NADH-linked substrate dependence of peroxide-induced respiratory inhibition and calcium efflux in isolated renal mitochondria

Peroxide-induced state 3 respiratory inhibition and Ca2+ efflux in isolated renal mitochondria exhibited a NADH-linked substrate dependence. ADP-stimulated respiratory rates in the presence of various concentrations of tert-butyl hydroperoxide (tBOOH, 0-1000 nmol/mg protein) were determined using glutamate, beta-hydroxybutyrate, or pyruvate as substrates. Pyruvate-driven respiration was most sensitive to inhibition (Ki approximately equal to 75 nmol of tBOOH/mg protein) followed by beta-hydroxybutyrate and glutamate (Ki approximately equal to 150 nmol of tBOOH/mg protein for each). Calcium (5-10 nmol/mg protein) potentiated tBOOH-induced respiratory inhibition using all three substrates. Mitochondrial Ca2+ efflux, induced by tBOOH, was most pronounced with pyruvate as substrate. Glutamate prevented Ca2+ efflux while the efflux rate with beta-hydroxybutyrate was intermediate between glutamate and pyruvate. The substrate-dependent pattern of tBOOH-induced NAD(P)H (NADH plus NADPH) and cytochrome b oxidation was similar to that seen for respiratory inhibition and Ca2+ efflux suggesting that NAD(P)H may be a common factor in both responses. Low tBOOH concentrations inhibited pyruvate dehydrogenase flux while higher concentrations enhanced pyruvate dehydrogenase flux and activation. The results are discussed in relation to currently proposed theories of reactive oxygen-induced respiratory inhibition, Ca2+ efflux, and reperfusion injury.     

Calcium in isolated mitochondria from the left ventricular wall

Summary The content of calcium per mg mitochondrial protein has been measured by conventional biochemical methods in myocardial tissue of some mammalian species. In addition, a method is presented for (1) the analysis of mitochondrial volumes in the same tissues and (2) calculating the amount of calcium in units of 10⁶ mitochondria.It appears that a highly significant correlation exists between the calcium content and the number of mitochondria, with a positive correlation coefficient of 0.92. The mean mitochondrial volume in fractions of the rabbit myocardium was found to be 1.3386 m³. Electron microscopic studies demonstrate pure mitochondrial fractions and only moderate structural alterations. The method described may represent a useful supplement for the estimation of calcium fluxes in mitochondria and of alterations in their volume, number and structure under conditions of myocardial ischemia.This work was supported by grants from the Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

An assessment of the calcium content of rat liver mitochondria in vivo.

The effect of systematically altering the isolation conditions on the total calcium content of mitochondria isolated from perfused rat liver was examined. We showed that, under most isolation conditions, significant redistributions of mitochondrial calcium occurred resulting in up to 5-fold changes of the total calcium content. Mitochondrial Ca2+ flux inhibitors such as Ruthenium Red and nupercaine were only partially effective in inhibiting such redistributions. We present evidence indicating that the total calcium content of rat liver mitochondria in situ may approximate 2 nmol X (mg of protein)-1.     

Active calcium sequestration by intestinal microsomes. Stimulation by increased calcium load

Calcium uptake by an endoplasmic reticulum-enriched membrane fraction isolated from rat small intestine was investigated using a rapid filtration technique. Calcium sequestration was stimulated by the presence of ATP and released by the calcium ionophore A23187. ATP stimulation of calcium uptake was dependent on the presence of magnesium, inhibited by vanadate, and refractory to calmodulin. Kinetic studies revealed a K0.5 for the ATP-stimulated uptake of 62.5 nM Ca and a Jmax of 1.4 nmol of Ca/mg protein X min. A high dietary calcium load stimulated maximal uptake by 80% with no change in affinity. The magnitude of maximal uptake and the high affinity of this transport system suggest that the endoplasmic reticulum may play a significant role in cytosolic calcium sequestration and that extracellular calcium leads to modulation of intracellular endoplasmic reticulum calcium buffering.     

Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a45Ca exchange technique in the presence of magnesium,phosphate, and ATP at 37°C

Summary The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37°C and in the presence of 2mm inorganic phosphate, 3mm ATP, 0.05 or 1.1mm free magnesium and a calcium buffer, nitrilotriacetic acid, was investigated using a⁴⁵Ca exchange technique. The amounts of⁴⁰Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of⁴⁵Ca exchange. At 0.05mm free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 m, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 m. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The⁴⁵Ca exchange data were found to be consistent with a system in which two compartments of exchangeable calcium are associated with the mitochondria. Changes in the concentration of inorganic phosphate did not significantly affect the⁴⁵Ca exchange curves, whereas an increase in the concentration of free magnesium inhibited exchange. The maximum rate of calcium outflow from the mitochondria was estimated to be 1.7 nmol/min per mg of protein, and the value ofK _0.5 for intramitochondrial exchangeable calcium to be about 1.6 nmol per mg of protein. Ruthenium Red decreased the fractional transfer rate for calcium inflow to the mitochondria while nupercaine affected principally the fractional transfer rates for the transfer of calcium between the two mitochondrial compartments. The use of the incubation conditions and⁴⁵Ca exchange technique described in this report for studies of the effects of agents which may alter mitochondrial calcium uptake or release (e.g., the pre-treatment of cells with hormones) is briefly discussed.     

Kinetics of mitochondrial calcium transport. II. A kinetic description of the sodium-dependent calcium efflux mechanism of liver mitochondria and inhibition by ruthenium red and by tetraphenylphosphonium

Sodium-dependent calcium efflux from rat liver mitochondria has been studied as a function of mitochondrial calcium loads (2 to 40 nmol/mg) and extramitochondrial sodium concentrations (5 to 40 mM). The resulting data can be fit to a terreactant model which exhibits simultaneous kinetics (i.e. both sodium and calcium must be bound simultaneously for transport to occur). The Hill coefficients for the calcium and sodium dependences were 1.0 +/- 0.1 and 2.0 +/- 0.2, respectively. The cooperativity of the sodium dependence allows the terreactant model to be reduced to a bireactant model in which the sodium concentration only appears mathematically as the square of the sodium concentration. The data then fit the relationship (Formula: see text) The experimentally determined value of Vmax is found to be 2.6 +/- 0.5 nmol/mg/min, and the load of calcium (KCa) and concentration of sodium (KNa) necessary to stimulate the efflux to half its maximal calcium-dependent activity and sodium-dependent activity, respectively, were 8.1 +/- 1.4 nmol of Ca2+/mg and 9.4 +/- 0.6 mM Na+. This sodium-dependent calcium efflux from liver mitochondria was inhibited by magnesium, by ruthenium red, and by tetraphenylphosphonium. Fifty percent inhibition was obtained at 1.0-1.5 mM magnesium, at 12 nmol of ruthenium red/mg of protein, and at 0.2 microM tetraphenylphosphonium.     

Calcium pools in sea urchin eggs: roles of endoplasmic reticulum and mitochondria in relation to fertilization.

A preparation of sea urchin eggs permeabilized with digitonin (40 microM for 2.5 min) was used to study the kinetic characteristics of the two cellular compartments suspected to play a key role in cellular calcium transfer during fertilization: an ATP-dependent Ca2+ pool (Km = 0.47 microM; Vm = 0.48 nmol/min.mg protein) probably located in the endoplasmic reticulum and a mitochondrial Ca2+ pool (Km = 1.50 microM; Vm = 0.12 nmol/min.mg protein). Fertilization triggered a decrease in the rate of ATP dependent uptake by the non-mitochondrial pool (Km = 0.59 microM; Vm = 0.15 nmol/min.mg protein) while it transiently increased the Ca2+ uptake into mitochondria (2 min post-fertilization: Km = 2.20 microM; Vm = 0.40 nmol/min.mg protein). Microanalysis studies performed on quickly frozen, freeze substituted and embedded eggs showed a transient Ca2+ enrichment of mitochondria soon after fertilization thus suggesting that mitochondria behave as a Ca2+ sink at fertilization. Results are discussed in relation to the role of endoplasmic reticulum and mitochondria in handling free calcium during the early period following sea urchin egg fertilization.     

Aspects of energy-linked calcium accumulation by rat heart mitochondria.

When intact rat heart mitochondria were pulsed with 150 nmol of CaCl2/mg of mitochondrial protein, only a marginal stimulation of the rate of oxygen consumption was observed. This result was obtained with mitochondria isolated in either the presence or absence of nagarse. In contrast, rat liver mitochondria under similar conditions demonstrated a rapid, reversible burst of respiration associated with energy-linked calcium accumulation. Direct analysis of calcium retention using 45Ca and Millipore filtration indicated that calcium was accumulated by heart mitochondria under the above conditions via a unique energy-dependent process. The rate of translocation by heart mitochondria was less than that of liver mitochondria; likewise the release of bound calcium back into the medium was also retarded. These results suggest that the slower accumulation and release of calcium is characteristic of heart mitochondria. The amound of calcium bound was independent of penetrant anions at low calcium concentrations. Above 100 nmol/mg of mitochondrial protein, the total calcium bound was increased by the presence of inorganic phosphate. Under nonrespiring conditions, a biphasic Scatchard plot indicative of binding sites with different affinities for Ca2+ was observed. The extrapolated constants are 7.5 nmol/mg bound with an apparent half-saturation value of 75 muM and 42.5 nmol/mg bound with half-saturation at 1.15 mM. The response of the reduced State 4 cytochrome b to pulsed additions of Ca2+ was used to calculate an energy-dependent half-saturation constant of 40 muM. When the concentration of free calcium was stabilized at low levels with Ca2+-EGTA buffers, the spectrophotometrically determined binding constant decreased two orders of magnitude to an apparent affinity of 4.16 X 10(-7) M. Primary of calcium transport over oxidative phosphorylation was not observed with heart mitochondria. The phosphorylation of ADP competed with Ca2+ accumulation, depressed the rates of cation transport, and altered the profile of respiration-linked H+ movements. Consistent with these result was the observation that with liver mitochondrial the magnitude of the cytochrome b oxidation-reduction shift was greater for Ca2+ than for ADP, whereas calcium responses never surpassed the ADP response in heart mitochondria. Furthermore, Mg2+ ingibited calcium accumulation by heart mitochondria while having only a slight effect upon calcium transport in liver mitochondria. The unique energetics of heart mitochondrial calcium transport are discussed relative to the regulated flux of cations during the cardiac excitation-relaxation cycle.     

Vascular smooth muscle mitochondria: Magnesium content and transport

Endogenous magnesium content and magnesium transport of isolated bovine vascular smooth muscle mitochondria were studied. Mitochondria isolated from atherosclerotic bovine arteries contained two to three times as much magnesium (178 nmol/mg of mitochondrial protein) as those isolated from normal arteries (67 nmol/mg of mitochondrial protein). Electron-opaque granules were visible in the unstained unfixed mitochondria and could be shown with electron probe analysis to consist of magnesium, calcium, and phosphorus. At concentrations of external Mg²⁺ from 0 to 6 mm, the vascular smooth muscle mitochondria exhibited respiratory substrate-supported release of Mg²⁺ as studied with metallochromic indicator, eriochrome blue, using dual-wavelength spectrophotometry. The maximal velocity of energized release (3 nmol of Mg²⁺/s/mg of mitochondrial protein) was observed at 4 mm external Mg²⁺ and the half-maximal transport occurred at 0.5 mm.     

Calcium and magnesium levels in isolated cardiac mitochondria from mice injected with isoproterenol

Summary Calcium (Ca) and Magnesium (Mg) are determined by atomic absorption flame spectrometry in isolated cardiac mitochondria from mice receiving subcutaneous injections of DL-isoproterenol HC1 (ISO), and in mitochondria of untreated controls. In the controls, mitochondria were isolated in the presence or absence of ruthenium red. On the absence of ruthenium red in the isolation medium, mitochondrial Ca levels increase by about 300%, while levels of Mg remain unchanged. Focal myocardial necrosis following a single ISO-injection is shown by electron microscopy. Ca and Mg levels are largely unaffected by a single dose of ISO until 24 h after the injection. A slight increase in Ca occurs in the 48 h samples. When multiple injections of ISO are given every 12th hour for 48 h, 72 h and 96 h, respectively, endogenous Ca and Mg increase significantly. It is suggested that this increase might be associated with ISO-induced cardiac hypertrophy rather than with the pharmacological effects of ISO per se.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

On the state of calcium ions in isolated rat liver mitochondria. III. Diversity of ruthenium red action on different calcium pools

Calcium efflux from isolated mitochondria on ruthenium red addition was shown to be biphasic. The rate of efflux from a slowly releasable pool was independent of preincubation. It could be saturated and in extrapolation revealed a maximal rate of 3.6 nmol/(min X mg protein). The efflux from a second, rapidly dischargeable pool was related to calcium added up to 300 nmol/mg protein when a final rate of 15 nmol/(min X mg protein) was reached. The magnitude of the latter pool depended on the time of preincubation in the presence of calcium and correlated with mitochondrial swelling. After ruthenium red addition, a further increase of this pool and spontaneous, destructive calcium release was prevented. Three conclusions are drawn from these results: On preincubation with calcium, part of the mitochondrial calcium develops into a rapidly dischargeable pool. This pool is responsible for mitochondrial alterations resulting in a spontaneous, destructive release of total calcium. Ruthenium red inhibits calcium release by discharging mitochondria from this destructive calcium pool. To avoid artefacts, mitochondrial parameters should be carefully controlled when ruthenium red-insensitive calcium efflux is studied.     

Subcellular Ca2+ transport in different areas of dog heart

Calcium transport and ATPase activities were determined in the heavy and mitochondrial fractions isolated from the left and right atria as well as ventricles of dogs. Ultrastructural distribution of these organelles in different areas of the myocardium was also examined. Calcium binding, calcium uptake, and calcium ATPase activities of the atrial microsomes were lower than those of the ventricles. On the other hand, mitochondrial calcium binding and uptake activities in the right atrium were higher than those in other areas. The mitochondrial total ATPase activities in the atria were also higher than those in the ventricles. Mitochondrial as well as microsomal yields from ventricles were significantly higher. Size and number of mitochondria in the ventricles were greater whereas no striking difference in the distribution of sarcoplasmic reticulum was apparent in different areas of the heart. Poorly developed calcium transport functions in the atrial microsomes may be one of the factors responsible for the generation of lower contractile force in this tissue in comparison with the ventricle.     

Ontogeny of mitochondrial calcium transport in spontaneously hypertensive (SHR) and WKY rats

The current studies were designed to investigate calcium uptake by intestinal jejunal sacs as well as in intestinal mitochondria of spontaneously hypertensive rats and their genetically matched WKY control rats. Kinetics of jejunal calcium uptake by jejunal sacs of adult SHR and WKY rats showed a significant decrease in Vmax of calcium uptake in SHR (227 +/- 24 versus 423 +/- 22 nmol.g tissue-1.3 min-1) compared to WKY rats P less than 0.001. To explore the intracellular handling of calcium by the intestinal mitochondria, calcium uptake was characterized by intestinal mitochondria before (suckling and weanling periods) and after (adult period) development of hypertension. Calcium uptake by intestinal mitochondria was driven by ATP in the presence of succinate as a respiratory substrate. Calcium uptake was stimulated several fold by the presence of ATP compared to no ATP conditions. Maximal calcium uptake occurred between 15-30 min and was significantly greater in adult SHR and WKY rats compared to corresponding values in weanling and suckling rats. Maximal ATP dependent calcium uptake in adult, weanling and suckling WKY rats was significantly greater compared to corresponding mean values in each age group in SHR (P less than 0.001). Oligomycin (10 micrograms/mg protein) inhibited calcium uptake partially. Ruthenium red (0.25 microM), 1 mM sodium azide and 0.5 mM dinitrophenol inhibited calcium uptake by more than 80% in both SHR and WKY rats. Kinetic parameters for ATP stimulated calcium uptake at 10 s revealed a Vmax of 0.56 +/- 0.6, 3.46 +/- 0.23 and 3.95 +/- 0.52 nmol/mg protein/10 s in suckling, weanling and adult WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the response of mitochondrial calcium transport to exogenous phosphate during development in flight muscle of the sheep blowfly Lucilla cuprina.

1. Ca2+ transport by mitochondria isolated from flight muscle of the sheep blowfly Lucilla cuprina does not occur in the absence of added P1. Maximum rates of transport are attained when about 2.5 mM-phosphate is present. 2. As mitochondria develop, high but not low phosphate concentrations begin to inhibit Ca2+ transport markedly; those isolated from 2-day-old flies for example, are inhibited by about 75% by 20 mM-phosphate. Maximum rates of transport, i.e. those measured in the presence of 2.5 mM-phosphate, begin to decline only when the fly is about 3 days old. 3. Mitochondrial phosphate transport activity does not change during development of the blowfly, but the endogenous concentration of the anion does. At emergence it is about 6nmol/mg of protein, increases to about 17 nmol/mg of protein at 2-3h and then rapidly declines to reach less than 5 nmol/mg of protein after 2 days of adult life. 4. Studies on the effect of phosphate on oxidation of alpha-glycerophosphate in the absence and presence of ADP reveal a lack of inhibition by high phosphate concentrations indicating that the anion does not influence Ca2+ transport by preventing the generation of the proton electrochemical gradient across the inner membrane. 5. It is concluded that the molecular assembly in the inner membrane of Lucilla mitochondria responsible for transporting Ca2+ is fully developed at emergence and remains so for at least 2-3 days of adult life. The possibility exists that Ca2+-transport activity in these mitochondria is controlled at least in part by P1.     

A method for determining the kinetic characteristics of Ca2+-transporting systems of subcellular structures in smooth muscle

A simple method is suggested to determine kinetic characteristics of the Ca2+ active transport systems in the smooth muscle. The use of this method has shown that the initial rate of Ca2+ accumulation in the myometrium mitochondria (57.5 nmol per 1 mg of protein/1 min) is 50 times higher than in the sarcolemma vesicles. The calcium capacity of mitochondria (254 nmol per 1 mg of protein) also exceeds essentially (36 times) that of the membrane vesicles. Meanwhile, the Ca2+-transporting systems of these two subcellular structures practically do not differ from each other in the magnitude of the cation semiaccumulation period (4-7 min).     

Intramitochondrial and extramitochondrial free calcium ion concentrations of suspensions of heart mitochondria with very low,plausibly physiological,contents of total calcium

The 2-oxoglutarate dehydrogenase of intact rat heart mitochondria is activated by Ca²⁺, with 50% activation at approximately 0.5 nmol of total Ca/mg of mitochondrial protein, in the presence of P_i and Mg²⁺. Mitochondrial Ca contents in excess of 2 nmol/mg of protein result in 100% activation of the enzyme. Investigation of Ca²⁺ release from the mitochondria using the metallochromic indicator Arsenazo III defines aS _0.5 of 5.4±0.4 nmol of Ca/mg of protein, when the endogenous Ca content of the mitochondria is progressively depleted with EGTA, prior to the initiation of the release process being studied. The subsequent determination of matrix free Ca²⁺ concentration by the null-point technique has allowed expression of these results in terms of free concentration rather than Ca content, with an activity coefficient of approximately 0.001 for matrix Ca²⁺. From the above, Ca²⁺ efflux from heart mitochondria is not saturated at the mitochondrial Ca contents or Ca²⁺ concentrations which give effective regulation of dehydrogenase activity. A consequence is that heart mitochondria do not buffer the pCa of the extramitochondrial medium at these Ca contents (<2 nmol/mg of protein), and this is shown in direct measurements of extramitochondrial pCa. This is taken to question the physiological significance of mitochondrial buffering of cytosolic free Ca²⁺ in normal heart.     

"Allosteric regulation" of calcium-uptake in rat liver mitochondria

During investigations of calcium uptake by rat liver mitochondria, at a buffered free calcium concentration of 2 microM, a considerable acceleration of calcium uptake was occasionally observed. From the following experiments it can be concluded that the acceleration occurred when mitochondria had become anaerobic, and hence deenergized, because they had been stored in the refrigerator for a while. Mitochondria which had become transitorily deenergized by blocking the respiratory chain with KCN, rotenone or antimycin showed an accelerated calcium uptake when the membrane potential necessary for calcium uptake was regenerated. This acceleration of calcium uptake was also seen when a potassium diffusion potential was induced by valinomycin in previously deenergized mitochondria. The velocity of calcium uptake in transitorily deenergized mitochondria increased irrespective of the presence of magnesium in the incubation medium. The activation of the Ca uniporter was reversible, and both processes, activation and deactivation, were time-dependent and developed within a time span of minutes. Oligomycin strongly inhibited the deactivation of the uniporter by ATP, hence the membrane potential is intrinsically effective and does not act via ATP. The altered kinetics of the Ca uniporter were responsible for the acceleration of calcium uptake which was measured at low calcium concentration with previously deenergized mitochondria. The dependence of the rate of calcium uptake on the concentration of calcium in the medium is hyperbolic in transitorily deenergized mitochondria [Km = 6.7 microM; V = 455 nmol/(min X mg protein)] and sigmoidal in normal ones. It is additionally independent of the presence of magnesium ions. We found Hill coefficients of 3.47 and 2.94 in experiments with and without magnesium, respectively. Correspondent kinetics, hyperbolic in deenergized and sigmoidal in normal mitochondria, were obtained when calcium uptake was not driven by the system of respiratory chain, but by the potassium diffusion potential induced by valinomycin. The alteration in the kinetics of the Ca uniporter has consequences in the range of physiological calcium levels, but mainly in pathological states of liver cells. These points are discussed.     

Adrenal glucocorticoids, adenine nucleotide translocation, and mitochondrial calcium accumulation.

The administration of dexamethasone to rats markedly diminished the initial rate and maximal extent of substrate-dependent calcium uptake in subsequently isolated liver mitochondria, and enhanced the release of calcium. The apparent Km for calcium transport was not altered by dexamethasone treatment and it ranged from 50 to 80 muM when an EDTA/Ca buffer system was used in the presence of magnesium, and 20 muM when an NTA/Ca buffer system without magnesium was employed. In contrast, when ATP was employed as the energy source, there was no significant difference in initial rate, Km, or the extent of calcium accumulation between mitochondria from control and dexamethasone-treated animals. Although mitochondria from dexamethasone-treated animal showed 15% less cytochrome c oxidase activity/mg of protein, overall respiratory capacity and ATP production from ADP were the same as in control mitochondria. However, mitochondria from dexamethasone-treated animals translocated ATP from inside to outside faster than those from control animals. When the ATP in the medium was depleted by glucose and hexokinase, both types of mitochondria retained essentially all the preloaded calcium until total ATP reached a critical level (7 approximately 5 mumol of ATP/mg of protein). When ATP content fell below this critical level, mitochondria released all the calcium quickly. Dexamethasone treatment increased the susceptibility of mitochondria to the depletion of ATP. These data indicate that the dexamethasone-induced decrease in maximal calcium transport and in calcium retention carrier system per se, but o an altered ability of the mitochondria to regulate intramitochondrial ATP content.     

Ca2+-mediated activation of phosphoenolpyruvate carboxykinase occurs via release of Fe2+ from rat liver mitochondria

The addition of calcium chloride to rat liver homogenates resulted in activation of phosphoenolpyruvate carboxykinase by as much as 50%. The enhanced activity was inhibited by quinolinic acid; it was not additive with activation by FeCl2, and stimulation was prevented by 1,10-phenanthroline. Activation by calcium was lost when the particulate fractions of liver were removed, but an activating system could be reconstituted with isolated mitochondria, purified P-enolpyruvate carboxykinase, and purified ferroactivator. Iron-loaded mitochondria were more responsive to calcium than controls. A release of Fe2+ from washed mitochondria could be detected spectrophotometrically when 25-75 nmol of Ca/mg of protein were added to the mitochondrial suspension. If Ca2+ was buffered with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the threshold of Ca2+ necessary for release of Fe2+ was approximately 10(-7) M, with peak response between 5 X 10(-7) and 10(-6) M. Total Fe2+ detected was normally 20-30 pmol of Fe2+/mg of protein. The synthetic activator of P-enolpyruvate carboxykinase, 3-aminopicolinic acid, as well as other picolinic acid derivatives, is capable of withdrawing Fe2+ associated with the mitochondrial fraction; after incubation with mitochondria, 3-aminopicolinate will activate phosphoenolpyruvate carboxykinase in the absence of exogenous metal.     

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.