Characterisation of Ca2+ transport activity by white adipose tissue mitochondria (original) (raw)
Ca2' transport in mitochondria isolated from rat white adipocytes has been examined and many of the properties found to be similar to those reported for mitochondria isolated from rat liver. Ca" transport is ruthenium red-sensitive (Ki -5 pmol . mg protein-'), the affinity for free Ca2' is high (Km -3.3 PM) and the V,, is 135 nmol Ca2+ .min-'.mg protein-' at 4°C with 0.2 mM Pi present. Ca" transport is stimulated by increasing the medium [Pi], and is inhibited when ATP or Mg2+ is added to the incubation system and in contrast to brown adipocyte mitochondria, Ca2' efflux is not promoted by Na+. White adipocyte mitochondria may play a role in the regulation of total cell calcium in this tissue.
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 1980
Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca 2÷ transport in this preparation were characterized and compared to those of (Ca 2÷ + Mg2+)-ATPase activity. The time course of Ca 2÷ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8--11.5 nmol Ca2÷/mg protein after 3--4 min of incubation. The rate of Ca 2÷ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca2÷/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent Km values of 100 pM and 7 #M for ATP concentration ranges above and below 50 pM, respectively. The apparent Km values for Mg 2÷ and Ca 2÷ were 132 pM and 0.36--0.67 #M, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal (Ca 2÷ + Mg~÷)-ATPase. The presence of potassium was required for maximum Ca 2÷ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca 2÷ transport and (Ca ~÷ + Mg2÷)-ATPase activity was K ÷ ~ Na ÷ = NH~ ~ Li ÷. Ca 2÷ transport and (Ca:++ Mg2÷)-ATPase activity were both inhibited 10--20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 ]~M p-chloromercuribenzene
Journal of Bioenergetics and Biomembranes, 1996
The role of the adenine nucleotide translocase on Ca2+ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 μM Ca2+, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca2+ release, even though it inhibits the ADP exchange reaction. However, when the Ca2+ concentration was increased to 150 μM, carboxyatractyloside did induce Ca2+ release, and ADP favored Ca2+ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It is suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.
Characteristics and possible functions of mitochondrial Ca2+ transport mechanisms
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
Mitochondria produce around 92% of the ATP used in the typical animal cell by oxidative phosphorylation using energy from their electrochemical proton gradient. Intramitochondrial free Ca 2+ concentration ([Ca 2+ ] m) has been found to be an important component of control of the rate of this ATP production. In addition, [Ca 2+ ] m also controls the opening of a large pore in the inner mitochondrial membrane, the permeability transition pore (PTP), which plays a role in mitochondrial control of programmed cell death or apoptosis. Therefore, [Ca 2+ ] m can control whether the cell has sufficient ATP to fulfill its functions and survive or is condemned to death. Ca 2+ is also one of the most important second messengers within the cytosol, signaling changes in cellular response through Ca 2+ pulses or transients. Mitochondria can also sequester Ca 2+ from these transients so as to modify the shape of Ca 2+ signaling transients or control their location within the cell. All of this is controlled by the action of four or five mitochondrial Ca 2+ transport mechanisms and the PTP. The characteristics of these mechanisms of Ca 2+ transport and a discussion of how they might function are described in this paper.
Sodium-induced calcium release from mitochondria in brown adipose tissue
Proceedings of the National Academy of Sciences, 1979
Coupled mitochondria of brown adipose tissue can accumulate Ca2+ if a substrate is present. The Ca2+ is released by addition of 20 mM Na+, but not by addition of K+ or choline +. Energy-dissipating Na+-induced Ca2+ cycling occurs maximally with 20 mM Na+ and 10 microM Ca2+. In brown adipocytes, the Ca2+ ionophore A23187 and the Na+ ionophore monensin increase respiration if substrate is added, and incubation in a low-Na+ buffer decreases norepinephrine-induced respiration. Thus Na+-induced Ca2+ release takes place in brown adipose tissue; released Ca2+ could have a regulatory or thermogenic role or both.
Mitochondria as all‐round players of the calcium game
The Journal of Physiology, 2000
Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca¥ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca¥, the possibility that physiological stimuli that raise the [Ca¥] of the cytoplasm could trigger major mitochondrial Ca¥ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude of the cytoplasmic [Ca¥] rises. The direct measurement of mitochondrial [Ca¥] with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation, the cytoplasmic Ca¥ signal is always paralleled by a much larger rise in [Ca¥] in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca¥ transport and novel, unexpected results have altered long-standing dogmas in the field of calcium signalling. Here we focus on four main topics: (i) the current knowledge of the functional properties of the Ca¥ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca¥ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca¥ sources; (iii) the physiological consequences of Ca¥ transport for both mitochondrial function and the modulation of the cytoplasmic Ca¥ signal; and (iv) evidence that alterations of mitochondrial Ca¥ signalling may occur in pathophysiological conditions.
The Journal of biological chemistry, 1988
Rapid, unidirectional Ca2+ influx was examined in isolated brown adipocytes by short incubations (30 s) with 45Ca2+. Ca2+ uptake was found to be large in the resting brown adipocyte, but was markedly inhibited when the cells were presented with norepinephrine. Specific alpha 1-adrenergic stimulation was without effect on Ca2+ uptake. The effect of norepinephrine (which had an EC50 of 140 nM) could be inhibited by beta-adrenergic blockade and could be mimicked by forskolin (an adenylate cyclase activator) and theophylline (a phosphodiesterase inhibitor). Exogenous free fatty acids such as octanoate and palmitate (classical stimulators of respiration in brown adipocytes) were also able to dramatically inhibit Ca2+ uptake by the cells. The artificial mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induced a large reduction in cellular Ca2+ uptake (even in the presence of the ATPase inhibitor oligomycin), and in the presence of FCCP the inhibitory effec...
Calcium and mitochondria: mechanisms and functions of a troubled relationship
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2004
Mitochondria promptly respond to Ca 2+ -mediated cell stimulations with a rapid accumulation of the cation into the matrix. In this article, we review (i) the basic principles of mitochondrial Ca 2+ transport, (ii) the physiological/pathological role of mitochondrial Ca 2+ uptake, (iii) the regulatory mechanisms that may operate in vivo, and (iv) the new targeted Ca 2+ probes that allowed the brediscoveryQ of these organelles in calcium signalling. D
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