Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2.

Rat pancreatic islets contain a Ca2+-activated and thiol-dependent transglutaminase (EC 2.3.2.13) comparable in activity with that found in rat liver, lung and spleen. The Ca2+-dependence of this enzyme is such that half-maximal velocity was obtained in the region of 40 microM. Preincubation of rat islets with primary-amine substrates of transglutaminase (monodansylcadaverine, methylamine, ethylamine, propylamine and cystamine) led to an inhibition of glucose-stimulated insulin release by these amines. Kinetic analysis of the competitive substrates methylamine, monodansylcadaverine, propylamine and ethylamine for their ability to inhibit islet transglutaminase activity indicated a potency that matched their ability to inhibit glucose-stimulated insulin release. When these amines were tested for their effects on glucose-stimulated protein synthesis and glucose utilization, the most potent inhibitor of insulin release, monodansylcadaverine, had no effect on either process at 100 microM. The amines cystamine, ethylamine, methylamine and propylamine had variable effects on these metabolic processes. For ethylamine, methylamine and propylamine, concentrations were found which inhibited glucose-stimulated insulin release in a manner which was found to be independent of their effects on either glucose oxidation or protein synthesis. Primary amines may therefore inhibit insulin release through their incorporation by islet transglutaminase into normal cross-linking sites. A role for protein cross-linking in the secretory mechanism is suggested.     

Tissue variation of mitochondrial oxidative phosphorylation efficiency in cold-acclimated ducklings

We investigated the oxidative phosphorylation efficiency of liver and gastrocnemius muscle mitochondria in thermoneutral and cold-acclimated ducklings. The yield of oxidative phosphorylation was lower in muscle than in liver mitochondria, a difference that was associated with a higher proton conductance in muscle mitochondria. Cold exposure did not affect oxidative phosphorylation efficiency or basal proton leak in mitochondria. We conclude that the basal proton conductance of mitochondria may regulate mitochondrial oxidative phosphorylation efficiency, but is not an important contributor to thermogenic processes in cold-acclimated ducklings.     

Effect of insulin on oxidative phosphorylation in the liver and heart mitochondria of adrenalectomized rats

The effect of insulin was studied as applied to the inhibited under conditions of adrenalectomy process of oxidative phosphorylation in the rat liver and heart mitochondria. It is established that adrenalectomy does not change oxidative activity of mitochondria but inhibits the process of phosphorylation, which results in the decreased values of the ADP/O coefficient and the respiratory control. Insulin administered to the adrenalectomized rats 3h before the experiments reduces the disturbed oxidative phosphorylation in mitochondria of the liver and heart by intensifying the degree of ADP phosphorylation.     

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.     

Regulation of thyroid hormones of the interaction of mitochondria with low-molecular weight cytoplasmic mediators, induced by phosphate- dependent transport of K+ and H+ ions through the mitochondrial inner membrane

In vivo thyroid hormones control the binding to mitochondria of low molecular weight water-soluble cytoplasmic mediators that are capable to induce oxidative phosphorylation uncoupling, by increasing the sensitivity of mitochondria to the effects of these mediators. In hyperthyroid rat liver mitochondria cytoplasmic mediators stimulate the phosphate-dependent transport of K+ and H+ in a greater degree than in liver mitochondria of control rats. The increase in the oxidative phosphorylation uncoupling by cytoplasmic mediators is one of mechanisms of thermogenesis stimulation by thyroid hormones.     

Regulation of oxidative phosphorylation, K+ ions transport and the volume of mitochondrial matrix by cytoplasmic glycopeptide and Ca2+ ions

A thermostable low molecular weight glycopeptide containing syalic acids, which uncouples mitochondrial oxidative phosphorylation, has been detected, isolated and purified from rat liver cytoplasm. In the presence of the glycopeptide, oxidative phosphorylation in rat liver mitochondria is uncoupled by low physiological concentrations of Ca2+, which otherwise do not have any appreciable effect on the mitochondria. Oxidative phosphorylation uncoupling by the glycopeptide is accompanied by an increase of the mitochondrial volume. This process has a limited amplitude and is regulated by changes in Ca2+ concentration in the extramitochondrial space. The glycopeptide has been shown to induce K+ transport across the inner mitochondrial membrane, this effect is enhanced by Ca2+.     

Effects of peginol C-2000 on respiration and oxidative phosphorylation in liver mitochondria of rats poisoned with butylcaptax]

The data on the effect of synthetic antioxidant Paginol C-2000 on respiration and oxidative phosphorylation in liver mitochondria of rats primed with butylcaptax are given. It is shown that intoxication of organism with butylcaptax in the dose of 1/10 LD50 during 5 days resulted in suppression of respiration and oxidative phosphorylation in rat liver mitochondria. It is found, that administration of Raginol C-2000 into organisms of primed animals leads to the reduction of succinate pathway of oxidation, that, in its turn, results in the normalization of functioning level of ATP-synthesizing system of mitochondria.     

Characteristics of biochemical processes of the liver in rats with various types of open-field behavior

Respiration and phosphorylation of liver mitochondria, lipid spectrum of the blood serum activity of hepatocytes main microsomal enzymes were studied in outbred rats with various emotionality. It was found that in low-emotional rats, the intensity of phosphorylation processes in liver mitochondria and enzymes activity of hematocytes microsomal oxidative system responsible for detoxication processes,-- was significantly higher than in low-emotional animals. Analogous differences were found in the lipid blood spectrum and in liver mitochondria antioxidant activity of animals with different emotionality. It is supposed that there is an inter-connection between animals behaviour in the open field and the character of biochemical processes in the liver.     

High phosphate requirement for oxidative phosphorylation and low affinity for phosphate transport in newborn rat liver mitochondria

Rat liver mitochondria are not fully functional at birth. The relationship between this deficiency and the affinity for phosphate, in oxidative phosphorylation or in phosphate transport, have been studied.The phosphate concentration necessary to observe maximal rate of succinate oxidation in the presence of ADP was higher for newborn than for adult rat liver mitochondria. After preincubation of newborn rat liver mitochondria with ATP, the rate of succinate oxidation in the presence of ADP increased with phosphate concentration similarly for newborn and adult rat liver mitochondria. The maximal rate of phosphate-acetate exchange, which is an indirect measure of the rate of phosphate transport across the mitochondrial membrane, was not significantly different for adult and newborn rat liver mitochondria. On the contrary the apparent affinity for phosphate was about ten-fold lower for newborn than for adult mitochondria.     

Averufin, an anthraquinone mycotoxin possessing a potent uncoupling effect on mitochondrial respiration.

Averufin and averufin dimethylether from Aspergillus versicolor were examined for their uncoupling effects on oxidative phosphorylation in isolated rat liver mitochondria to get insight into the mode of toxic action of averufin. Averufin uncoupled oxidative phosphorylation, causing 50% uncoupling at about 1.5 microM with respect to the decrease in P/O ratio. Averufin dimethylether did not uncouple but inhibited state 3 respiration of mitochondria, which was not released by either 2,4-dinitrophenol or averufin.     

Averufin, an anthraquinone mycotoxin possessing a potent uncoupling effect on mitochondrial respiration

Averufin and averufin dimethylether from Aspergillus versicolor were examined for their uncoupling effects on oxidative phosphorylation in isolated rat liver mitochondria to get insight into the mode of toxic action of averufin. Averufin uncoupled oxidative phosphorylation, causing 50% uncoupling at about 1.5 microM with respect to the decrease in P/O ratio. Averufin dimethylether did not uncouple but inhibited state 3 respiration of mitochondria, which was not released by either 2,4-dinitrophenol or averufin.     

High phosphate requirement for oxidative phosphorylation and low affinity for phosphate transport in newborn rat liver mitochondria

Rat liver mitochondria are not fully functional at birth. The relationship between this deficiency and the affinity for phosphate, in oxidative phosphorylation or in phosphate transport, have been studied. The phosphate concentration necessary to observe maximal rate of succinate oxidation in the presence of ADP was higher for newborn than for adult rat liver mitochondria. After preincubation of newborn rat liver mitochondria with ATP, the rate of succinate oxidation in the presence of ADP increased with phosphate concentration similarly for newborn and adult rat liver mitochondria. The maximal rate of phosphate-acetate exchange, which is an indirect measure of the rate of phosphate transport across the mitochondrial membrane, was not significantly different for adult and newborn rat liver mitochondria. On the contrary the apparent affinity for phosphate was about ten-fold lower for newborn than for adult mitochondria.     

The hydrophobic cationic cyanine dye inhibits oxidative phosphorylation by inhibiting ADP transport, not by electrophoretic transfer, into mitochondria

The effect of the divalent cationic cyanine dye tri-S-C4(5) on oxidative phosphorylation in rat liver mitochondria was examined. The dye at about 100 n mols per mg mitochondrial protein inhibited state 3 respiration and ATP synthesis almost completely. However, it had no effect on submitochondrial particles, like other hydrophobic cations. The dye inhibited the transport of ADP into mitochondria mediated by the adenine nucleotide translocator. Thus, the inhibition of oxidative phosphorylation by the cationic dye was concluded to be due to its action on the adenine nucleotide translocator, not to its electrophoretic transfer into the inner space of mitochondria according to the inside-negative electrochemical potential.     

Toxicity of emestrin,a new macrocyclic dithiodioxopiperazine mycotoxin,to mitochondrial function

The effect of emestrin, a new macrocyclic epidithiodioxopiperazine mycotoxin from severalEmericella species, on mitochondrial reactions was studied using isolated rat liver mitochondria to gain insight into the molecular mechanism for itsin vivo toxicity to rat and mouse. Emestrin was found to inhibit ATP synthesis in mitochondria causing an uncoupling of oxidative phosphorylation and a depression of respiration in isolated mitochondria. In addition to these effects on mitochondrial respiration, emestrin elicited a dratsic structural alteration (swelling) of mitochondria as observed in thein vivo system. The mitochondrial swelling was significantly enhanced by the subsequent addition of calcium ion. Emestrin B, in which dithio group is replaced by trithio group, exerted an uncoupling effect on oxidative phosphorylation without accompanying such depressive effect on state 3 respiration as observed for emestrin.     

Changes in the stimulation of mitochondrial respiration by adrenaline depending upon the dose

Effects of low and high 25 and 100 micrograms per 100 g of body weight doses of adrenaline on respiration and oxidative phosphorylation in rat liver mitochondria are compared. The high dose of adrenaline is shown to decrease activation of respiration and phosphorylation typical of the low doses. This decrease is caused by inhibition of succinate dehydrogenase and is accompanied by uncoupling of respiration and phosphorylation in mitochondria.     

Effect of visible light on the ATP synthetase function of mitochondria

Dependence of oxidative phosphorylation in rat liver mitochondria on redox state of endogenous flavoenzymes was studied. Regulation of the redox state of endogenous flavins in mitochondria was performed by visible light illumination of lambda = 380 nm in oxygen free as well as in oxygenated media. A decrease of oxidative phosphorylation was obtained at full reduction of flavins (respiratory control and ATP synthesis were measured); this effect was partially recovered when flavin was reoxidized by oxygen. Results of ESR measurements of the signal gl = 2.00 associated with semireduced flavins were in accordance with the appearance of diamagnetically completely reduced form of flavin. These facts may be considered as evidence of the regulative role of flavins in oxidative phosphorylation.     

Streptolydigin, an Inhibitor of Oxidative Phosphorylation in Rat Liver Mitochondria

Streptolydigin interferes with oxidative phosphorylation in rat liver mitochondria. The agent acts primarily as an uncoupler of respiration-associated phosphorylation but also impairs respiration to various degrees depending on the substrate. Streptolydigin partially inhibits electron flow at a point past the cytochrome b and prior to the cytochrome c reduction site. Streptolydigin also inhibits the function of the enzyme ribonucleic acid polymerase in whole bacterial cells and cell-free systems. The streptolydigin concentrations that cause effective inhibition of ribonucleic acid polymerase in cell-free systems are approximately 10 times less than those required to inhibit oxidative phosphorylation in mitochondria.     

Interaction of benzoquinones with mitochondria interferes with oxidative phosphorylation characteristics

Studies with four benzoquinones, viz. juglone, embelin, maesaquinone and maesanin, on rat liver mitochondria oxidative phosphorylation have been carried out. Three of the benzoquinones are uncouplers in the order juglone greater than maesoquinone greater than embelin, while maesanin is an inhibitor of electron transport and oxidative phosphorylation.     

Effects of ATP on various steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria

Preincubation of newborn rat liver mitochondria with ATP increases their state 3 respiration rate [J. K. Pollak (1975) Biochem. J. 150, 477-488; J. R. Aprille, and G. K. Asimakis (1980) Arch. Biochem. Biophys. 201, 564-575]. To determine which reactions contribute to control the rate of succinate oxidation with and without prior exposure to ATP, the effects of inhibitors specific for various reactions were studied. The adenine nucleotide translocator does not control the respiration in newborn more than in the adult mitochondria. The supply of reducing equivalents to the respiratory chain is an important step controlling the rate of oxidative phosphorylation by mitochondria from newborn rat liver, especially after preincubation with ATP. On the contrary, titrations with oligomycin show that the preincubation with ATP markedly decreases the control exerted by the ATPase-ATP synthase complex. That the rate of ATP synthesis is one of the steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria is in striking contrast to the behavior of adult rat liver mitochondria. Other differences include a greater permeability to protons and a marked increase in sensitivity to mersalyl, indicating an easier accessibility of the proteins involved in oxidative phosphorylation to the thiol reagent.     

The Inhibitory Effect of 3-Nitropropionate on the Oxidative Phosphorylation in Rat Mitochondria

The effect of 3-nitropropionate (3-NPA)on oxidative phosphorylation by using mitochondria prepared from both rat liver and brain were investigated in connection with the toxicity of this material. It was found that 3-NPA inhibited oxidative phosphorylation. In this inhibition, the uptake of inorganic phosphate was blocked but the oxygen uptake was not influenced at all. Furthermore, increase in ATPase activity of intact mitochondria was shown by the addition of 3-NPA. Results showed that 3-NPA disturbed oxidative phosphorylation as an uncoupler. However, the degree of inhibition by 3-NPA was not so high in comparison with other well-known uncouplers.

Thus the toxicity of 3-NPA is not due to the inhibition of oxidative phosphorylation. 3-NPA also does not affect on cytochrome oxidase activity.