Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2.
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ABSTRACT:
SUBMITTER: Lukacs GL
PROVIDER: S-EPMC1148585 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
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Project description: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+.
| S-EPMC1149608 | biostudies-other
Project description: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.
| S-EPMC1133721 | biostudies-other
Project description: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.
| S-EPMC1158169 | biostudies-other
Project description: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.
| S-EPMC1186568 | biostudies-other
Project description:1. The mitochondrial content of active (dephospho) pyruvate dehydrogenase (PDHA) was found to be severalfold higher at an extramitochondrial Ca2+ concentration of 2 microM (pCa6) than at pCa7. The nature of the respiratory substrate did not affect this finding. 2. This Ca2+-dependence was shown in state-4 and 50%-state-3 conditions [see Chance & Williams (1956) Adv. Enzymol. 17, 65-134], but was absent in the presence of excess ADP (state 3). 3. Na+ and Mg2+ ions shifted the pCa value required for a maximal PDHA content to lower values. This was attributed to a stimulation of mitochondrial Ca2+ egress and an inhibition of uptake, respectively. Na+ ions diminished pyruvate dehydrogenase phosphate phosphatase activity in mitochondria which had been extensively depleted of Ca2+ ions by incubation with EGTA, raising the possibility of a direct inhibitory effect of Na+ ions, unrelated to Ca2+ movements. 4. Mg2+ ions lowered the mitochondrial PDHA content at pCa 6.24 and 6.48, but had only minimal effects in the presence of EGTA. 5. The effects of P1 and bicarbonate ions on PDHA content were also studied, as possible effectors of mitochondrial Ca2+ transport. Bicarbonate ions abolished the response to Ca2+ ions, by generating maximal values of PDHA content, but such a response was still observed when physiological concentrations of both P1 and bicarbonate were used. 6. The pCa of the medium in the range 6.33 to over 7 affected PDHA content, with only very minor changes in state-4 rates of O2 uptake and no change in [ATP]/[ADP] ratio or in mitochondrial [NADH]/[NAD+] ratio, provided that Mg2+ ions were present. Thus the effect of Ca2+ ions on PDHA content is unlikely to be mediated by changes in [ATP]/[ADP] and [NADH]/[NAD+] ratio and is more likely to be direct. Equally, changes in the [acetyl-CoA]/[CoA] ratio in response to Ca2+ ions when the substrate was pyruvate were the converse of those required to mediate changes in interconversion, and are probably secondary to changes in PDHA content.
| S-EPMC1162807 | biostudies-other
Project description:Superparamagnetism is a phenomenon caused by quantum effects in magnetic nanomaterials. Zero-valent metals with diameters below 5 nm have been suggested as superior alternatives to superparamagnetic metal oxides, having greater superspin magnitudes and lower levels of magnetic disorder. However, synthesis of such nanometals has been hindered by their chemical instability. Here we present a method for preparing air-stable superparamagnetic iron nanoparticles trapped between thermally reduced graphene oxide nanosheets and exhibiting ring-like or core-shell morphologies depending on iron concentration. Importantly, these hybrids show superparamagnetism at room temperature and retain it even at 5 K. The corrected saturation magnetization of 185 Am(2) kg(-1) is among the highest values reported for iron-based superparamagnets. The synthetic concept is generalized exploiting functional groups of graphene oxide to stabilize and entrap cobalt, nickel and gold nanoparticles, potentially opening doors for targeted delivery, magnetic separation and imaging applications.
| S-EPMC5027615 | biostudies-literature
Project description: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.
| S-EPMC1148273 | biostudies-other
Project description:The work examines the kinetic parameters of Ca2+ uptake via the mitochondrial calcium uniporter complex (MCUC) and the opening of the Ca2+-dependent permeability transition pore (MPT pore) in the liver and heart mitochondria of rats with high resistance (HR) and low resistance (LR) to acute hypoxia. We found that the rate of Ca2+ uptake by mitochondria of the liver and heart in HR rats is higher than that in LR rats, which is associated with a higher level of the channel-forming subunit MCU in liver mitochondria of HR rats and a lower content of the dominant-negative channel subunit MCUb in heart mitochondria of HR rats. It was shown that the liver mitochondria of HR rats are more resistant to the induction of the MPT pore than those of LR rats (the calcium retention capacity of liver mitochondria of HR rats was found to be 1.3 times greater than that of LR rats). These data correlate with the fact that the level of F0F1-ATP synthase, a possible structural element of the MPT pore, in the liver mitochondria of HR rats is lower than in LR rats. In heart mitochondria of rats of the two phenotypes, no statistically significant difference in the formation of the MPT pore was revealed. The paper discusses how changes in the expression of the MCUC subunits and the putative components of the MPT pore can affect Ca2+ homeostasis of mitochondria in animals with originally different tolerance to hypoxia and in hypoxia-induced tissue injury.
| S-EPMC7023097 | biostudies-literature
Project description:1. Rates of Ca2+ inflow across the hepatocyte plasma membrane in the presence of vasopressin were estimated by using quin2. 2. Plots of the rate of Ca2+ inflow as a function of the intracellular quin2 concentration reached a plateau at about 1.7 mM intracellular quin2. Ca2+ inflow was inhibited by 60% in the presence of 400 microM-verapamil. 3. A plot of the rate of Ca2+ inflow as a function of the concentration of extracellular Ca2+ ([Ca2+]o) was biphasic. The second (slower) phase showed no sign of saturation at values of [Ca2+]o up to 5 mM. It is concluded that, in the presence of vasopressin, Ca2+ flows into the liver cell by two different processes, one of which is not readily saturated by Ca2+o. 4. The effect of the replacement of extracellular NaCl by choline or tetramethylammonium chloride on cellular Ca2+ movement was found to depend on the presence or absence of intracellular quin2. 5. In cells loaded with quin2 and incubated in the presence of choline or tetramethylammonium chloride, a small decrease in the basal intracellular free Ca2+ concentration ([Ca2+]i) was observed, and the increase in [Ca2+]i caused by the addition of vasopressin was considerably diminished when compared with cells incubated in the presence of NaCl. In cells loaded with quin2, replacement of NaCl by choline chloride caused a decrease in Ca2+ inflow in the presence of vasopressin, as measured by using quin2 or 45Ca2+ exchange, whereas no change in Ca2+ inflow was observed in the absence of vasopressin. 6. In cells not loaded with quin2, replacement of NaCl by choline chloride did not alter Ca2+ inflow either in the presence or in the absence of vasopressin. 7. It is concluded that (i) Ca2+ inflow through the basal and receptor-activated Ca2+ inflow systems does not involve the inward movement of Ca2+ in exchange for Na+ or the induction of Ca2+ inflow by intracellular Na+, and (ii) the presence of both intracellular quin2 and extracellular choline or tetramethylammonium chloride (in place of NaCl) inhibits Ca2+ inflow through the receptor-activated Ca2+ inflow system but not through the basal Ca2+ inflow system, and inhibits the release of Ca2+ from intracellular stores.
| S-EPMC1133547 | biostudies-other
Project description:Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.
| S-EPMC1221255 | biostudies-other