Ca2+ Efflux in Mitochondria from the YeastEndomyces magnusii
2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês
10.1074/jbc.m103685200
ISSN1083-351X
AutoresYulia I. Deryabina, Elena N. Bazhenova, Nils‐Erik L. Saris, R. A. Zvyagilskaya,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoCalcium release pathways in Ca2+-preloaded mitochondria from the yeastEndomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca2+ release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca2+ and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca2+ efflux set in after uptake of ∼150 nmol of Ca2+/mg of protein. The rate of this efflux was proportional to the Ca2+ load and insensitive to aeration, protonophorous uncouplers, and Na+ions. Ca2+ efflux was inhibited by La3+, Mn2+, Mg2+, tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca2+efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca2+ efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na+-independent pathway, in many ways similar to the one in mammalian mitochondria. Calcium release pathways in Ca2+-preloaded mitochondria from the yeastEndomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca2+ release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca2+ and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca2+ efflux set in after uptake of ∼150 nmol of Ca2+/mg of protein. The rate of this efflux was proportional to the Ca2+ load and insensitive to aeration, protonophorous uncouplers, and Na+ions. Ca2+ efflux was inhibited by La3+, Mn2+, Mg2+, tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca2+efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca2+ efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na+-independent pathway, in many ways similar to the one in mammalian mitochondria. inner mitochondrial membrane transmembrane potential carbonyl cyanidem-chlorophenylhydrazone cyclosporin A mitochondrial permeability transition tetraphenylphosphonium Calcium plays an important role as an intracellular messenger in signal transduction (1Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2268) Google Scholar, 2Mooren F.C. Kinne R.K.H. Biochim. Biophys. Acta. 1998; 1406: 127-151Crossref PubMed Scopus (51) Google Scholar). Mitochondrial Ca2+ uptake is important in relaying a signal for stimulation of respiration and oxidative phosphorylation (3McCormack J.G. Denton R.M. Biochem. Soc. Trans. 1993; 21: 793-799Crossref PubMed Scopus (72) Google Scholar, 4Kavanagh N.I. Ainscow E.K. Brand M.D. Biochim. Biophys. Acta. 2000; 1457: 57-70Crossref PubMed Scopus (70) Google Scholar), which has encouraged study of mitochondrial Ca2+ handling (5Pozzan T. Rizzuto R. Eur. J. Biochem. 2000; 267: 5267-5273Crossref PubMed Scopus (47) Google Scholar). Mitochondria are receiving increasing attention also due to their central role in the mechanism of cell death (6Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (659) Google Scholar). Animal mitochondria take up Ca2+ by a uniport mechanism and release it by a number of different mechanisms, including Na+ antiport, a sodium-independent pathway, and via opening of a cyclosporin A-sensitive pore (for review, see Ref.7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). Yeast mitochondria generally lack a Ca2+ uptake pathway of any significant physiological relevance (8Balcavage W.X. Lloyd J.L. Mattoon J.R. Ohnishi T. Scarpa A. Biochim. Biophys. Acta. 1973; 305: 41-51Crossref PubMed Scopus (30) Google Scholar, 9Tanida I. Hasegawa A. Iida H. Ohya Y. Anraku Y. J. Biol. Chem. 1995; 270: 10113-10119Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Instead, a vacuolar V-ATPase and a Ca2+/H+ antiporter regulate cytosolic [Ca2+] (10Förster C. Kane P.M. J. Biol. Chem. 2000; 275: 38245-38253Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, we have found that tightly coupled mitochondria from the yeast Endomyces magnusii are able to take up Ca2+ by a uniport mechanism, although the apparent Km is rather high, 150–180 μm (11Zvyagilskaya R.A. Leikin Y.N. Kozhokaru N.L. Kotelnikova A.V. Dokl. Akad. NAUK SSSR. 1983; 269: 1233-1240Google Scholar, 12Leikin Y.N. Votyakova T.V. Bazhenova E.N. Zvyagilskaya R.A. Kotelnikova A.V. Biochemistry (Moscow). 1987; 52: 676-682Google Scholar). Ca2+ uptake at low concentrations can, however, as in animal mitochondria, be substantially increased by micromolar concentrations of polyamines (13Votyakova T.V. Bazhenova E.N. Zvjagilskaya R.A. FEBS Lett. 1990; 261: 139-141Crossref PubMed Scopus (18) Google Scholar, 14Votyakova T.V. Bazhenova E.N. Zvjagilskaya R.A. J. Bioenerg. Biomembr. 1993; 25: 569-574Crossref PubMed Scopus (19) Google Scholar, 15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Also ADP, Ca2+ itself, and a high intramitochondrial NADH/NAD+ ratio stimulate mitochondrial Ca2+ uptake (15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Bazhenova E.N. Deryabina Y.I. Zvyagilskaya R.A. Dokl. Akad. NAUK SSSR. 1997; 353: 1-3Google Scholar). In the presence of all these physiological modulators, the initial rate of Ca2+ uptake is quite high (up to 2 μmol/min/mg of protein), and the mitochondrial Ca2+-buffering capacity is remarkably high. Ca2+ uptake by E. magnusii mitochondria is thus potentially important in Ca2+ homeostasis and signal transduction (15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Bazhenova E.N. Deryabina Y.I. Zvyagilskaya R.A. Dokl. Akad. NAUK SSSR. 1997; 353: 1-3Google Scholar). The Ca2+ efflux pathways are also of interest in this context. In this study, we have characterized the mitochondrial Ca2+ efflux mechanism, which shares many of its characteristics with the sodium-independent Ca2+ efflux mechanism of mitochondria from non-excitable mammalian cells (19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar). The yeast E. magnusii strain VKM Y261 was grown in glycerol-containing semisynthetic medium as described previously (20Zvyagilskaya R.A. Selenshchikova V.A. Uralskaya L.A. Kotelnikova A.V. Biochemistry (Moscow). 1981; 46: 3-10Google Scholar). Cells were harvested at the late exponential growth phase (10–13 g (wet weight)/liter). Mitochondria were isolated by the method developed in our laboratory (17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The oxygen consumption in mitochondrial suspensions was monitored polarographically with a Clark-type electrode in medium containing 0.6 m mannitol, 1 mm Tris phosphate (pH 7.4), 1 mm EDTA, 20 mm pyruvate, 5 mm malate, and mitochondria corresponding to 0.5 mg/ml protein. The mitochondrial preparations were well coupled, showing respiratory control ratios of ∼4 under these conditions. ADP/oxygen ratios were close to the theoretical maximum. Respiratory control and ADP/oxygen ratios were calculated as described by Chance and Williams (21Chance B. Williams G.R. Nature. 1955; 175: 1120-1121Crossref PubMed Scopus (268) Google Scholar). Mitochondria were fully active for at least 4 h after preparation. Ca2+ uptake was assayed by the murexide method employing dual wavelength photometry at 507–540 nm with a Hitachi 557 spectrophotometer. Unless otherwise specified in the figure legends, the incubation medium contained 0.6 m mannitol, 2 mm Tris phosphate or 20 mm Tris acetate (pH 7.4), 20 mm Tris pyruvate, 5 mm malate, 50 μm murexide, and mitochondria corresponding to 0.5 mg/ml protein. The inner mitochondrial membrane transmembrane potential (ΔΨ)1 was measured at the wavelength pair 523/555 nm with 7 μm safranin (22Åkerman K.E.O. Wikström M.K.F. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar). Mitochondrial swelling was monitored by recording changes in absorbance at 540 nm. Protein was assayed by the method of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216357) Google Scholar) with bovine serum albumin as the standard. Mannitol, sorbitol, EDTA, EGTA, bovine serum albumin, ADP, spermine, valinomycin, nigericin, atractyloside, CCCP, Tris, pyruvate, malate, murexide, and CaCl2 were purchased from Sigma. Coomassie G-250, NADH, and safranin were from Serva. A23187 was from Roche Molecular Biochemicals. Dithiothreitol was from Reanal. Cyclosporin A (CsA) was a kind gift of Novartis. In the presence of Pi, yeast mitochondria were able to take up almost all of the Ca2+ added in successive aliquots (up to 600 nmol of Ca2+/mg of protein) and to retain it until the oxygen was exhausted after 5–10 min of incubation (Fig. 1, trace a). Anaerobiosis induced a rapid collapse of ΔΨ (Fig. 1,trace c), the driving force of Ca2+uptake by the calcium uniporter. The rate of Ca2+ uptake was much faster than that of efflux induced by anaerobiosis (Fig. 1), which is in accordance with the ability of the mitochondria to reduce the [Ca2+] to very low levels at steady state. On the other hand, upon collapse of ΔΨ, efflux could be by reversal of the uptake mechanism, the calcium uniporter. Intensive aeration of the incubation medium fully reestablished ΔΨ, prevented spontaneous Ca2+ release, and elicited re-uptake of the Ca2+ released (Fig. 1, trace a). Aeration before each Ca2+ addition also prevented Ca2+ release (Fig. 1, trace b) and a drop in ΔΨ (trace d), thus increasing the Ca2+-buffering capacity of the yeast mitochondria. The efflux rate was not affected by the addition of CsA (Fig. 1,trace a). Since no Ca2+ efflux was observed in respiring yeast mitochondria in the presence of Pi, acetate was used instead as a permeant anion. The Ca2+ efflux pathways in animal mitochondria are not influenced by acetate and thus can be studied in the presence of this anion, the calcium salt of which is soluble (24Rizzuto R. Bernardi P. Favoron M. Azzone G.F. Biochem. J. 1987; 246: 271-277Crossref PubMed Scopus (50) Google Scholar). In yeast mitochondria, Ca2+ efflux ensued after uptake of ∼75 μm Ca2+ (Fig.2, trace a). Ca2+ release was not due to a decline in ΔΨ (Fig. 2,trace b), and it was insensitive to micromolar concentrations of CsA (data not shown). The efflux was thus spontaneous and not due to anaerobiosis or induction of the mitochondrial permeability transition (MPT). The rate of Ca2+ efflux was proportional to the Ca2+ load (Fig.3 A). It was inhibited half-maximally by Pi at 0.3 mm (Fig.3 B).Figure 3Effect of Ca2+ load (A) and of Pi (B) on the rate of Ca2+ efflux in yeast mitochondria. The medium was the same as that described in the legend to Fig. 2, but without murexide. In A and B, 100 and 500 μm Ca2+ were added, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In acetate medium, Ca2+ uptake would cause mitochondrial swelling due to accumulation of calcium acetate in the matrix, whereas efflux would result in contraction. Fig.4 shows the effect of various agents on the contraction due to spontaneous Ca2+ efflux in mitochondria respiring on pyruvate + malate. In mitochondria to which 100 μm Ca2+ had been added, the addition ofN-ethylmaleimide only slightly slowed contraction, whereast-butyl hydroperoxide, oxalacetate, atractyloside, and the uncoupling agent CCCP in the presence of EGTA were without any effect (Fig. 4). In animal mitochondria, efflux of Ca2+ can be studied by inhibiting the uniporter with ruthenium red (25Moore C.L. Biochem. Biophys. Res. Commun. 1971; 42: 298-305Crossref PubMed Scopus (471) Google Scholar, 26Igbavboa U. Pfeiffer D.R. J. Biol. Chem. 1988; 263: 1405-1412Abstract Full Text PDF PubMed Google Scholar). However, theE. magnusii mitochondrial calcium uniporter is not inhibited by ruthenium red (12Leikin Y.N. Votyakova T.V. Bazhenova E.N. Zvyagilskaya R.A. Kotelnikova A.V. Biochemistry (Moscow). 1987; 52: 676-682Google Scholar) and may even be stimulated by it (16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar). In the acetate medium, there was a spontaneous net efflux of accumulated Ca2+ (Fig. 2), but this did not exclude a simultaneous uptake. We therefore examined whether La3+, a competitive inhibitor of mitochondrial Ca2+ transport (27Reed K.C. Bygrave F.L. Biochem. J. 1974; 140: 143-155Crossref PubMed Scopus (288) Google Scholar), could be used as an inhibitor of the uniporter. However, although Ca2+ uptake was sensitive to low concentrations of La3+ (Fig. 5 A,trace b), efflux was also slightly inhibited, more so at higher concentrations (trace a). Half-maximal inhibition of Ca2+ uptake was attained at ∼25 μm La3+. The effects of La3+ were thus different for uptake and efflux of Ca2+. The rate of sodium-independent Ca2+ efflux in animal mitochondria can be modulated by a number of cations (7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar, 28Gunter T.E. Gunter K.K. Sheu S.-S. Gavin C.E. Am. J. Physiol. 1994; 267: C313-C339Crossref PubMed Google Scholar), including Mg2+(29Rottenberg H. Marbach M. Biochim. Biophys. Acta. 1990; 1016: 77-86Crossref PubMed Scopus (56) Google Scholar), Mn2+ (30Allshire A. Bernardi P. Saris N.-E.L. Biochim. Biophys. Acta. 1985; 807: 202-209Crossref PubMed Scopus (55) Google Scholar, 31Gavin C.E. Gunter K.K. Gunter T.E. Neurotoxicology (Little Rock). 1999; 20: 445-453PubMed Google Scholar), Ba2+ (32Lukács G.L. Fonyó A. Biochim. Biophys. Acta. 1985; 809: 160-166Crossref PubMed Scopus (17) Google Scholar), Sr2+ (33Saris N.-E.L. Bernardi P. Biochim. Biophys. Acta. 1983; 725: 19-24Crossref PubMed Scopus (21) Google Scholar), polyamines (19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar, 30Allshire A. Bernardi P. Saris N.-E.L. Biochim. Biophys. Acta. 1985; 807: 202-209Crossref PubMed Scopus (55) Google Scholar), and tetraphenylphosphonium (TPP+) (28Gunter T.E. Gunter K.K. Sheu S.-S. Gavin C.E. Am. J. Physiol. 1994; 267: C313-C339Crossref PubMed Google Scholar) as well as by ionophores (19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar) and hypo-osmolarity (34Novgorodov S.A. Yaguzhinsky L.A. FEBS Lett. 1985; 183: 47-51Crossref PubMed Scopus (8) Google Scholar). We therefore examined the effect of several of these agents on Ca2+ efflux in E. magnusii mitochondria. Ca2+ efflux was inhibited by Mn2+ (Fig.5 B, trace a) and by Mg2+(Fig. 5 C, trace a). These cations also affected the rate of Ca2+ uptake and efflux with a different concentration dependence (Fig. 5, B and C). Thus, 200 μm Mn2+ inhibited Ca2+ efflux by 80% (Fig. 5 B, trace a), whereas Ca2+ uptake was inhibited by only 50% (trace b). Mg2+ at 1.5 mm almost totally blocked Ca2+ efflux (Fig.5 C, trace a) and inhibited Ca2+ uptake by only 50% (trace b). In the case of Mg2+ (Fig. 5 C), the concentration dependence of inhibition of Ca2+ uptake and efflux was even more strikingly different. TPP+ is a potent and apparently specific inhibitor of the sodium-independent Ca2+ efflux pathway in animal mitochondria (28Gunter T.E. Gunter K.K. Sheu S.-S. Gavin C.E. Am. J. Physiol. 1994; 267: C313-C339Crossref PubMed Google Scholar). We also found that in yeast mitochondria, TPP+ strongly inhibited Ca2+efflux (Fig. 5 D), with a maximal inhibitory effect at 10 μm and a half-maximal effect at 1.8 μm(calculated in Dixon plots), which is close to theKi value obtained for liver mitochondria (28Gunter T.E. Gunter K.K. Sheu S.-S. Gavin C.E. Am. J. Physiol. 1994; 267: C313-C339Crossref PubMed Google Scholar). Uptake of Ca2+ was inhibited only slightly (10%) by 10 μm TPP+ (data not shown). Ca2+efflux in yeast mitochondria was almost totally inhibited by 1.2 mm Pi (Fig. 3 B), a concentration that was found to be optimal in sustaining Ca2+ uptake (11Zvyagilskaya R.A. Leikin Y.N. Kozhokaru N.L. Kotelnikova A.V. Dokl. Akad. NAUK SSSR. 1983; 269: 1233-1240Google Scholar). Spermine increased the rate of Ca2+ efflux when added both before (Fig. 6, trace c) and after (trace a) Ca2+, with no effect upon ΔΨ (traces a and b). The spermine-induced Ca2+ release was nonlinear and similar to that seen after the addition of the Ca2+ ionophore A23187 (Fig. 2). The addition of A23187 after spermine induced further Ca2+release (Fig. 6, trace a). The rate of Ca2+ efflux in yeast mitochondria was not affected by the redox state of pyridine nucleotides and was only slightly inhibited by micromolar concentrations of ADP (data not shown). Preincubation with 4 mm NADH to greatly reduce intramitochondrial pyridine nucleotides had no effect (data not shown), whereas these substances have been shown to stimulate Ca2+uptake (17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Swelling of mammalian mitochondria in hypotonic medium was found to stimulate the sodium-independent Ca2+ efflux pathway, presumably because of stretching of the inner mitochondrial membrane (24Rizzuto R. Bernardi P. Favoron M. Azzone G.F. Biochem. J. 1987; 246: 271-277Crossref PubMed Scopus (50) Google Scholar, 34Novgorodov S.A. Yaguzhinsky L.A. FEBS Lett. 1985; 183: 47-51Crossref PubMed Scopus (8) Google Scholar, 35Bernardi P. Azzone G.F. Eur. J. Biochem. 1983; 134: 377-383Crossref PubMed Scopus (58) Google Scholar). Likewise, in yeast mitochondria, a significantly (2.5-fold) enhanced Ca2+ efflux was observed when mitochondria were incubated in hypotonic medium (Fig.7 A, trace b). To elucidate the mechanism of the efflux pathway, we examined the effects of CCCP (a protonophorous uncoupler), nigericin (an ionophore acting via K+/H+ antiport), and valinomycin (a K+ ionophore). Ca2+ efflux was only partially inhibited (30%) by 20–200 nm CCCP (data not shown), whereas little effect (if any) was seen on the associated contraction of mitochondria (Fig. 4). Ca2+ efflux was completely inhibited by 100 nm nigericin (the medium was supplemented with 0.5 mm KCl) (Fig. 7 B, trace a). Nigericin, which equilibrates K+ and H+gradients across the inner mitochondrial membrane and is thereby able to partially transform ΔpH into ΔΨ, slightly increased ΔΨ at most. The addition of 100 nm valinomycin (the medium was supplemented with 4 mm KCl) caused dissipation of ΔΨ and increased the rate of Ca2+ efflux (Fig. 7 B,trace b). In the presence of Pi, E. magnusii mitochondria are capable of massive uptake of Ca2+ (see Fig. 1). In animal mitochondria, Pi has been reported to induce the release of accumulated Pi (36Saris N.-E.L. Soc. Sci. Fenn.; Comment. Phys.-Math. 1963; 28: 1-77Google Scholar), which is now known to be due to induction of MPT (7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). Massive accumulation was observed in animal mitochondria in the presence of both Piand ATP due to precipitation of calcium phosphates (for review, see Ref. 37Lehninger A.L. Carafoli E. Rossi C.S. Adv. Enzymol. 1967; 29: 259-320PubMed Google Scholar) and inhibition of MPT (7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). In the absence of adenine nucleotides, Ca2+ uptake by rat liver mitochondria is also stimulated by Pi, and the sodium-independent efflux is inhibited, which was interpreted as being due to lowering of matrix [Ca2+] by its binding to Pi (19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar). In Pi-depleted liver mitochondria, the sodium-independent Ca2+ efflux rate is stimulated 15-fold over that in the presence of Pi and is potently inhibited by the addition of Pi (38Zoccarato F. Nicholls D. Eur. J. Biochem. 1982; 127: 333-338Crossref PubMed Scopus (106) Google Scholar). Stimulation of Ca2+ uptake by Pi and inhibition of efflux have also been described in microsomes and were also interpreted as being due to binding of Ca2+ by Pi (39Fulceri R. Bellomo G. Gamberucci A. Romani A. Benedetti A. Biochem. J. 1993; 289: 299-306Crossref PubMed Scopus (25) Google Scholar). The same mechanism may account for the effect of Pi on Ca2+ efflux in E. magnusii mitochondria. The efflux of accumulated Ca2+ in acetate medium could occur by a reversal of the calcium uniporter upon lowering of ΔΨ, by specific efflux mechanisms such as the Ca2+/2Na+ antiporter (40Crompton M. Künzi M. Carafoli E. Eur. J. Biochem. 1977; 79: 549-558Crossref PubMed Scopus (184) Google Scholar) and the Ca2+/n H+ antiporter (7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar, 19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar, 28Gunter T.E. Gunter K.K. Sheu S.-S. Gavin C.E. Am. J. Physiol. 1994; 267: C313-C339Crossref PubMed Google Scholar) in animal mitochondria, or by reversible opening of the MPT pore. However, the efflux rate was not influenced by uncoupling amounts of CCCP (Fig.4) or Na+ (data not shown), which excludes the uniporter and the Ca2+/2Na+ antiporter mechanisms. This is borne out by data showing that the uptake and efflux pathways differ in their sensitivities to La3+ (Fig. 5 A) and modulators such as Mn2+ (Fig. 5 B), Mg2+ (Fig. 5 C), TPP+ (Fig.5 D), and spermine (Fig. 6). Efflux was only slightly inhibited by ADP (data not shown), whereas uptake was potently stimulated (with a half-maximal effect at 3–5 μm and at a maximal effect at 25 μm) (15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Bazhenova E.N. Deryabina Y.I. Zvyagilskaya R.A. Dokl. Akad. NAUK SSSR. 1997; 353: 1-3Google Scholar). In addition, the efflux rate was not influenced by preincubation of the mitochondrial suspension with 4 mm NADH, which ensures a high redox state of the intramitochondrial pyridine nucleotides in yeast mitochondria (41Zvyagilskaya R.A. Zelenshchikova V.A. Burbaev D.S. Dokl. Akad. NAUK SSSR. 1982; 263: 491-493Google Scholar), although Ca2+ uptake is stimulated under these conditions (17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In animal mitochondria, ADP inhibits and atractyloside stimulates Ca2+ release mediated by the MPT pore, presumably by affecting the conformation of the adenine nucleotide carrier (7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). InE. magnusii mitochondria, Ca2+ efflux was only slightly affected by ADP (data not shown). The addition of atractyloside, pro-oxidants, N-ethylmaleimide, or small amounts of CCCP or oxidation of pyridine nucleotides by oxalacetate had little effect (if any) on the rate of mitochondrial contraction associated with Ca2+ efflux (Fig. 4). All these findings indicate that there is no Ca2+-stimulated opening of a MPT pore in these mitochondria. This conclusion is strongly supported by the absence of inhibition by CsA and by the massive accumulation of Ca2+ in the presence of Pi(Fig. 1) that would induce MPT in animal mitochondria (7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). No MPT of this type has been found in Saccharomyces cerevisiae mitochondria (42Jung D.W. Bradshaw P.C. Pfeiffer D.R. J. Biol. Chem. 1997; 272: 21104-21112Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Mg2+ ions are believed to stabilize the inner mitochondrial membrane by modulating ion transport and inhibiting the Ca2+-activated phospholipase A2 and MPT (43Saris N.-E.L. Mervaala E. Karppanen H. Khawaja J.A. Lewenstam A. Clin. Chim. Acta. 2000; 294: 1-26Crossref PubMed Scopus (1149) Google Scholar), the latter not being of relevance in the case of yeast mitochondria as discussed above. The mechanism of the inhibitory action of Mn2+ on Ca2+ efflux has not been conclusively established. It seems likely that in E. magnusii mitochondria, Mg2+ and Mn2+ exert their action from the cytosolic side since these mitochondria have only a low-capacity transport system for Mg2+ (44Zvyagilskaya R.A. Votyakova T.V. Bazhenova E.N. Mikrobiologia. 1987; 56: 543-548PubMed Google Scholar), and the calcium uniporter supports slow transport of Sr2+, Mn2+, or Ba2+ only at high millimolar concentrations (data not shown). Furthermore, these cations exert their inhibitory action without any lag. Spermine is well known as an important modulator of the Ca2+ transport system in animal mitochondria, stimulating electrogenic Ca2+ uptake (45Saris N.-E.L. Wikström M.K.F. Seppälä A.J. FEBS Symp. 1969; 17: 363-368Google Scholar, 46Nicchitta C.V. Williamson J.R. J. Biol. Chem. 1984; 259: 12978-12983Abstract Full Text PDF PubMed Google Scholar, 47Karadzhov Y.S. Kudzina L.Y. Zinchenko V.P. Biofizika. 1988; 33: 1001-1005PubMed Google Scholar, 48Rustenbeck I. Reiter H. Lenzen S. Biochem. Mol. Biol. Int. 1996; 38: 1003-1011PubMed Google Scholar, 49Jensen J.R. Lynch G. Baudry M. J. Neurochem. 1987; 48: 765-772Crossref PubMed Scopus (61) Google Scholar) and mostly inhibiting Ca2+ efflux (29Rottenberg H. Marbach M. Biochim. Biophys. Acta. 1990; 1016: 77-86Crossref PubMed Scopus (56) Google Scholar, 46Nicchitta C.V. Williamson J.R. J. Biol. Chem. 1984; 259: 12978-12983Abstract Full Text PDF PubMed Google Scholar, 49Jensen J.R. Lynch G. Baudry M. J. 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The mechanism of the stimulation of the efflux rate by spermine may be an allosteric change in the conformation of the transporter or/and an effect of binding of spermine to negatively charged membrane groups, thus favoring the binding and dissociation of Ca2+ from the membrane. It is also possible that Ca2+ itself stimulates the transporter by the same allosteric effect on the transporter, which would explain the stimulation of efflux after uptake over a certain threshold. Nigericin inhibited Ca2+ efflux in the presence of 0.5 mm K+ under conditions that did not change ΔΨ, whereas valinomycin stimulated efflux in the presence of 4 mm K+ (Fig. 7 B). Under these conditions, valinomycin caused a decrease in ΔΨ and swelling (Fig.4). Since there should be no appreciable pH gradient in the presence of acetate, and a drop in ΔΨ induced by CCCP did not change the efflux rate (Fig. 4), it seems likely that the inhibition is due to reduction of the mitochondrial volume. The valinomycin-induced stimulation of efflux could then be due to swelling (Fig. 4) induced by accumulation of potassium acetate in the matrix. Taken together, these data indicate that Ca2+ efflux inE. magnusii mitochondria is primarily mediated by a sodium-independent pathway. This could be a passive Ca2+/n H+ antiporter, with ΔpH as a driving force, as in liver mitochondria. Whether indeed these transporters are related at the molecular level requires further studies. The data presented demonstrate, to our knowledge for the first time, that mitochondria from a yeast species are endowed with independent systems for influx and efflux of Ca2+. Fig.8 summarizes the modulation of Ca2+ uptake and efflux pathways in E. magnusii mitochondria and their possible physiological implications. An increase in the cytosolic Ca2+ concentration would activate Ca2+ uptake by respiring mitochondria via the Ca2+ uniporter, with ΔΨ generated across the inner mitochondrial membrane as the driving force (7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). This would establish a higher steady-state concentration of free Ca2+ required for the activation of Ca2+-sensitive matrix enzymes, particularly of Ca2+-sensitive, NAD+-dependent dehydrogenases supplying reducing equivalents to the respiratory chain (3McCormack J.G. Denton R.M. Biochem. Soc. Trans. 1993; 21: 793-799Crossref PubMed Scopus (72) Google Scholar). In our preliminary experiments, we found that the pyruvate dehydrogenase complex inE. magnusii mitochondria was activated by submicromolar [Ca2+]. The Ca2+ that accumulated in the matrix would be buffered by binding to Pi and other substances. When Ca2+ uptake exceeds the matrix Ca2+-binding capacity, as in the absence of added Pi, an excessive rise in matrix [Ca2+] would trigger Ca2+ efflux primarily by a sodium-independent mechanism, i.e. electroneutral Ca2+/2H+ antiport, or by activation of a Ca2+-specific channel (7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). The result would be an increase in medium [Ca2+], but the ensuing increased rate of Ca2+ uptake in the Ca2+ cycling would not be enough to lower ΔΨ (Fig. 2). In conclusion, these data demonstrate that E. magnusii mitochondria have a specific Ca2+ efflux pathway that is activated by elevated matrix free [Ca2+]. In many respects, it is similar to the sodium-independent Ca2+efflux pathway in mitochondria from non-excitable mammalian cells. Thus, in both types of mitochondria, mitochondrial Ca2+efflux is modulated by the same substances. The efflux in E. magnusii was spontaneous and not due a decline in ΔΨ or induction of MPT. We are grateful to Dr. Jahangir A. Khawaja (Helsinki) for checking the English in this manuscript.
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