Donata Branca

Generation, control, and processing of cellular calcium signals.

Referee: Guiseppe Inesi, M.D., Ph.D., Professor and Chairman, School of Medicine, Dept. of Biochemistry and Molecular Biology, Univeristy of Maryland, Baltimore.

Influence of the anesthetic 2,6-diisopropylphenol on the oxidative phosphorylation of isolated rat liver mitochondria.

Isolated rat liver mitochondria have been incubated in the presence of the general anesthetic 2,6-diisopropylphenol (0-100 microM) and the efficiency of oxidative phosphorylation has been evaluated by measuring the respiratory rates, the rates of ATP synthesis or hydrolysis and the magnitude of the transmembrane electrical potential. The results obtained indicate that: (a) in mitochondria energized either by succinate or by ATP, 2,6-diisopropylphenol decreased the transmembrane electrical potential and increased the rates of either electron transfer or ATP hydrolysis; (b) in succinate-energized mitochondria 2,6-diisopropylphenol, at concentrations causing substantial depression of the transmembrane electrical potential, did not modify either the rate of phosphorylation of added ADP or the rate of ADP-stimulated respiration: (c) in succinate-energized mitochondria 2,6-diisopropylphenol caused a concentration-dependent inhibition of the uncoupler-stimulated rate of succinate oxidation. These findings suggest that under the experimental conditions reported 2,6-diisopropylphenol affected the generation and/or maintenance of the transmembrane electrical potential while leaving unchanged the coupling between the electron flow in the respiratory chain and the synthesis of ATP.

Uncoupling effect of the general anesthetic 2,6-diisopropylphenol in isolated rat liver mitochondria

2,6-Diisopropylphenol, a general anesthetic, was previously reported to reduce the transmembrane electrical potential in isolated rat liver mitochondria without affecting the rate of ATP production. This effect appeared to contrast with the generally accepted chemiosmotic mechanism for oxidative phosphorylation. In this study we further examined the influence of 2,6-diisopropylphenol on the production of ATP by isolated mitochondria and we studied its effect on the permeability of the inner mitochondrial membrane to protons. In order to clarify the effects of 2,6-diisopropylphenol on mitochondrial ATP production the activities of the adenine nucleotide translocator and the ATP synthetase were evaluated. The results obtained indicate that the depression of the transmembrane electrical potential elicited by 2,6-diisopropylphenol decreased the activity of the ATP synthetase (as expected in the chemiosmotic model for energy coupling), but not that of the adenine nucleotide translocator. The decrease of the ATP synthetase activity, however, did not result in an apparent inhibition of the overall rate of ATP production in isolated mitochondria due to the rate-limiting effect of the adenine nucleotide translocator in this process. Moreover 2,6-diisopropylphenol was found to increase the permeability to protons of the inner mitochondrial membrane; this effect became more marked as the pH of the incubation medium was increased, demonstrating that it involved the dissociated form of 2,6-diisopropylphenol. These observations suggested that 2,6-diisopropylphenol affected oxidative phosphorylation by acting as a mild protonophore and that its effectiveness was limited by the low fraction of phenol dissociated at near-physiological pH.

Influence of the anesthetic 2,6-diisopropylphenol (propofol) on isolated rat heart mitochondria

The influence of the anesthetic 2,6-diisopropylphenol on isolated rat heart mitochondria has been investigated at a range of concentrations encompassing high and low clinical values. Low clinical concentrations of the anesthetic appeared unable to affect both oxidative phosphorylation and calcium homeostasis. 2,6-diisopropylphenol at high clinical levels decreased both the transmembrane electrical potential and the synthesis of ATP, while leaving mitochondrial calcium homeostasis unaffected. The results obtained suggest that isolated heart mitochondria are substantially insensitive to low clinical concentrations of 2,6-diisopropylphenol, thus largely excluding the possibility that mitochondrial alterations might be involved in the cardiac depression induced by this anesthetic.

Involvement of long-chain acyl CoA in the antagonistic effects of halothane and L-carnitine on mitochondrial energy-linked processes

Incubation of rat liver mitochondria in the presence of halothane induced a consistent impairment of mitochondrial oxidative phosphorylation without significantly affecting the steady-state of transmembrane electrical potential. These alterations of mitochondrial energy-linked processes were associated with a consistent accumulation of long-chain acyl CoA. Addition of L-carnitine partially prevented the effects of halothane on oxidative phosphorylation and completely abolished the halothane-induced long-chain acyl CoA accumulation. The possibility is discussed that the damaging action of halothane on mitochondrial functions might be partially ascribed to the noxious action of the excess of long-chain acyl CoA induced the anesthetic.

L-carnitine effect on halothane-treated mitochondria.

Addition of halothane to the incubation medium is shown to lower respiratory control and transmembrane potential and to increase ATPase activity in isolated rat liver mitochondria. Evidence is presented that L-carnitine is able to substantially decrease the negative effects of halothane on the energy-linked processes of mitochondria. The effects of halothane and the protective action of L-carnitine are discussed in the light of a possible involvement of long-chain acyl CoA in the unpairing of mitochondrial energy-linked functions.

Alteration of mitochondrial bioenergetics due to intravenous injection of a perfluorocarbon emulsion.

Wistar albino rats were intravenously injected with 1 ml of an oxyphoretic emulsion of perfluorobutylfurane and killed 3, 7 or 30 days later. Mitochondria isolated from the liver and kidneys of treated rats showed a small decrease in the transmembrane electrical potential and a substantial depression of the rates of both ATP synthesis and ADP-stimulated respiration. These alterations in mitochondrial oxidative phosphorylation appear to be induced by perfluorocarbon and/or tensioactive molecules interacting with hydrophobic cell structures.

The inhibition of calcium efflux from rat liver mitochondria by halogenated anesthetics

The halogenated anesthetics halothane, enflurane and isoflurane inhibit the calcium efflux induced by Ruthenium Red in isolated rat liver mitochondria. The extent of the inhibition is higher for enflurane (approximately 50%) than for either isoflurane (approximately 35%) or halothane (approximately 15%), and does not increase significantly between 0.1 and 0.6-1.0 mM anesthetic. Both the mitochondrial respiratory rate and transmembrane electrical potential are unaffected by the halogenated anesthetics concentrations capable to inhibit the efflux of calcium.

Uptake and metabolic rate of liposome-entrapped [U-14C]glutamate in rats

Abstract The metabolic fate of [U-14C]glutamate administered intravenously to rats in aqueous solutions or entrapped in vescicles of phospholipids extracted from rat brain has been studied. When [U-14C]glutamate was administered entrapped in liposomes, higher serum radioactivity levels and higher radioactivity uptake by liver and brain were observed. Rats treated with [U-14C]glutamate entrapped in liposomes exhibited an increased glutamate oxidation shown by increased expired [14C]CO2 and a modified elimination of the radioactivity in urine.

Biochemical and morphological observations on rat liver and kidneys six months after intravenous injection of a perfluorocompound emulsion.

The histological appearance of liver and kidneys and the energy metabolism of isolated liver and kidney mitochondria were evaluated in rats 6 months after intravenous administration of 1 ml of a perfluorocompound emulsion. Both liver and kidney specimens showed neither significant histological alteration nor the presence of intracytoplasmic perfluorocompound particles. A substantial depression of the rate of ATP synthesis was observed both in liver and kidney isolated mitochondria (with respect to control mitochondria) although the magnitude of the transmembrane electrical potential was unaltered. The depression of ATP synthesis in mitochondria isolated from perfluorocompound-treated rats appeared then unrelated to the presence of perfluorocompound micelles within the cells, and might result from the interaction of either the perfluorocompound or the emulsifying agent with the mitochondrial ATP synthetase.