Measurement of matrix free Mg2+ concentration in rat heart mitochondria by using entrapped fluorescent probes.

1. The concentration of free Mg2+ ([Mg2+]m) within the matrix of isolated rat heart mitochondria was measured after loading of the mitochondria with the fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2. No detectable change in total mitochondrial magnesium content occurred during loading with the indicators. Apparent Kd values for Mg2+ of 3.7 mM and 2.3 mM were obtained for mag-indo-1 and mag-fura-2 respectively within mitochondria permeabilized to bivalent cations with ionomycin and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These values are 2.7- and 1.8-fold greater respectively than those obtained for the free acid forms of the dyes in incubation medium. 2. Based on the above Kd values, mitochondrial matrix Mg2+ concentrations were found to lie in the range 0.8-1.5 mM in the absence, or immediately after the addition, of a respiratory substrate. 3. Incubation of mitochondria in the presence of respiratory substrate, but in the absence of external Mg2+, led to a time-dependent decline in [Mg2+]m to about half the initial values after 5 min. This was accompanied by a fall in the total mitochondrial magnesium content from 12.7 to 7.0 nmol/mg of protein. 4. ADP (0.5 mM), ATP (0.5 mM) or 10 mM-NaCl had no significant effect on the fall in [Mg2+], whereas 1 microM-nigericin blocked, and 0.3 microM-valinomycin accelerated, the fall. 5. External Mg2+ concentrations above 1 mM progressively inhibited and reversed the decline in free and total mitochondrial Mg2+.     

Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria

The entrapment of the Ca2+-sensitive fluorescence indicators fura-2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ [( Ca2+]m) [(1987) Biochem J. 248, 609-613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura-2-loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half-maximal and maximal activation were attained at 0.8 and 1.6 microM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.     

Studies on mitochondrial Ca2+-transport and matrix Ca2+ using fura-2-loaded rat heart mitochondria

Rat heart mitochondria were incubated for 5 min at 30 degrees C and at approx. 40 mg protein.ml-1 and in the presence of 10 microM fura-2/AM. This allowed the entrapment of free fura-2 within the mitochondrial matrix and its use as a probe for Ca2+, but without affecting the apparent viability of the mitochondria. Parallel measurements of the activities of the intramitochondrial Ca2+-sensitive enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, allowed an assessment of their sensitivity to measured free Ca2+ within intact mitochondria incubated under different conditions; the enzymes responded to matrix Ca2+ over the approximate range 0.02-2 microM with half-maximal effects at about 0.3-0.6 microM Ca2+. Effectors of Ca2+-transport across the inner membrane (e.g., Na+, Mg2+, Ruthenium red, spermine) did not appear to affect these ranges, but did bring about expected changes in Ca2+ distribution across this membrane. Significantly, when mitochondria were incubated in the presence of physiological concentrations of both Na+ and Mg2+, and at low extramitochondrial Ca2+ (less than 400 nM), there was a gradient of Ca2+ (in:out) of less than unity; at higher extramitochondrial [Ca2+] (but still within the physiological range) the gradient was greater than unity indicating a highly cooperative nature of transmission of the Ca2+ signal into the matrix under such conditions.     

Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2

The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.     

Fluorescence measurements of free Ca2+ concentration in human erythrocytes using the Ca2+-indicator fura-2

We report here the use of the fluorescent Ca2+-chelator fura-2 to directly measure free Ca2+ concentration within intact human erythrocytes and the influence of viscosity on the fluorescence of this probe. The bright fluorescence of fura-2 has permitted the use of low concentrations of indicator and cells, thus minimizing the screening effect and the intrinsic fluorescence of haemoglobin. Erythrocytes (10(8) cells/ml) were loaded with 0.5 microM fura-2AM then diluted at 10(7) cells per ml for measurements. The extracellular signal was suppressed by addition of manganese ions just before recording spectra. Under these conditions, a blood sample of 100 microliter was sufficient for analysis. To study the influence of viscosity on fura-2 fluorescence, gelatin and polyvinylpyrrolidone at various concentrations were added to a physiological buffer to perform fura-2-Ca fluorescence standard curves. Fluorescence intensities and the apparent affinity constant for Ca2+ were modified by viscosity. When intra-erythrocytic viscosity was simulated with 21 g/l polyvinylpyrrolidone to obtain a mean viscosity of 14 mPa.s similar to that observed in human erythrocytes, the mean value of free Ca2+ concentration measured in erythrocytes from healthy subjects was 78 +/- 16 nM (mean +/- S.D., n = 29).     

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.     

Measurement of free Ca2+ changes and total Ca2+ release in a single striated muscle fibre using the fluorescent indicator quin 2

The fluorescent Ca2+ indicator, quin 2, has been used in isolated striated muscle fibres. There is a distinct quin 2 fluorescence peak at lambda 500 nm upon excitation at lambda 339 nm after axial injection of the potassium salt of quin 2, pH 7.1. Single voltage-clamp or current clamp electrical stimulation resulted in a distinct transient change in the fluorescence at lambda 500 nm which was not observed at lambda 400 nm, the peak of the fibre autofluorescence. Ca2+ buffering is marked at high quin 2 concentrations (greater than or equal to 400 microM) producing a slow decay of force and fluorescence. At lower concentrations (8-30 microM) of quin, the decay of force is within the range observed in non-injected control fibres. A Kd of 457 nM at 5 mM free Mg2+ suggests an upper resting free Ca2+ concentration of 310 nM at 12 degrees C.     

Heterogeneity of mitochondrial matrix free Ca2+: resolution of Ca2+ dynamics in individual mitochondria in situ

The role of mitochondria inCa²⁺ homeostasis is controversial.We employed the Ca²⁺-sensitive dyerhod 2 with novel, high temporal and spatial resolution imaging toevaluate changes in the matrix freeCa²⁺ concentration of individualmitochondria([Ca²⁺]_m)in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 µM serotonin (5-HT) evoked modest cytosolicCa²⁺ transients[cytosolic freeCa²⁺ concentration([Ca²⁺]_cyt)<500 nM; measured with fura 2] and triggered contractions inshort-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca²⁺]_m)in only 4% of cells. This revealed heterogeneity in the responses ofindividual mitochondria, all of which stained with MitoTracker GreenFM. In contrast, stimulation with 100 µM ATP evoked large cytosolicCa²⁺ transients (>1,000 nM) andinduced pronounced, reversible elevation of[Ca²⁺]_m(measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca²⁺ uptake usually lagged behindthe cytosolic Ca²⁺ transient peakby 3-5 s, and[Ca²⁺]_mdeclined more slowly than did bulk[Ca²⁺]_cyt.The uptake delay may prevent mitochondria from interfering with rapidsignaling events while enhancing the mitochondrial response to large,long-duration elevations of[Ca²⁺]_cyt.The responses of arterial myocytes to modest physiological stimulationdo not, however, depend on such marked changes in [Ca²⁺]_m.     

The regulation of extramitochondrial steady state free Ca2+ concentration by rat insulinoma mitochondria

For the study of Ca2+ handling by mitochondria of an insulin secretory tissue, a method for the isolation of functionally intact insulinoma mitochondria is described. The mitochondria had a respiratory control ratio of 6.3 +/- 0.3 with succinate as a substrate. The regulation of extramitochondrial [Ca2+]o concentration by suspensions of insulinoma mitochondria was studied using Ca2+-selective minielectrodes. The mitochondria were found to maintain an ambient free Ca2+ concentration of about 0.3 and 0.9 microM in the absence or presence of Mg2+ (1 mM), respectively. The addition of Na+ resulted in a dose-dependent (half-maximal 4 mM Na+) increase in steady state [Ca2+]o. Na+ accelerated the ruthenium red-induced Ca2+ efflux, suggesting the existence of a Ca2+/2Na+ antiporter, as described in mitochondria of excitable tissues. Experiments were performed to study the effects of various agents on the steady state extramitochondrial free Ca2+. cAMP, 3-isobutyl-1-methylxanthine, and NADH were found to have no effect, whereas phosphoenolpyruvate induced a net Ca2+ efflux, the kinetic of which suggests deleterious effects on mitochondrial functions. A small decrease in pH (0.1 unit) of the incubation buffer resulted in an increase of the extramitochondrial Ca2+ steady state that was reversible upon restoration of the pH to its initial value. In conclusion, insulinoma mitochondria were able to maintain an extramitochondrial [Ca2+]o steady state in the submicromolar range that was markedly influenced by the ionic composition of the incubation medium. Thus, mitochondria may play a role in the regulation of cellular calcium homeostasis and insulin release.     

The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration.

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2--3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.     

The use of the Ca2(+)-sensitive intramitochondrial dehydrogenases and entrapped fura-2 to study Sr2+ and Ba2+ transport across the inner membrane of mammalian mitochondria

In extracts of rat heart mitochondria, Sr2+ mimicked the activatory effects of Ca2+ on the Ca2(+)-sensitive intramitochondrial enzymes, pyruvate dehydrogenase phosphate phosphatase, isocitrate dehydrogenase (NAD+), and 2-oxoglutarate dehydrogenase, but at about tenfold higher concentrations (effective range approximately 1-100 muM) in each case. Ba2+ had no effect on extracted phosphatase, but did mimic the effect of Ca2+ on the other two enzymes with effective concentration ranges similar to those of Sr2+; as with Ca2+ and Sr2+, effective Ba2+ ranges were slightly (2-3-fold) raised by increases in ATP/ADP. In intact uncoupled rat heart mitochondria, the effects of Sr2+ and Ba2+ on the pyruvate and 2-oxoglutarate dehydrogenases were essentially similar to their effects in extracts. In fully coupled rat heart or liver mitochondria, the effective concentration ranges of extramitochondrial Sr2+, leading to activation of the matrix enzymes, were always approximately tenfold higher than those for Ca2+ under all conditions. Ba2+ did not affect pyruvate dehydrogenase in coupled mitochondria, but was shown to activate 2-oxoglutarate dehydrogenase in heart or liver mitochondria, and also isocitrate dehydrogenase (NAD+) in the latter; effective concentration ranges for extramitochondrial Ba2+ were approximately 100-fold greater than those for Ca2+, and like those for Ca2+ and Sr2+, were affected markedly by Mg2+ and spermine (which inhibit and promote mitochondrial Ca2+ uptake, respectively) but, in contrast to Ca2+ and Sr2+, they were hardly affected at all by Na+ (which promotes mitochondrial Ca2+ egress). Ba2+ effects were also blocked by ruthenium red (an inhibitor of mitochondrial Ca2+ uptake), but not so effectively as its blockage of the effects of Sr2+ and Ca2+. Ba2+ and Sr2+ both mimicked the inhibitory effects of extramitochondrial Ca2+ on the Na+/Ca2+ exchanger, but only Sr2+ could mimic Ca2+ in exchanging for internal Ca2+ by this mechanism. Both Sr2+ and Ba2+ changed the fluorescent properties of fura-2 or indo-1 in a similar manner to Ca2+, but with higher kd values. In fura-2-loaded rat heart mitochondria, increases in matrix Sr2+ and Ba2+ and the effects of the transport effectors could be readily demonstrated.     

Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

Modulation of Ca2+ efflux from heart mitochondria.

The efflux of Ca2+ from rat heart mitochondria has been examined by using Ruthenium Red to inhibit active uptake after predetermined loadings with Ca2+. The efflux is proportional to the internal Ca2+ load; it is increased by Na+ applied when the mitochondria are respiring and this effect is inhibited by oligomycin. The efflux of Ca2+ is diminished by ATP and by ADP, with the latter the more effective. Both active uptake and efflux of Ca2+ are slowed by bongkrekic acid; this action has a time lag. The lower efflux found with the nucleotides and with bongkrekic acid seems to correspond to the more condensed state seen in the electron microscope when these agents are applied [Stoner & Sirak (1973) J. Cell Biol. 56, 51-64, 65-73]. The results are discussed in relation to the less-permeable state being contingent upon nucleotide binding to the membrane.     

Quantitative measurements of the cytosolic Ca2+ activity within isolated guinea pig nerve-endings using entrapped arsenazo III and quin2

The absorbance changes of intrasynaptosomally entrapped arsenazo III have been converted into values of free Ca²⁺ concentration by correcting for the nonlinear response of arsenazo III at different concentrations of the dye as well as for changes in internal pH. An average resting value for free Ca²⁺ concentration around 0.4 μM is obtained. Depolarization with veratridine or gramicidin increases this value to around 3 μM. Measurements of cytosolic free Ca²⁺ with the quin2 method gives much lower values in similar conditions. The release of prelabelled [¹⁴C]noradrenaline from the nerve-endings is maximally activated when the internal free Ca²⁺ concentration rises as measured with arsenazo III to about 4 μM when titrated with increasing concentrations of ionophore A23187.     

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.