperoxide [14-19]. A number of studies have shown that the permeability barrier for Ca 2+ release is maintained through the stabilization of the A T P / A D P translocase in the matrix side conformation [15,18,23]. Thus, carboxyatractyloside by stabilizing the translocase in the cytosolic side can induce the release of Ca 2÷ in mammalian mitochondria. However, evidence was presented in this study that CAT failed to induce Ca 2÷ release in Euglena mitochondria (Fig. 4). This lack of CAT effect cannot be ascribed to a diminished A T P / A D P translocase sensitivity to CAT, considering that CAT effectively inhibited ADP-stimulated respiration and ATP hydrolysis in Euglena mitochondria. In addition, cyclosporin A also failed to block the Ca 2÷ release induced by Cd 2÷ and Hg 2+, despite the presence of significant cyclosporin-A-sensitive cyclophilin activity. To this regard, it has been proposed that cyclophilin [23,43] transforms the A T P / A D P translocase into an open pore for ion flux, whereas cyclosporin A by interacting with cyclophilin induces the pore closure. Thus,

A. Uribe et al. /Biochirnica et Btophysica Acta 1186 (1994) 107-116

the data with CAT and heavy metals suggest that Ca 2÷ release in Euglena mitoehondria, in contrast to that found in mammalian mitochondria, is regulated by factors other than the A T P / A D P translocase. Pathways for Ca 2+ release and membrane permeability transition insensitive to cyclosporin A have also been described for mammalian mitochondria [42,44]. Sr 2+ does not replace Ca 2÷ for the opening of the cyclosporin-A-sensitive pore in mammalian mitochondria [1,2]. However in Euglena mitochondria both Ca 2+ and Sr 2+ were released and membrane potential collapsed by the addition of Cd 2+ and Hg 2÷ (Figs. 5 and 6). Clearly, the pathway for cation efflux in Euglena mitochondria is not of the same nature as that present in mammalian mitochondria. An intriguing question for bacterial, plant and other respiratory chain systems containing alternative pathways has been the elucidation of the physiological role of such alternative respiratory chains. These cyanideresistant alternative pathways become more apparent under stress conditions in bacteria [45,46] and Euglena [20,27], Isolation and reconstitution of bacterial cytochrome o, one of the alternative terminal oxidases, showed that it was capable of H ÷ pumping and generation of a membrane potential [47,48]. Euglena mitochondria also have a cyanide-resistant alternative respiratory chain [7,20,26-28], which has not known physiological role as yet: efforts to obtain ATP synthesis supported by the alternative pathway have been unsuccessful [7]. However, as shown in this study, Euglena mitochondria were able to take up Ca 2+ through an energy-dependent, uncoupler-sensitive reaction in the presence of either antimycin or cyanide (Fig. 7). Thus, this interesting finding suggests that the alternative pathway of Euglena mitochondria may drive the accumulation of Ca 2÷ in conditions where the functioning of the cyanide-sensitive pathway is compromised. References [1] Nicholls, D.G. and Akerman, K. (1982) Biochim. Biophys. Acta 683, 57-88. [2] Gunter, T.E. and Pfeiffer, D.R. (1990) Am. J. Physiol. 258, C755-C786. [3] Martins, I.S. and Vercesi A.E. (1985) Biochem. Biophys. Res. Commun. 129, 943-948. [4] Ferguson, I.B., Reid, M.S. and Romani, R.J. (1985) Plant Physiol. 77, 877-880. [5] Martins, I.S., Carnieri, E.G.S. and Vercesi, A.E. (1986) Biochim. Biophys. Acta 850, 49-56. [6] Moreno-S~inchez, R. and Raya, J.C. (1987) Plant Sci. [7] Uribe, A. and Moreno-S~inchez, R. (1992) Plant Sci. [8] Docampo, R. and Vercesi, A.E. (1989) J. Biol. Chem. 264, 108-111. [9] Vercesi, A.E., Macedo, D,V., Lima, S.A., Gadelha, F.R. and Docampo, R. (1990) Mol. Biochem. Parasitol. 42, 119-124.

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