Loop-L bulbar ventricular malformation.II. Surgical treatment

In vitro Mn2+ decreases respiration at metabolic state III and at the uncoupled state. Pretreatment with Mn2+ decreased also ADP to oxygen ratio in rat liver and brain mitochondria. The mechanism of manganese toxicity involving suppression of substrate oxidation and decrease of oxidative phosphorylation efficiency in brain mitochondria is discussed.     

Respiration and oxidative phosphorylation in the apicomplexan parasite Toxoplasma gondii

Respiration, oxidative phosphorylation, and the mitochondrial membrane potential (DeltaPsi) of tachyzoites of the apicomplexan parasite Toxoplasma gondii were assayed in situ using very low concentrations of digitonin to render their plasma membrane permeable to succinate, ADP, safranin O, and other small molecules. The rate of basal respiration was slightly increased by digitonin when the cells were incubated in medium containing succinate. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was oxidized by antimycin A-poisoned mitochondria. The addition of ADP after TMPD/ascorbate also resulted in phosphorylating respiration. The antitoxoplasmosis drug atovaquone, at a very low concentration (0.03 microM), totally inhibited respiration and disrupted the mitochondrial membrane potential. Atovaquone was shown to inhibit the respiratory chain of T. gondii and mammalian mitochondria between cytochrome b and c1 as occurs with antimycin A1. Phosphorylation of ADP could not be obtained in permeabilized tachyzoites in the presence of either pyruvate, 3-oxo-glutarate, glutamate, isocitrate, dihydroorotate, alpha-glycerophosphate, or endogenous substrates. Although ADP phosphorylation was detected in the presence of malate, this activity was rotenone-insensitive and was probably due to the conversion of malate into succinate through a fumarate reductase activity that was detected in mitochondrial extracts. Together these results provide the first direct biochemical evidence that the respiratory chain and oxidative phosphorylation are functional in apicomplexan parasites, although the terminal respiratory pathway is different from that in the mammalian host.     

Genetic-demographic characteristics of farming regions and small cities of the Tomsk district

The effect of the herbicide 4,6-dinitro-o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10-50 microM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium, in the presence of valinomycin. At higher concentrations (> 50 microM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca(2+)-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca(2+)-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.     

Changes in adenine nucleotide and mitochondrial metabolism of the kidney of burned rats and their relation to insulin

In rats with third-degree burns, the blood glucose level increased remarkably, with a concomitant suppression of insulin secretion from the pancreas after an oral glucose load. The energy charge (ATP + 1/2 ADP/ATP + ADP + AMP) levels of the kidney decreased to 0.659 as compared with 0.858 of controls at 8 hr after the burn (p less than 0.001). The phosphorylative activity of the kidney mitochondria fell to one third of controls at 8 hr after the burn (p less than 0.001), and that of heart mitochondria decreased to approximately 70% (p less than 0.005); the fall in liver and brain was less remarkable. The decrease in mitochondrial phosphorylative activity was accompanied by a reduction in the respiratory control ratio, P/O ratio, and state 3 respiration. The concentrations of cytochrome a(+a3) in the kidney mitochondria decreased to 69.9% of controls at 8 hr after the burn (p less than 0.001), those of cytochrome b to 82.6%, and those of cytochrome c + c1 to 75.3% (p less than 0.001). The decreased energy charge and oxidative phosphorylation of the kidney in burned rats were remarkably restored by subcutaneous administration of insulin. It is suggested that a reduction in insulin secretion from the pancreas may play an important role in initiating an impairment of adenine nucleotide and mitochondrial metabolism of the kidney.     

Linoleic acid-induced activity of plant uncoupling mitochondrial protein in purified tomato fruit mitochondria during resting, phosphorylating, and progressively uncoupled respiration

An uncoupling protein was recently discovered in plant mitochondria and demonstrated to function similarly to the uncoupling protein of brown adipose tissue. In this work, green tomato fruit mitochondria were purified on a self-generating Percoll gradient in the presence of 0.5% bovine serum albumin to deplete mitochondria of endogenous free fatty acids. The uncoupling protein activity was induced by the addition of linoleic acid during the resting state, and in the progressively uncoupled state, as well as during phosphorylating respiration in the presence of benzohydroxamic acid, an inhibitor of the alternative oxidase and with succinate (+ rotenone) as oxidizable substrate. Linoleic acid strongly stimulated the resting respiration in fatty acid-depleted mitochondria but had no effect on phosphorylating respiration, suggesting no activity of the uncoupling protein in this respiratory state. Progressive uncoupling of state 4 respiration decreased the stimulation by linoleic acid. The similar respiratory rates in phosphorylating and fully uncoupled respiration in the presence and absence of linoleic acid suggested that a rate-limiting step on the dehydrogenase side of the respiratory chain was responsible for the insensitivity of phosphorylating respiration to linoleic acid. Indeed, the ADP/O ratio determined by ADP/O pulse method was decreased by linoleic acid, indicating that uncoupling protein was active during phosphorylating respiration and was able to divert energy from oxidative phosphorylation. Moreover, the respiration rates appeared to be determined by membrane potential independently of the presence of linoleic acid, indicating that linoleic acid-induced stimulation of respiration is due to a pure protonophoric activity without any direct effect on the electron transport chain.     

Evaluation of birth cohort patterns in population disease rates

1. The effects of piroxicam, a nonsteroidal anti-inflammatory drug, on rat liver mitochondria were investigated in order to obtain direct evidence about a possible uncoupling effect, as suggested by a previous work with the perfused rat liver. 2. Piroxicam increased respiration in the absence of exogenous ADP and decreased respiration in the presence of exogenous ADP, the ADP/O ratios and the respiratory control ratios. 3. The ATPase activity of intact mitochondria was increased by piroxicam. With 2,4-dinitrophenol uncoupled mitochondria, inhibition was observed. The ATPase activity of freeze-thawing disrupted mitochondria was insensitive to piroxicam. 4. Swelling driven by the oxidation of several substrates and safranine uptake induced by succinate oxidation were inhibited. 5. The results of this work represent a direct evidence that piroxicam acts as an uncoupler, thus, decreasing mitochondrial ATP generation.     

Defects at center P underlie diabetes-associated mitochondrial dysfunction

Detailed respiration studies on isolated liver mitochondria from streptozotocin-induced diabetic Sprague-Dawley rats revealed a disease-associated decrease in the ADP/O ratio, a marker for mitochondrial ability to couple the consumption of oxygen to the phosphorylation of ADP. This decrease was observed following induction of respiration with glutamate/malate, succinate, or duroquinol, which enter the electron transport chain selectively at complexes I (NADH dehydrogenase), II (succinate dehydrogenase), or III (cytochrome bc1 complex), respectively. These data, coupled with studies using respiratory inhibitors (most importantly antimycin A and myxothiazol), localize at least a portion of this defect to a single site within the electron transport chain (center P in the Q-cycle portion of complex III). These results suggest that liver mitochondria from diabetic animals may generate increased levels of reactive oxygen species at the portion of the electron transport chain already established as the major site of mitochondrial free radical generation. The reduction in the ADP/O ratio occurred in mitochondria that do not have overt defects in the respiratory control ratio or in State 3 and State 4 respiration. The data in this paper suggest that defects in center P of the electron transport chain likely increase mitochondrial exposure to oxidants in the diabetic. This data may partially explain the evidence of altered exposure and/or response to reactive species in mitochondria from diabetics. This work thus provides further clues to the interaction between oxidative stress and diabetes-associated mitochondrial dysfunction.     

The dependence on the extramitochondrial ATP/ADP-ratio of the oxidative phosphorylation in mitochondria isolated by a new procedure from rat skeletal muscle

A simple method for preparation of rat skeletal muscle mitochondria is presented using gentle mechanical homogenization in a syringe and nagarse treatment (EC 3.4.4.16). This method enables the preparation of skeletal muscle mitochondria, whose outer membrane is intact to 95%. Furthermore, with mitochondria prepared by this method the regulation of respiration and phosphorylation by the extramitochondrial ATP/ADP-ratio can be demonstrated. In accordance to rat liver and heart mitochondria and to mitochondria of rabbit reticulocytes, the regulation by the extramitochondrial ATP/ADP-ratio lies in the range from 5 (corresponding to 98% of the maximum respiration) to 100 (corresponding to state 4). At extramitochondrial ATP/ADP-ratios from 0.01 to 1 the respiration rate is nearly constant (maximum rate of respiration).     

Expression of specific white adipose tissue genes in denervation-induced skeletal muscle fatty degeneration

To determine whether cationic uncouplers of oxidative phosphorylation induce permeability transition in mitochondria, the effects of the divalent cationic sulfhydryl cross-linker copper-o-phenanthroline (Cu(OP)2) and the cyanine dye tri-S-C4(5) on rat liver mitochondria were examined. Like Ca2+, they accelerated mitochondrial respiration with succinate and induced mitochondrial swelling when inorganic phosphate (Pi) was present in the incubation medium. The acceleration of respiration and swelling were inhibited by the SH-reagent N-ethylmaleimide, and by the specific permeability transition inhibitor cyclosporin A (CsA). In addition, these cations, like Ca2+, induced release of ADP entrapped in the mitochondrial matrix space, and the morphological change of mitochondria induced by these cations was essentially the same as that induced by Ca2+. It is concluded that the uncoupling actions of Cu(OP)2 and tri-S-C4(5) are due to induction of permeability transition in the inner mitochondrial membrane.     

Respiration and ion permeability of the inner membrane in rat "sodium" liver mitochondria

Ion permeability of internal membrane and a respiration in isolated rat liver mitochondria, further related to as "sodium ones", were studied following replacement of K+ ions for Na+ ones in the mitochondrial matrix. As compared with the control ("potassium mitochondria"), state 4 respiration in the sodium mitochondria, energized by succinate, was shown to be enhanced in KCl or sucrose media. Oxygen consumption rates in the sodium mitochondria, being in state 3 or stimulated by 2,4-dinitrophenol, were lower than rates for the control mitochondria. This effect was much pronounced in the sucrose medium. The coefficients, characterizing the distribution of 137Cs between mitochondria and the medium, were lower for the sodium mitochondria than for the control in the presence of 2.5 mM succinate and 10(-8) M valinomycin. In comparison with the control, a more extensive swelling for the sodium mitochondria was found, first, in the medium containing 25 mM K-acetate and 100 mM sucrose for succinate-energized mitochondria, and second, in the medium containing 125 mM NH4NO3 without mitochondrial energization. Changes disclosed in respiration, swelling and coefficients of 137Cs distribution for the sodium mitochondria are supposed to be caused by non-uniform effects of Na+ and K+ ions on the water structure of mitochondrial matrix, ion permeability of internal membrane, and the activity in oxidative phosphorylation enzymes.     

Features of noradrenaline stimulation of rat liver mitochondria respiration by ADP and calcium ions

A single administration of a physiological dose of noradrenaline to animals. in contrast to adrenaline, stimulates the respiration of mitochondria not only under oxidation of FAD-dependent Krebbs cycle substrate of the succinase but also HAD-dependent substrate of alpha-ketoglutarate. In the both cases the phosphorylation rate increases, since the action of noradrenaline, separating the respiration and oxidative phosphorylation, was not found. Noradrenaline increases the capacity of mitochondria to more actively absorb calcium ions under oxidation of succinate than under that of alpha-ketoglutarate.     

Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).     

Effect of superoxide in air on structural organization and phosphorylating respiration of mitochondria

The effect of negative air ions generated by a Tchijevsky lustre was studied in rat liver mitochondria in homogenate under conditions providing preservation of their native structural organization in the form of associations. Rapid formation of large associations from intermediate and small associations occurs after the homogenate was put into medium pretreated by negative air ions (NAI). Under the influence of a flow of NAI on mitochondria stored on ice for 2-3 h formation of giant associations is observed. Associations of mitochondria are not dissipated by stirring with a magnetic stirrer for several minutes. The influence of medium pretreated with NAI or flow of NAI on homogenate improves weakened respiratory energy control and restores stimulation of phosphorylating respiration by adrenalin administration. The improvement of phosphorylation in mitochondria by NAI is perhaps caused by more complete preservation of both their native structural organization and related physiological regulation of mitochondrial processes in isolated preparations.     

Oxidative phosphorylation in intact hepatocytes: quantitative characterization of the mechanisms of change in efficiency and cellular consequences

Two mechanisms may affect the yield of the oxidative phosphorylation pathway in isolated mitochondria: (i) a decrease in the intrinsic coupling of the proton pumps (H+/2e- or H+/ATP), and (ii) an increase in the inner membrane conductance (proton or cation leak). Hence three kinds of modifications can occur and each of them have been characterized in isolated rat liver mitochondria (see preceding chapter by Rigoulet et al.). In intact isolated hepatocytes, these modifications are linked to specific patterns of bioenergetic parameters, i.e. respiratory flux, mitochondrial redox potential, DY, and phosphate potential. (1) The increase in H+/ATP stoichiometry of the mitochondrial ATP synthase, as induced by almitrine [20], leads to a decrease in mitochondrial and cytosolic ATP/ADP ratios without any change in the protonmotive force nor in the respiratory rate or redox potential. (2) In comparison to carbohydrate, octanoate metabolism by beta-oxidation increases the proportion of electrons supplied at the second coupling site of the respiratory chain. This mimics a redox slipping. Octanoate addition results in an increased respiratory rate and mitochondrial NADH/NAD ratio while protonmotive force and phosphate potential are almost unaffected. The respiratory rate increase is associated with a decrease in the overall apparent thermodynamic driving force (2deltaE'o - ndeltap) which confirms the 'redox-slipping-like' effect. (3) An increase in proton conductance as induced by the protonophoric uncoupler 2,4-dinitrophenol (DNP) leads to a decrease, as expected, in the mitochondrial NADH/NAD and ATP/ ADP ratios and in deltapsi while respiratory rate is increased. Thus, each kind of modification (proton leak, respiratory chain redox slipping or increase in H+/ATP stoichiometry of ATPase) is related to a specific set of bioenergetic parameters in intact cells. Moreover, these patterns are in good agreement with the data found in isolated mitochondria. From this work, we conclude that quantitative analysis of four bioenergetic parameters (respiration rate, mitochondrial NADH/ NAD ratio, protonmotive force and mitochondrial phosphate potential) gives adequate tools to investigate the mechanism by which some alterations may affect the yield of the oxidative phosphorylation pathway in intact cells.     

Modulation of oxidative phosphorylation by Mg2+ in rat heart mitochondria

The effect of varying the Mg2+ concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg2+ concentration. Half-maximal stimulation (K0.5) of ATP synthesis by Mg2+ was obtained with 0.13 +/- 0.02 mM (n = 7) with succinate (+rotenone) and 0.48 +/- 0.13 mM (n = 6) with 2-oxoglutarate (2-OG) as substrates. Similar K0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in Pi concentration promoted a decrease in the K0.5 values of ATP synthesis, membrane potential formation and state 4 respiration for Mg2+ with 2-OG, but not with succinate. These results indicate that 2-OGDH is the main step of oxidative phosphorylation modulated by Mg2+ when 2-OG is the oxidizable substrate; with succinate, the ATP synthase is the Mg2+-sensitive step. Replacement of Pi by acetate, which promotes changes on intramitochondrial pH abolished Mg2+ activation of 2-OGDH. Thus, the modulation of the 2-OGDH activity by Mg2+ has an essential requirement for Pi (and ADP) in intact mitochondria which is not associated to variations in matrix pH.     

Mitochondrial effects of L-ropivacaine, a new local anesthetic

The effects of the local anesthetics ropivacaine and bupivacaine were investigated on isolated rat liver mitochondria. The efficiency of oxidative phosphorylation was evaluated by measuring the rates of respiration and ATP synthesis and the magnitude of the transmembrane electrical potential (deltapsi). Bupivacaine did not alter the ADP-stimulated respiration but strongly affected the resting respiration, which was more than doubled at 0.6 mM. In addition, it decreased the transmembrane electrical potential, and the ATP synthesis rate (deltapsi was less than 100 mV at 0.6 mM). Ropivacaine did not alter the ADP-stimulated respiration, and the resting respiration seemed to be substantially unaffected up to 1.2 mM; a slight increase was observed at 1.8 and 2.4 mM. The transmembrane potential was decreased by anesthetic concentrations higher than 1.2 mM and ATP synthesis was consequently affected. The findings suggest that ropivacaine is less toxic than bupivacaine, in rat liver mitochondria.     

The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase

We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibers. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 11 mM glucose as substrate, and supplemented with either 20 mM beta-guanidinopropionic acid (beta-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31 P-NMR, Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, beta-GPA-P, GA-P and GA-P over a similar time course (approximately 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK, Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K(m) and increased Vmax vs control), but none by GA, mGPA or beta-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.     

Mitochondria uncoupling by a long chain fatty acyl analogue

Mitochondria uncoupling by fatty acids in vivo is still questionable, being confounded by their dual role as substrates for oxidation and as putative genuine uncouplers of oxidative phosphorylation. To dissociate between substrate and the uncoupling activity of fatty acids in oxidative phosphorylation, the uncoupling effect was studied here using a nonmetabolizable long chain fatty acyl analogue. beta,beta'-Methyl-substituted hexadecane alpha,omega-dioic acid (MEDICA 16) is reported here to induce in freshly isolated liver cells a saturable oligomycin-insensitive decrease in mitochondrial proton motive force with a concomitant increase in cellular respiration. Similarly, MEDICA 16 induced a saturable decrease in membrane potential, proton gradient, and proton motive force in isolated liver and heart mitochondria accompanied by an increase in mitochondrial respiration. Uncoupling by MEDICA 16 in isolated mitochondria was partially suppressed by added atractyloside. Hence, fatty acids may act as genuine uncouplers of cellular oxidative phosphorylation by interacting with specific mitochondrial proteins, including the adenine nucleotide translocase.     

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside. 2. [35S]carboxyatractyloside and [3H]bongkrekic acid bind to potato mitochondria with high affinity (Kd = 10 to 20 nM, n=0.6-0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [3H]atractyloside binds to potato mitochondria with a much lower affinity (Kd=0.45 muM) than carboxyatractyloside or bongkrekic acid. 3. Bound [3H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [35S]carboxyatractyloside by bongkrekic acid and of bound [3H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP. 4. Bongkrekic acid competes with [35S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [35S]carboxyatractyloside binding. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.     

Urban-rural difference in cereal consumption by people in Shandong Province, China

The influence of the 1,4-dihydropyridines (DHPs), water-soluble glutapyrone available as sodium, potassium and ammonium salts of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP-4-carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2-propoxyethyl 2,6-dimethyl-4-(2-difluoromethoxyphenyl)-1,4-DHP-3,5-dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high-amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self-aggregation of mitochondria isolated in salt media after ATP-induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10-100 microM) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration-dependent manner (2.5-50 microM) increased (two-fold at 20-50 microM) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP- and succinate-induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP-induced contraction amplitude (two-fold at 0.5-5.0 mM), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre-exposure to 2.5 mM glutapyrone resulted in at least a 10-fold inhibition of the reversible aggregation rate in the presence of 99 and 198 microM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT-induced and some other mitochondrial myopathies.