RESUMO
Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), 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. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Saccharomycetales/metabolismo , Acetatos/metabolismo , Meios de Cultura , Transporte de Íons , Fosfatos/metabolismoRESUMO
Ca2+-release pathways from Ca2+-preloaded mitochondria of the yeast Endomyces 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. Intensive aeration of the mitochondrial suspension rapidly inhibited the efflux of Ca2+ and induced its reuptake. The Ca2+ release was not affected by cyclosporin A, an inhibitor of the nonselective permeability transition of mammalian mitochondria. With acetate as the permeant anion, a spontaneous net Ca2+ efflux began after uptake of about 75% of the added cation. The rate of this efflux was insensitive to cyclosporin A, aeration, and Na+ and was proportional to the Ca2+ load. The Ca2+ release was inhibited by La3+, Mn2+, Mg2+, TPP+, and nigericin (in the presence of KCl) and activated by spermine and hypotonicity. We conclude that Ca2+ efflux from preloaded E. magnusii mitochondria is very similar to the Na+-independent specific pathway for Ca2+ release operative in mitochondria from nonexcitable mammalian tissues.
Assuntos
Cálcio/metabolismo , Saccharomycetales/metabolismo , Transporte de Íons/efeitos dos fármacos , Metais/farmacologia , Mitocôndrias/metabolismo , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Saccharomycetales/efeitos dos fármacos , Sódio/metabolismo , Espermina/farmacologiaRESUMO
The Ca2+ uptake by mitochondria from the yeast Endomyces magnusii has earlier been found to be driven by the membrane potential and to be stimulated by spermine. It thus functions in a similar fashion as the animal mitochondrial calcium uniporter. Here, it is shown that the uptake is stimulated, i.e., Ca2+ can be accumulated from lower [Ca2+], under hypotonic conditions. Ruthenium Red, an inhibitor of the animal uniporter, under certain conditions, stimulates the yeast uniporter. The mechanism of the stimulation by hypotonicity and Ruthenium Red is discussed.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Mitocôndrias/metabolismo , Rutênio Vermelho/farmacologia , Saccharomycetales/metabolismo , Cálcio/metabolismo , Canais de Cálcio , Transporte de Íons/efeitos dos fármacos , Pressão Osmótica , Espermina/farmacologiaRESUMO
The Ca2+ transport system of Endomyces magnusii mitochondria has been shown previously to be activated by spermine. Here we report it to be regulated also by low, physiological ADP concentrations, by the intramitochondrial NADH/NAD+ ratio, and by Ca2+ ions. The combination of all these physiological modulators induced high initial rates of Ca2+ uptake and high Ca2+-buffering capacity of yeast mitochondria, enabling them to lower the medium [Ca2+] to approximately 0.2 microM. The mechanisms of stimulation by these agents are discussed.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Saccharomycetales/metabolismo , Difosfato de Adenosina/metabolismo , Atractilosídeo/farmacologia , Transporte de Íons , Mitocôndrias/efeitos dos fármacos , NAD/metabolismoRESUMO
Spermine, spermidine, and magnesium ions modulate the kinetic parameters of the Ca2+ transport system of Endomyces magnusii mitochondria. Mg2+ at concentrations up to 5 mM partially inhibits Ca2+ transport with a half-maximal inhibiting concentration of approximately 0.5 mM. In the presence of 2 mM MgCl2, the S0.5 value of the Ca2+ transport system increases from 220 to 490 microM, which indicates decreased affinity for the system. Spermine and spermidine exert an activating effect, having half-maximal concentrations of 12 and 50 microM, respectively. In the case of spermine, the S0.5 value falls to 50-65 microM, which implies an increase in the transport system affinity for Ca2+. Both Mg2+ and spermine cause a decrease of the Hill coefficient, giving evidence for a smaller degree of cooperativity. Spermine and spermidine enable yeast mitochondria to remove Ca2+ from the media completely. In contrast, Mg2+ lowers the mitochondrial buffer capacity. When both Mg2+ and spermine are present in the medium, their effects on the S0.5 value and the free extramitochondrial Ca2+ concentration are additive. The ability of spermine and Mg2+ to regulate yeast mitochondrial Ca2+ transport is discussed.
Assuntos
Cálcio/metabolismo , Magnésio/metabolismo , Poliaminas/metabolismo , Saccharomycetales/metabolismo , Transporte Biológico/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Poliaminas/farmacologia , Saccharomycetales/efeitos dos fármacosRESUMO
Spermine at concentrations of 12-100 microM considerably activates the Ca2+ transport system of the Endomyces magnusii yeast mitochondria. As a result, in the presence of spermine the mitochondria are able to decrease extramitochondrial Ca2+ to the physiological level. At Ca2+ concentrations up to 200 microM, spermine enhances the initial rate of Ca2+ uptake (a half-maximal effect at 12 microM spermine). The Ca2+ concentrations required for half-maximal Ca2+ uptake rate to be achieved were 160 and 60 microM Ca2+ without and with spermine, respectively. Spermidine is shown to be less effective (a half-maximal effect at 50-100 microM spermidine). The polyamines do not change the parameters of energy coupling of mitochondria. The data obtained enabled the yeast mitochondria to be considered to take part in regulation of cytoplasmic and matrix Ca2+.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Saccharomycetales/metabolismo , Espermina/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Cadaverina/farmacologia , Mitocôndrias/efeitos dos fármacos , Putrescina/farmacologia , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/ultraestrutura , Espermidina/farmacologiaRESUMO
Endomyces magnusii mitochondria were shown to be incapable of active Mg2+ transport at 0.1--16 mM concentrations. As was found using the inhibition analysis, when magnesium ions are added to the mitochondria once the phosphorylation cycle is over, the respiration is stimulated because adenylate kinase and H+-ATPase (Mg2+-dependent enzymes) are activated.