Regulation of oxidative phosphorylation in intact mammalian heart in vivo

A dynamic computer model of oxidative phosphorylation in intact heart was developed by modifying the model of oxidative phosphorylation in intact skeletal muscle published previously. Next, this model was used for theoretical studies on the regulation of oxidative phosphorylation in intact heart in vivo during transition between different work intensities. It is shown that neither a direct activation of ATP usage alone nor a direct activation of both ATP usage and substrate dehydrogenation, including the calcium-activated tricarboxylate acid cycle dehydrogenases, can account for the constancy of [ADP], [PCr], [P(i)] and [NADH] during a significant increase in oxygen consumption and ATP turnover encountered in intact heart in vivo. Only a direct activation of all oxidative phosphorylation complexes in parallel with a stimulation of ATP usage and substrate dehydrogenation enabled to reproduce the experimental data concerning the constancy of metabolite concentrations. The molecular background of the differences between heart and skeletal muscle in the kinetic behaviour of the oxidative phosphorylation system is also discussed.     

Regulation of the degree of coupling of oxidative phosphorylation in intact rat liver

The degree of coupling of oxidative phopshorylation q was determined in isolated perfused livers and in livers in vivo from fed and fasted rats. This determination of q was based on a simple nonequilibrium-thermodynamic representation of the major reactions of cytosolic adenine nucleotides, and made use of the measured cytosolic concentrations of adenine nucleotides, phosphate, and lactate/pyruvate ratios in extracted livers. The deviations of the measured values from the theoretically predicted ones at different mass action ratios of the adenylate kinase reaction showed that the basic assumptions of the model, including linearity between flows and thermodynamic forces, were fulfilled in intact liver within the experimental error. The degree of coupling was higher in livers from fed rats than in livers from fasted rats. In particular, the determined values of q were close to the theoretical degrees of coupling qecp and qecf which allow maximization of output power and output flow of oxidative phosphorylation for fed and fasted states, respectively, at optimal efficiency and minimal energy costs. This finding indicates that conductance matching between the load and phosphorylation is fulfilled in vivo. Moreover, it was found that fatty acids lower the degree of coupling in a concentration-dependent manner. This suggested that in livers in the fasted state q is decreased due to elevated fatty-acid levels. Thus fatty acids could act as metabolic regulators of the degree of coupling, enabling the cell to optimize efficiency of oxidative phosphorylation under different metabolic regimes.     

Regulation of mitochondrial oxidative phosphorylation through cell signaling

The mitochondrial oxidative phosphorylation (OxPhos) system plays a key role in energy production, the generation of free radicals, and apoptosis. A lack of cellular energy, excessive radical production, and dysregulated apoptosis are found alone or in combination in most human diseases, including neurodegenerative diseases, stroke, cardiovascular disorders, ischemia/reperfusion, and cancer. In the context of its relevance to human disease, this article reviews current knowledge about the regulation of OxPhos with a focus on cell signaling and discusses identified phosphorylation sites with the aid of crystal structures of OxPhos complexes. Several recent studies have shown that all OxPhos components can be phosphorylated; even the small electron carrier cytochrome c is tyrosine phosphorylated in vivo. We propose that in higher organisms, in contrast to bacteria, cell signaling pathways are the main regulator of energy production, triggered for example by hormones. Pathways that have been identified to act on OxPhos include protein kinases A and C and growth factor activated receptor tyrosine kinase signaling. Present knowledge about kinases and phosphatases that execute signals at the level of the mitochondrial OxPhos system, and newly emerging concepts, such as the translocation of kinases to the mitochondria upon stimulation of a signaling pathway, are discussed.     

Theoretical studies on the regulation of oxidative phosphorylation in intact tissues

The theoretical studies on the regulation of oxidative phosphorylation that were performed with the aid of kinetic models of this process are overviewed. A definition of the regulation of the flux through a metabolic pathway is proposed and opposed to the control exerted by particular enzymes over this flux. Different kinetic models of oxidative phosphorylation proposed in the literature are presented, of which only the model proposed by myself and co-workers was extensively used in theoretical studies on the regulation and compensation in the oxidative phosphorylation system. These theoretical studies have led to the following conclusions: (1) in isolated mitochondria, an increase in the activity of an artificial ATP-using system stimulates mitochondria mainly via changes in [ADP], while changes in [ATP] and [P(i)] play only a minor role; (2) in non-excitable tissues (e.g. liver), hormones (acting via some cytosolic factor(s)) activate directly both ATP usage and at least some enzymes of the ATP-producing block; (3) in excitable tissues (e.g. skeletal muscle), neural signals stimulate (via some cytosolic factor(s)) in parallel all the steps of oxidative phosphorylation together with ATP usage and substrate dehydrogenation; (4) the decrease in the flux through cytochrome oxidase caused by a decrease in oxygen concentration is, at least partially, compensated by a decrease in Delta p and increase in the reduction level of cytochrome c. A theoretical prediction is formulated that there should exist and be observable a universal cytosolic factor/regulatory mechanism which directly activates (at least in excitable tissues) all complexes of oxidative phosphorylation during an increased energy demand.     

Regulation of oxidative phosphorylation through parallel activation

When the mechanical work intensity in muscle increases, the elevated ATP consumption rate must be matched by the rate of ATP production by oxidative phosphorylation in order to avoid a quick exhaustion of ATP. The traditional mechanism of the regulation of oxidative phosphorylation, namely the negative feedback involving [ADP] and [Pi] as regulatory signals, is not sufficient to account for various kinetic properties of the system in intact skeletal muscle and heart in vivo. Theoretical studies conducted using a dynamic computer model of oxidative phosphorylation developed previously strongly suggest the so-called each-step-activation (or parallel activation) mechanism, due to which all oxidative phosphorylation complexes are directly activated by some cytosolic factor/mechanism related to muscle contraction in parallel with the activation of ATP usage and substrate dehydrogenation by calcium ions. The present polemic article reviews and discusses the growing evidence supporting this mechanism and compares it with alternative mechanisms proposed in the literature. It is concluded that only the each-step-activation mechanism is able to explain the rich set of various experimental results used as a reference for estimating the validity and applicability of particular mechanisms.     

Regulation of the reaction rate of ATP synthesis in intact mitochondria

High concentrations of respiration inhibitors are known to sharply decrease the membrane potential in mitochondria. The effect of relatively low concentrations of oxidative phosphorylation inhibitors on the value of membrane potential of intact mitochondria and on the rate of respiration and phosphorylation as well was studied. It has been found that within a certain concentration range the inhibitors of oxidative phsophorylation--malonic acid, sodium cyanide m-chlorophenyl hydrozonecarbonylcyanide, sharply decrease the phosphorylation rate (by 70 divided by 90%) but do not practically a affect the membrane potential value of intact mitochondria in the state 3 according to Chance.     

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.     

Regulation of oxidative phosphorylation in mitochondria of epididymal bull spermatozoa

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.     

Protamine inhibition of the oxidative phosphorylation in intact, cytochrome c-depleted and restored mitochondria.

On the basis of polarographic data it is shown that protamine has a biphasic effect on the respiration of intact mitochondria. At lower protamine concentrations respiration is stimulated and this combined with a decrease of the respiratory control index; at higher ones respiration is inhibited and respiratory control is lost. In cytochrome c-depleted and restored mitochondria protamine effect on oxidative phosphorylation is only inhibitory. Increasing cytochrome c concentrations restore respiration in protamine-treated cytochrome c depleted mitochondria but not the respiratory control. Binding of cytochrome c to mitochondria is studied by determining from Scatchard plots the number of high affinity binding sites (n) and their stability constants (K). In absence of protamine in intact mitochondria n = 2.7 and K = 4.67-10(6) M-1; in cotochrome c depleted mitochondria n = 4.7 and K = 5.16-10(6) M-1. In both types of mitochondria protamine decreases significantly n as well as K. These data show that protamine may affect oxidative phosphorylation by causing desorption of cytochrome c from the inner mitochondrial membrane.     

Regulation of oxidative phosphorylation in the inner membrane of rat liver mitochondria by calcium ions

The effect of accumulation of Ca2+ at physiological concentrations (10(-8)-10(-6) M) on the rates of ATP synthesis and hydrolysis in rat liver mitochondria was studied. An addition of 5 x 10(-7) M Ca2+ resulted in the maximal rates of synthesis and hydrolysis of ATP. Decrease in the concentration of Ca2+ to 10-8 M or its increase to 5 x 10(-6) M inhibited oxidative phosphorylation and ATP hydrolysis. It was found that the rate of oxidative phosphorylation correlated with the phosphorylation level of a 3.5-kD peptide in the mitochondrial inner membrane on varying the Ca2+ concentration. The possible regulation of oxidative phosphorylation in mitochondria by Ca2+ is discussed.     

Regulation of oxidative phosphorylation in mitochondria by external free Ca2+ concentrations

The rate of oxidative phosphorylation was studied in rat liver mitochondria incubated with free Ca2+ concentrations that range from 10(-9) to 5 X 10(-6) M. The highest rate was observed between 0.5-1.0 microM Ca2+. ATP synthesis was measured by polarographic and spectrophotometric techniques and by uptake of radioactive inorganic phosphate. The concentration of Ca2+ at which maximal rates of ATP synthesis take place is modified by Mg2+ and phosphate. The dependence of oxidative phosphorylation on Ca2+ was observed with alpha-ketoglutarate, glutamate + malate, and succinate, but not with beta-hydroxybutyrate. At 10(-9) M Ca2+ there is a continuous exit of endogenous Ca2+, while with 10(-6) M Ca2+, intramitochondrial Ca2+ levels remained constant throughout time. Apparently the control of the level of internal Ca2+ by external Ca2+ modulates the rate of oxidative phosphorylation. Uncoupler-stimulated respiration also depends on Ca2+ concentration, even though at 10(-9) to 10(-6) M Ca2+ the rate of oxidative phosphorylation is lower than the rate of uncoupled respiration. The contribution of the ADP/ATP carrier and the ATP synthase to the kinetic regulation of ATP synthesis at 10(-9) and 10(-6) M Ca2+ was evaluated by titrations with carboxyatractyloside and oligomycin, respectively. The contribution of the carrier and the synthase to the regulation of the final rate of ATP synthesis was different at the two concentrations of Ca2+; therefore, the concentration of extramitochondrial Ca2+ influences the overall kinetics of oxidative phosphorylation.     

Regulation of oxidative phosphorylation as a possible method of normalizing cerebral metabolism

Experiments were performed to study the effect of chronic emotional painful stress on oxidative phosphorylation in different structures of rat brain at varying times of the development as well as after pretreatment with psychotropic agents. During the stage of excess catabolism, stress was demonstrated to dramatically inhibit and dissociate oxidative phosphorylation. This led to the impairment of macroerg synthesis and to the reduction of the brain macroerg content. Prophylactic administration of the derivatives of nicotinic acid and GABA markedly stimulated oxidative phosphorylation making it return to the initial level. Mebicar and meprobamate were less powerful. Chlorodiazepoxide aggravated stressful effects on tissue respiration and oxidative phosphorylation. It has been demonstrated that energy metabolism of the brain may return to normal at the expense of stimulation of oxidative phosphorylation.