Ca2+ ions, an allosteric activator of calcium uptake in rat liver mitochondria

In a previous investigation, I have shown that the kinetics of the Ca uniporter change fundamentally when mitochondria have transitorily lost their membrane potential. The sigmoidal kinetics, usually observed in liver mitochondria, became almost hyperbolic. This means an increase in the affinity for calcium, and hence a considerable acceleration of Ca uptake in the range of low, e.g., physiological calcium concentration. In this investigation I show that extramitochondrial calcium released from the deenergized mitochondria causes the allosteric activation of the Ca uniporter. The dependence of the allosterical activation on the extramitochondrial Ca2+ concentration and on time is described. It is also reported that it is possible to activate allosterically the Ca uniporter of energized mitochondria by a short-term elevation of the extramitochondrial Ca2+ concentration. The process of activation is reversible. It is quickly reversed by the addition of chelators for Ca2+, and it is slowly reversed when the activating Ca2+ has to be removed by the mitochondrial Ca uniporter, though the bulk of extramitochondrial calcium is taken up by it very quickly. Several kinetics of the Ca uniporter are described. The implications of continually changing kinetics of the Ca uniporter are considered for carbon tetrachloride intoxication and the action of alpha 1-adrenergic agonists in liver cells.     

Spermine, another specific allosteric activator of calcium uptake in rat liver mitochondria

Calcium uptake in rat liver mitochondria is accelerated by spermine. At a concentration of 2 microM Ca2+ and 1 mM Mg2+ a maximal, 10-fold activation by 1.2 mM spermidine was obtained; a half-maximal activation was attained with 0.2 mM spermine. Spermidine was far less effective than spermine whereas putrescine was ineffective. The acceleration of Ca uptake at low, physiological Ca2+ concentrations is related to the altered kinetics of the Ca uniporter. Corresponding to the alteration by high Ca2+ concentrations previously described, the kinetics changed from sigmoidal in the absence to nearly hyperbolic in the presence of spermine. Mg2+ behaves as an allosteric inhibitor. This phenomenon of the allosteric activation of Ca uptake could not be observed in heart mitochondria.     

Properties of a new calcium ion antagonist on cellular uptake and mitochondrial efflux of calcium ions.

Compound YS 035 [NN-bis-(3,4-dimethoxyphenethyl)-N-methylamine] is a new synthetic compound capable of inhibiting Ca2+ uptake by different cells. The inhibition of Ca2+ uptake by muscle cells isolated from chicken embryo is dose-dependent in the compound YS 035 concentration range 10-30 microM. The new compound also inhibits Ca2+ entry into rat brain synaptosomes and less effectively into baby-hamster kidney cells. Compound YS 035 partially inhibits the slow Ca2+ release induced by Ruthenium Red and the rapid Na+-dependent efflux from heart mitochondria. The inhibition of the Na+/Ca2+ exchange appears to be of a non-competitive type with an apparent Ki of 28 microM. The new Ca2+ antagonist totally inhibits the Ca2+ efflux from liver mitochondria induced by Ruthenium Red, but it does not affect the release induced by uncoupler, respiratory inhibitor or chelator, nor the mitochondrial ATP synthesis and membrane potential. The properties shown by the new compound indicate it to be a Ca2+ antagonist and a useful tool for studies on the mitochondrial Ca2+ transport.     

Alkalinization within sarcoplasmic reticulum during the uptake of calcium ions.

The pH indicator, bromothymol blue, was incorporated into sarcoplasmic reticulum vesicles which bind more than 90% of the total added dye. The sequestered dye does not respond to changes in external pH upon addition of acid to the medium, since the decrease of absorbance at 616 nm is very slow. The absorbance of sequestered dye at 616 nm increases suddenly after triggering the transport of Ca²⁺ by ATP at a rate much higher than that of Ca²⁺ uptake, and declines when Ca²⁺ has been accumulated. When the uptake of Ca²⁺ is followed in the presence of oxalate, the absorbance of the indicator declines after the first phase of Ca²⁺ uptake. The results suggest that a transient alkalinization occurs rapidly inside the vesicles and reflects the formation of a transmembrane proton gradient responsible for sustaining the Ca²⁺ transport.     

Anion/calcium ion ratios and proton production in some mitochondrial calcium ion uptakes.

The uptake of Ca2+ by liver mitochondria, when phosphate movement is inhibited, occurs when Co2 is present and not in its absence. Uptake of Ca2+ to form CaCO3 yields 2H+/Ca2+. Heart mitochondria, when phosphate movement is inhibited, will take up Ca2+ with the exact equivalent of hydroxybutyrate, lactate or acetate. By providing a carrier for Cl- with tributyltin, a stoicheiometric uptake of Cl- with the Ca2+ takes place. The uptakes appear to occur without significant pH change; there appears to be no CO2-dependent uptake into heart mitochondria. Oxygenation of anaerobic heart mitochondria, in the presence of an inhibitor of phosphate movement and of generation of phosphate from internal ATP, does not yield significant change of external acidity in relation to the amount of O2 added. Use of Bromothymol Blue as an indicator of the distribution of a weak acid anion confirms that the transient nature of the response of the dye distribution to Ca2+ is connected with movement of endogenous phosphate. Bromothymol Blue accumulated in response to Ca2+ is discharged when entry of the Ca2+ (in the presence of mersalyl) is mediated with nigericin. It is concluded that Ca2+ uptakes will occur alternatively with the equivalent of anions or in exchange for endogenous K+ and that proton production is connected with the changes of ionization of phosphate (unless phosphate movement is inhibited) and in liver mitochondria with the hydration of CO2.     

Nutritional effects on mitochondrial bioenergetics. Alterations in calcium uptake by rat liver mitochondria

The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se.     

Modification by calcium ions of adenine nucleotide translocation in rat liver mitochondria

1. Added Ca(2+) stimulates the translocation of ATP by isolated rat liver mitochondria. 2. The apparent K(m) for added Ca(2+) in stimulating the translocation of 200mum-ATP is approx. 160mum (75mum ;free' Ca(2+)). 3. The greatest stimulation of ATP translocation by Ca(2+) occurs at the lower concentrations of ATP. 4. Sr(2+) (and to a lesser extent Ba(2+)) can replace Ca(2+) whereas Mg(2+) and Mn(2+) have only little ability to stimulate ATP translocation. 5. Translocation of dATP is also stimulated by Ca(2+) whereas that of ADP is stimulated to only a relatively small degree. 6. Studies with metabolic inhibitors and uncouplers provide evidence that stimulation by Ca(2+) and by uncouplers is additive and that the mechanism of Ca(2+) stimulation does not seem to involve the high-energy intermediate of oxidative phosphorylation. 7. In the presence of Ca(2+), ATP is able to effectively compete with ADP for translocation. 8. Added K(+) further enhances the ability of Ca(2+) to stimulate ATP translocation. 9. These findings are discussed in relation to the potential involvement of Ca(2+) in modifying enzymic reactions involved in the regulation of cell metabolism.     

The nature of the electron spin resonance signal during aerobic uptake of Mn2+ in mitochondria from rat liver

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high-energy membrane sites or to complexes of Mn2+ with inorganic phosphate. Identification of the spin exchange signal with a Mn-Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N-ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Pi is generated in the matrix, in the presence of N-ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal. The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50-60 nmol X mg protein-1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations. The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93-99] that the spin exchange signal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.     

Respiration-dependent calcium ion uptake by two preparations of cardiac mitochondria. Effects of palmitoyl-coenzyme A and palmitoylcarnitine on calcium ion cycling and nicotinamide nucleotide reduction state

Ca²⁺ uptake and the effect of the uptake inhibitors palmitoyl-CoA and palmitoylcarnitine were examined in two preparations of dog cardiac mitochondria. Mitochondria prepared by using the Nagarse technique was 2.5-fold more active in respiration-dependent Ca²⁺ uptake than were mitochondria isolated by using the Polytron procedure. Palmitoyl-CoA and palmitoylcarnitine inhibited Ca²⁺ uptake in both preparations uncompetitively, with K_i,app 0.4 and 20μm. Ca²⁺-uptake rates were related to, or influenced by, the concentration of mitochondrial reduced nicotinamide nucleotides, with uptake slowing as this concentration decreased. When most of the nicotinamide nucleotides was oxidized, Ca²⁺ release and respiratory stimulation were observed. In the presence of Ruthenium Red and palmitoyl-CoA, oxidation of nicotinamide nucleotides was abolished and the time to Ca²⁺ release was shortened corresponding to the time of onset of nicotinamide nucleotide oxidation in the absence of Ruthenium Red. The results suggest that NAD(P)H oxidation in the presence of rotenone was a consequence of Ca²⁺ re-uptake and that net Ca²⁺ release could be observed as reduced nicotinamide nucleotide concentrations declined. Although nicotinamide nucleotide oxidation occurred in the presence of rotenone, it was not linked in an apparent manner to acyl-group metabolism (palmitoylcarnitine was less effective than palmitoyl-CoA). Therefore either a by-pass of the rotenone block or a direct interaction of NAD(P)H with the Ca²⁺-uptake process was possible. Loss of NADH occurred before respiratory stimulation, and this loss may relate to decreased coupling efficiency at sites 2 and 3 of the respiratory chain, as suggested by others [Bhuvaneswaran & Wadkins (1978) Biochem. Biophys. Res. Commun. 82, 648–654].     

Calcium ion accumulation and volume changes of isolated liver mitochondria. Reversal of calcium ion-induced swelling

1. The excessive accumulation of Ca²⁺ by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led to extensive swelling and release of accumulated Ca²⁺ from the mitochondria. When the Ca²⁺ was removed from the medium by chelation with ethylene glycol bis(aminoethyl)tetra-acetate, the swelling was reversed in a respiration-dependent contraction. The contracted mitochondria were shown to have regained some degree of respiratory control. 2. The respiration-dependent contraction could be supported by electron transport through a restricted portion of the respiratory chain, and by substrates donating electrons at different levels in the respiratory chain. 3. Respiratory inhibitors appropriate to the substrate present completely inhibited the contraction. Uncoupling agents, and the inhibitors oligomycin and atractyloside, were without effect. 4. When the reversal of swelling had been prevented by respiratory inhibitors, the addition of ATP induced a contraction of the mitochondria. In the absence of added chelating agent the contraction was very slow. The ATP-induced contraction was completely inhibited by oligomycin and atractyloside, was incomplete in the presence of uncoupling agents and was unaffected by respiratory inhibitors. 5. The relationship between the energy requirements of respiration-dependent contraction and the requirements of ion transport and other contractile systems are discussed.