Rate Of Mitochondrial Swelling Research Articles

Mitochondrial cerebral dysfunction in rats with scopolamine-induced neurodegeneration under enalapril effect

Objective. Neurodestructive diseases are characterized by complex pathobiochemical cascades in the neuron, which cause disturbances in energy metabolism and the formation of mitochondrial dysfunction. The renin-angiotensin system plays an important role in the physiological functioning of mitochondria, the excessive activity of which increases the risk of neurodegenerative diseases of the brain. Although angiotensin-converting enzyme inhibitors are now considered as means of prevention and treatment of ischemic lesions of the central nervous system, their corrective properties in the development of central neurodegeneration continue to be refined. The objective of our study was investigation of enalapril effect, as an angiotensin-converting enzyme inhibitor, in case of mitochondrial dysfunction of the cerebral cortex and hippocampus of rats under conditions of scopolamine-induced neurodegeneration reproducing development of Alzheimer’s disease in the experiment.Material and methods. Scopolamine hydrochloride (Sigma, USA) was injected in rats through the peritoneum at a dose of 1 mg/kg for 27 days to simulate Alzheimer’s disease. Starting from the 28th day of the experiment, enalapril was introduced through the peritoneum at a dose of 1 mg/kg, once a day for 14 days.
 Results. Under conditions of scopolamine-induced Alzheimer’s disease in the mitochondrial fraction of the cerebral cortex and hippocampus of rats free radical oxidation of lipids and proteins ncreases, and activity of Krebs cycle enzymes decreases – α-ketoglutarate dehydrogenase and succinate dehydrogenase; light dispersion decreases and a relative rate of mitochondrial swelling increases. After enalapril administration for 14 days to rats with scopolamine-induced Alzheimer’s disease the content of products reacting with 2-thiobarbituric acid and protein oxidation modification decreases in the mitochondrial fraction of the cerebral cortex and hippocampus; in both examined structures, the activity of catalase, αketoglutarate dehydrogenase, succinate dehydrogenase increases, and superoxide dismutase – only in the cerebral cortex; a gradual decrease of light dispersion and relative rate of mitochondrial swelling occurs.Conclusion. Improvement of the antioxidant system state and energy supply of mitochondria, decreased intensity of mitochondrial swelling in the cerebral cortex and hippocampus of rats with scopolamine-induced Alzheimer’s disease are indicative of the protective properties of enalapril.

Gradual cold acclimation induces cardioprotection without affecting β-adrenergic receptor-mediated adenylyl cyclase signaling.

Novel strategies are needed that can stimulate endogenous signaling pathways to protect the heart from myocardial infarction. The present study tested the hypothesis that appropriate regimen of cold acclimation (CA) may provide a promising approach for improving myocardial resistance to ischemia/reperfusion (I/R) injury without negative side effects. We evaluated myocardial I/R injury, mitochondrial swelling, and β-adrenergic receptor (β-AR)-adenylyl cyclase-mediated signaling. Male Wistar rats were exposed to CA (8°C, 8 h/day for a week, followed by 4 wk at 8°C for 24 h/day), while the recovery group (CAR) was kept at 24°C for an additional 2 wk. The myocardial infarction induced by coronary occlusion for 20 min followed by 3-h reperfusion was reduced from 56% in controls to 30% and 23% after CA and CAR, respectively. In line, the rate of mitochondrial swelling at 200 μM Ca2+ was decreased in both groups. Acute administration of metoprolol decreased infarction in control group and did not affect the CA-elicited cardiprotection. Accordingly, neither β1-AR-Gsα-adenylyl cyclase signaling, stimulated with specific ligands, nor p-PKA/PKA ratios were affected after CA or CAR. Importantly, Western blot and immunofluorescence analyses revealed β2- and β3-AR protein enrichment in membranes in both experimental groups. We conclude that gradual cold acclimation results in a persisting increase of myocardial resistance to I/R injury without hypertension and hypertrophy. The cardioprotective phenotype is associated with unaltered adenylyl cyclase signaling and increased mitochondrial resistance to Ca2+-overload. The potential role of upregulated β2/β3-AR pathways remains to be elucidated.NEW & NOTEWORTHY We present a new model of mild gradual cold acclimation increasing tolerance to myocardial ischemia/reperfusion injury without hypertension and hypertrophy. Cardioprotective phenotype is accompanied by unaltered adenylyl cyclase signaling and increased mitochondrial resistance to Ca2+-overload. The potential role of upregulated β2/β3-adrenoreceptor activation is considered. These findings may stimulate the development of novel preventive and therapeutic strategies against myocardial ischemia/reperfusion injury.

Developmental and sex differences in cardiac tolerance to ischemia-reperfusion injury: the role of mitochondria 1.

Age and sex play an essential role in the cardiac tolerance to ischemia-reperfusion injury: cardiac resistance significantly decreases during postnatal maturation and the female heart is more tolerant than the male myocardium. It is widely accepted that mitochondrial dysfunction, and particularly mitochondrial permeability transition pore (MPTP) opening, plays a major role in determining the extent of cardiac ischemia-reperfusion injury. We have observed that the MPTP sensitivity to the calcium load differs in mitochondria isolated from neonatal and adult myocardium, as well as from adult male and female hearts. Neonatal and female mitochondria are more resistant both in the extent and in the rate of mitochondrial swelling induced by high calcium concentration. Our data further suggest that age- and sex-dependent specificity of the MPTP is not the result of different amounts of ATP synthase and cyclophilin D: neonatal and adult hearts, similarly as the male and female hearts, contain comparable amounts of MPTP and its regulatory protein cyclophilin D. We can speculate that the lower sensitivity of MPTP to the calcium-induced swelling may be related to the higher ischemic tolerance of both neonatal and female myocardium.

Calcium-Induced Mitochondrial Permeability Transitions: Parameters of Ca2+ Ion Interactions with Mitochondria and Effects of Oxidative Agents.

We evaluated the parameters of Ca2+-induced mitochondrial permeability transition (MPT) pore formations, Ca2+ binding constants, stoichiometry, energy of activation, and the effect of oxidative agents, tert-butyl hydroperoxide (tBHP), and hypochlorous acid (HOCl), on Ca2+ -mediated process in rat liver mitochondria. From the Hill plot of the dependence of MPT rate on Ca2+ concentration, we determined the order of interaction of Ca2+ ions with the mitochondrial sites, n = 3, and the apparent Kd = 60 ± 12µM. We also found the apparent Michaelis-Menten constant, Km, for Ca2+ interactions with mitochondria to be equal to 75 ± 20µM, whereas that in the presence of 300µM tBHP was 120 ± 20µM. Using the Arrhenius plots of the temperature dependences of apparent mitochondrial swelling rate at various Ca2+ concentrations, we calculated the activation energy of the MPT process. ΔEa was 130 ± 20kJ/mol at temperatures below the break point of the Arrhenius plot (30-34 °C) and 50 ± 9kJ/mol at higher temperatures. Ca2+ ions induced rapid mitochondrial NADH depletion and membrane depolarization. Prevention of the pore formation by cyclosporin A inhibited Ca2+-dependent mitochondrial depolarization and Mg2+ ions attenuated the potential dissipation. tBHP (10-150µM) dose-dependently enhanced the rate of MPT opening, whereas the effect of HOCl on MPT depended on the ratio of HOCl/Ca2+. The apparent Km of tBHP interaction with mitochondria in the swelling reaction was found to be Km = 11 ± 3µM. The present study provides evidence that three calcium ions interact with mitochondrial site with high affinity during MPT. Ca2+-induced MPT pore formations due to mitochondrial membrane protein denaturation resulted in membrane potential dissipation. Oxidants with different mechanisms, tBHP and HOCl, reduced mitochondrial membrane potential and oxidized mitochondrial NADH in EDTA-free medium and had an effect on Ca2+-induced MPT onset.

The difference in myocardial injuries and mitochondrial damages between asphyxial and ventricular fibrillation cardiac arrests

IntroductionVentricular fibrillation (VF) and asphyxia account for most cardiac arrests but differ in cardiac arrest course, neurologic deficit, and myocardial damage. In VF resuscitation, cardiac mitochondria were known to be damaged via excess generation of reactive oxygen species. This study evaluated the difference of cardiac mitochondrial damages between VF and asphyxial cardiac arrests. MethodsIn the VF + electrical shock (ES) group, VF was induced and untreated for 5 minutes, followed by 1 minute of cardiopulmonary resuscitation (CPR) and 1 ES of 5 J. Animals were killed immediately after ES. In the asphyxia group, cardiac arrest was induced by airway obstruction, and then pulselessness was maintained for 5 minutes, followed by 1 minute of CPR. The animals were killed immediately after CPR. The histology and ultrastructural changes of myocardium and complex activities and respiration of mitochondria were evaluated. The mitochondrial permeability transition pore opening was measured based on mitochondrial swelling rate. ResultsThe histopathologic examinations showed myocardial necrosis and mitochondrial damage in both cardiac arrests. Instead of regional damages of myocardium in the VF + ES group, the myocardial injury in the asphyxia group distributed diffusely. The asphyxia group demonstrated more severe mitochondrial damage than the VF + ES group, which had a faster mitochondrial swelling rate, more decreased cytochrome c oxidase activity, and more impaired respiration. ConclusionsBoth VF and asphyxial cardiac arrests caused myocardial injuries and mitochondrial damages. Asphyxial cardiac arrest presented more diffuse myocardial injuries and more severe mitochondrial damages than VF cardiac arrest.

Ascorbic acid mitigates the myocardial injury after cardiac arrest and electrical shock

To examine the effects of ascorbic acid (AA) administrated during cardiopulmonary resuscitation (CPR) on the myocardial injury in a rat model of ventricular fibrillation (VF) and electrical shock (ES). VF was induced in male Wistar rats and left untreated for 5min, followed by 1min of CPR, and then one ES of 5J. At the start of CPR, animals received either intravenous administration of AA (100mg/kg) or Tempol (30mg/kg), two antioxidants, or 0.9% saline (VF+ES group). After ES, animals were immediately killed. Myocardial lipoxidation was determined by malondialdehyde (MDA) assay. The histology and ultrastructural changes of myocardium were also evaluated. The mitochondrial permeability transition pore (mPTP) opening was measured based on the mitochondrial swelling rate. The complex activities and respiration of mitochondria were assessed, too. Increased myocardial injury and mitochondrial damage in the VF+ES group were noted. AA and Tempol alleviated such damages. Both AA and Tempol improved accelerated mitochondrial swelling; decreased complex activities and respiratory dysfunction occurred in the VF+ES group. The animals receiving AA and Tempol during CPR had better successful resuscitation rates and 72-h survival than the VF+ES group. Intravenous administration of AA and Tempol at the start of CPR may reduce lipid peroxidation and myocardial necrosis, diminish mitochondrial damage, facilitate resuscitation, and improve outcomes after VF+ES.

Protective Effects of Baicalin against Ischemia/Reperfusion Injury in Rat Liver

The cytoprotective properties of baicalin (1), a flavonoid glycoside isolated from Scutellaria baicalensis, have been investigated against injury to the liver caused by ischemia/reperfusion (I/R). Rats were subjected to 60 min of ischemia followed by 5 h of reperfusion, and 1 was administered intraperitoneally 24 and 1 h before ischemia. Following I/R, the levels of serum alanine aminotransferase and hepatic lipid peroxidation were elevated, whereas the hepatic glutathione content was decreased, with these changes attenuated by 1. Serum levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were markedly increased by I/R, but suppressed by 1. Baicalin attenuated increases in inducible nitric oxide synthase, cyclooxygenase (COX)-2, and TNF receptor 1-associated protein expression and augmented an increase in heme oxygenase-1 (HO-1). The increase in TNF-α, IL-6, and, COX-2 mRNA expression was attenuated by 1, while the increase in HO-1 mRNA expression was augmented. Nuclear factor-κB nuclear localization was inhibited by 1, and this compound limited the rate of mitochondrial swelling and the activation of caspases-3 and -8 observed in I/R rats. Rats treated with 1 had markedly fewer apoptotic cells than I/R rats. It was concluded that baicalin (1) exhibits antioxidant, anti-inflammatory, and antiapoptotic effects, which protect against hepatocellular I/R-induced damage.

Membrane Superficial Charge Modification Affects Miotochondrial Permeabilization by Derivatives of the Polycationic Peptide Btm-P1

Polycationic peptides demonstrate antimicrobial and anticancer properties. Earlier we designed, on the basis of the protoxin Cry11Bb, a 26-aa polycationic peptide BTM-P1, which demonstrated ionophoric and antimicrobial activities. It could be modified in the future to enhance anticancer action. In this work we found that the reverse peptide, BTM-RP1, has one order of magnitude lower capacity than BTM-P1 to permeabilize rat liver mitochondria. The activity of BTM-RP1 was increased by its modifications with tryptophane attached to its N-terminal (BTM-WRP1) or C-terminal (BTM-RP1W). The similar modifications of BTM-P1 peptide did not increase, or even decreased (BTM-P1W) the peptide activity. All these peptides, designed by us, were synthesized by Gen Script Company (USA) (>90% purity). When 10 μM cationic fluorescent probe safranin O, but not endogenous NAD(P)H fluorescence, was used as indicator of mitochondrial energization, the inner membrane potential markedly recovered after a decrease caused by each of 3 serial additions of 1 μM BTM-RP1. We also found that safranin O significantly decreased the rate of mitochondrial swelling induced by BTM-RP1 or by its tryptophane derivates. These data suggest that the superficial electrical charge of biomembranes, in addition to the trans-membrane potential, significantly affects the membrane permeabilization and selectivity in cell killing by polycationic peptides. We conclude that agents modifying superficial electrical charge of biological membranes could be used to influence the peptide cytotoxicity and selectivity. (Colciencias grant #111840820380 and the National University of Colombia grant #20101007930).

Influence of cholesterol on the formation of palmitate/Ca2+-activated pores in mitochondria and liposomes

The influenc of cholesterol on the formation of the mitochondrial cyclosporin A (CsA)-insensitive palmitate/Ca(2+)-activated pore has been studied. It has been established that increasing the cholesterol level in mitochondrial membranes results in an increase in the of rate of mitochondrial swelling induced by palmitic acid (> or = 20 microM) and Ca2+ (30 microM). This effect is not related to changes in the functional activity of organelles since cholesterol did not influence the mitochondrial respiration in different metabolic states. At the same time, it was shown that the palmitate/Ca(2+)-induced permeabilization of cholesterol-containing azolectin liposomes was Stronger than that of azolectin liposomes. It was found that, in the liposomal membrane, the Ca(2+)-induced phase separation of palmitic acid into distinct membrane domains takes place. The presence of cholesterol in membranes increases the extent of segregation.

Cytoprotective effects of melatonin against necrosis and apoptosis induced by ischemia/reperfusion injury in rat liver

Melatonin protects against organ ischemia; this effect has mainly been attributed to the antioxidant properties of the indoleamine. This study examined the cytoprotective properties of melatonin against injury to the liver caused by ischemia/reperfusion (I/R). Rats were subjected to 60 min of ischemia followed by 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. The serum aminotransferase activity and lipid peroxidation levels were increased markedly by hepatic I/R, which were suppressed significantly by melatonin. In contrast, the glutathione content, which is an index of the cellular redox state, and mitochondrial glutamate dehydrogenase activity, which is a maker of the mitochondrial membrane integrity, were lower in the I/R rats. These decreases were attenuated by melatonin. The rate of mitochondrial swelling, which reflects the extent of the mitochondrial permeability transition, was higher after 5 hr of reperfusion but was attenuated by melatonin. Melatonin limited the release of cytochrome c into the cytosol and the activation of caspase-3 observed in the I/R rats. The melatonin-treated rats showed markedly fewer apoptotic (TUNEL positive) cells and DNA fragmentation than did the I/R rats. These results suggest that melatonin ameliorates I/R-induced hepatocytes damage by inhibiting the level of oxidative stress and the apoptotic pathway. Consequently, melatonin may provide a new pharmacological intervention strategy for hepatic I/R injuries.

Derivatives of 1,4-dihydropyridines as modulators of ascorbate-induced lipid peroxidation and high-amplitude swelling of mitochondria, caused by ascorbate, sodium linoleate and sodium pyrophosphate

A group of 26 2,6-dimethyl-3,5-disubstituted and 2,6-dimethyl-3,4, 5-trisubstituted-1,4-dihydropyridines (1,4-H(2)Py=1,4-DHPs) and five related pyridines were studied as inhibitors of rat liver mitochondrial swelling and O(2) uptake by ascorbic acid-dependent lipid peroxidation (LP) and as modulators of mitochondrial swelling induced by Na(+)-linoleate or Na(+)-pyrophosphate. 1,4-DHPs studied include 4-unsubstituted and 4-methyl- and 4-phenyl-substituted 3, 5-dialkoxycarbonylderivatives of 2,6-dimethyl-1,4-DHP with variations in alkoxy chain length and composition, 4-unsubstituted and 4-methyl-, 4-aryl- and 4-pyridyl-substituted 3, 5-dianilidocarbonylderivatives, and a structurally related group of 3,5-dipyridylamidocarbonylderivatives. Many 1,4-DHPs possess marked antioxidant (AO) and membrane stabilizing activity, expressed as the mitochondrial swelling (deltaA(520)/t) and/or O(2) uptake rate decrease (V(0)/V) as well as prolongation of the induction period (tau/tau(0)) of mitochondrial swelling and/or O(2) uptake at ascorbic acid-dependent LP of rat liver mitochondria. 4-Unsubstituted 3,5-dialkoxycarbonyl-2,6-dimethyl-1,4-DHPs, as well as 4-unsubstituted or those possessing lipophylic 4-aryl- groups 3, 5-diamido-2,6-dimethyl-1,4-DHPs, reveal marked AO and membrane stabilizing properties. Oxidized (heteroaromatized) derivatives have minimal activity. Perhaps 1,4-DHPs preferably act as antioxidants on stages of initiation and prolongation of LP chain reactions at low concentrations: IC(50) (when V(0)/V or tau/tau(0)=2) are 0.1 microM to 100 microM. At 100 microM 3,5-di-p-hydroxyphenoxycarbonyl- and 3, 5-di-p-tolyloxycarbonyl-2,6-dimethyl-1,4-DHPs, as well as 3, 5-diethoxycarbonyl-2,6-dimethylpyridine (oxidized form of Hantzsch ester) and 3,5-diamyloxycarbonyl-2,6-dimethylpyridine, alter the mitochondrial swelling rate in the presence of natural protonophore Na(+)-linoleate (0.063 mM and 0.125 mM). 3,5-Di-n-butyloxycarbonyl-2, 6-dimethyl-1,4-DHP at 100 microM completely stops mitochondrial swelling in the presence of 0.8 mM Na(+)-pyrophosphate. In the presence of many of the 1,4-DHPs, the lipid peroxidation process was inhibited. However, the swelling process could be prolonged, promoted, accelerated or inhibited-depending on 1,4-DHPs structure, concentration, the type of initiators of the swelling process and the medium composition.

Nature of the masking of nucleotide-binding sites in brown adipose tissue mitochondria. Involvement of endogenous adenosine triphosphate.

Binding of the fluorescent nucleotide derivative 2'-O-dansyl GTP and purine nucleotides to brown adipose tissue mitochondria from hamster was studied. 2'-O-Dansyl GTP binds with enhanced fluorescence to the uncoupling protein (UCP) in the mitochondria, similar to the isolated protein. The fluorescence signal showed biphasic fast and slow increases. Treatment of the mitochondria with an anion exchanger (Dowex) increased the total fluorescence but decreased the slower phase. The biphasic fluorescence response was restored by incubation with only 1 microM ATP, indicating that residual bound ATP may be responsible for the observed slow phase. The binding of [14C]GTP and GDP also increased after Dowex treatment. The dissociation of bound [14C]ATP but not of bound [14C]ADP was slow and apparently limited the binding assays. Short incubation (5 min) resulted in a curvature of the Scatchard plot, where the 'high-affinity sites' correspond to the free UCP sites; GDP had apparently higher affinity than GTP. Dowex treatment and incubation for 60 min produced a more linear Scatchard plot. Under such conditions, one measures the maximal UCP-binding sites (1.2 mumol/g protein); GTP exhibited higher affinity (Kd = 0.64 microM) than GDP (Kd = 3.1 microM). Acute cold adaptation (40 min at 4 degrees C) of hamsters caused an increase by over 40% of [14C]GTP binding, as compared to the control warm-(28 degrees C)-adapted animals. Dowex treatment completely abolishes this unmasking/masking effect, where both mitochondria had identical binding capacity and affinity for GTP. The inhibition by purine nucleotides of H+ transport as measured by potassium-acetate-induced mitochondrial swelling was dependent on the incubation time. Diphosphates inhibited faster and triphosphates required longer incubation (10 min) but inhibited more strongly. A linear correlation between the mitochondrial swelling rate and GDP binding was observed for mitochondria with depleted endogenous ATP or with added ATP. These data indicate that residual bound ATP from the tissue is responsible for the masking phenomenon.

Nature of the Masking of Nucleotide‐Binding Sites in Brown Adipose Tissue Mitochondria

Binding of the fluorescent nucleotide derivative 2′‐O ‐dansyl GTP and purine nucleotides to brown adipose tissue mitochondria from hamster was studied. 2′‐O ‐Dansyl GTP binds with enhanced fluorescence to the uncoupling protein (UCP) in the mitochondria, similar to the isolated protein. The fluorescence signal showed biphasic fast and slow increases. Treatment of the mitochondria with an anion exchanger (Dowex) increased the total fluorescence but decreased the slower phase. The biphasic fluorescence response was restored by incubation with only 1 μM ATP, indicating that residual bound ATP may be responsible for the observed slow phase. The binding of [14C]GTP and GDP also increased after Dowex treatment. The dissociation of bound [14C]ATP but not of bound [14C]ADP was slow and apparently limited the binding assays. Short incubation (5 min) resulted in a curvature of the Scatchard plot, where the ‘high‐affinity sites’ correspond to the free UCP sites; GDP had apparently higher affinity than GTP. Dowex treatment and incubation for 60 min produced a more linear Scatchard plot. Under such conditions, one measures the maximal UCP‐binding sites (1.2 μmol/g protein); GTP exhibited higher affinity (Kd= 0.64 μM) than GDP (Kd= 3.1 μM). Acute cold adaptation (40 min at 4°C) of hamsters caused an increase by over 40% of [14C]GTP binding, as compared to the control warm‐(28°C)‐adapted animals. Dowex treatment completely abolishes this unmasking/masking effect, where both mitochondria had identical binding capacity and affinity for GTP. The inhibition by purine nucleotides of H+ transport as measured by potassium‐acetate‐induced mitochondrial swelling was dependent on the incubation time. Diphosphates inhibited faster and triphosphates required longer incubation (10 min) but inhibited more strongly. A linear correlation between the mitochondrial swelling rate and GDP binding was observed for mitochondria with depleted endogenous ATP or with added ATP. These data indicate that residual bound ATP from the tissue is responsible for the masking phenomenon.

Undesirable feature of safranine as a probe for mitochondrial membrane potential

This communication describes experiments showing that safranine, at the concentrations usually employed as a probe of mitochondrial membrane potential, causes significant undesirable side effects on Ca 2+ transport by liver mitochondria. The major observations are: (i) safranine potentiates the spontaneous Ca 2+ release from liver mitochondria induced by phosphate or acetoacetate. This is paralelled by potentiation of the release of state-4 respiration and of the rate of mitochondrial swelling, indicating a generalized effect of the dye on the mitochondrial membrane; (ii) the efflux of mitochondrial Ca 2+ stimulated by hydroperoxide is irreversible in the presence of safranine even if membrane stabilizers such as Mg 2+ and ATP are present. It is concluded that the use of safranine to monitor the changes in membrane potential during Ca 2+ transport by mitochondria should be avoided or special care be taken.

Permeability Properties of the Inner Membrane of Mung Bean Mitochondria and Changes during Energization

The permeability properties of the inner membrane of mung bean mitochondria were studied by osmotic swelling techniques. Rapid mitochondrial swelling was observed in isotonic ammonium phosphate, which indicated that an active phosphate/hydroxyl antiporter was present. The phosphate carrier was specifically inhibited by sulfhydryl reagents. Mitochondria did not swell in isotonic ammonium salts of malate, succinate, or fumarate, either in the presence or absence of 10 millimolar phosphate. Additionally, no swelling was observed in ammonium citrate upon addition of malate plus phosphate. Consequently, no evidence was obtained with the osmotic swelling technique for a coupled exchange of phosphate for dicarboxylic acids across the membrane.On the basis of valinomycin-induced swelling in potassium salts, suggestions were obtained that chloride, sulfate, and phosphate anions permeated more rapidly than acetate anions. Sulfhydryl reagents increased the rate of valinomycin-induced swelling in potassium phosphate, but had no effect on swelling in potassium acetate.Tributyltin induced a low rate of mitochondrial swelling in KCl in the absence of substrates, which indicated the presence of a low activity (Na(+)) K(+)/H(+) antiporter in the membrane. In the presence of NADH, rapid swelling, followed by a contraction, was observed upon addition of tributyltin. Swelling was insensitive to uncouplers, whereas contraction was uncoupler-sensitive. O(2) uptake (state 4) was greatest (3- to 4-fold stimulation) during the contraction phase, which indicated that the observed contraction was coupled to the pH gradient formed during electron transport. The results suggested that energization increased the activity of the (Na(+)) K(+)/H(+) antiporter in the inner mitochondrial membrane.

Inhibition by local anaesthetics of anion transport in isolated rat heart mitochondria

(1) (1)The effects of nupercaine and other local anaesthetics on anion transporters in isolated rat heart mitochondria were investigated at temperatures of 25° or 30°. The concentrations of nupercaine which inhibited by 50 per cent the rates of pyruvate-stimulated oxygen utilisation in (a) the presence and (b) the absence of α-cyano-3-hydroxycinnamate, (c) ADP-stimulated oxygen utilisation in the presence of atractyloside, (d) succinate-stimulated oxygen utilisation in the presence of phenylsuccinate (e) phosphate-induced swelling were (a) 0.02, (b) 0.4, (c) 0.3, (d) 1.1 and (e) 0.6 mM, respectively. (2) (2)Low concentrations of nupercaine (10–50 μM) increased by about 10 per cent the rates of ADP- and succinate-stimulated oxygen utilisation measured at 30° in the presence of atractyloside and phenylsuccinate, respectively. (3) (3)Inhibition by nupercaine of pyruvate-stimulated oxygen utilisation in the presence of α-cyano-3-hydroxycinnamate was found to be non-competitive with respect to pyruvate. (4) (4)Butacaine and tetracaine inhibited pyruvate-stimulated oxygen utilisation, but were less effective than nupercaine. Procaine and xylocaine had little effect. (5) (5)[2- 14C]Pyruvate uptake was inhibited by nupercaine with 50% inhibition given by 0.2 and 0.6 mM nupercaine at 25° and 4° respectively. (6) (6)The concentrations of nupercaine which inhibited pyruvate-stimulated oxygen utilisation in the presence of 2-cyano-3-hydroxycinnamate had no effect on the rate of mitochondrial swelling or the acceptor control ratio of the mitochondria. (7) (7)It is concluded that the pyruvate transporter is markedly more susceptible to the inhibitory effects of nupercaine than are the adenine nucleotide. dicarboxylate or phosphate ion transporters.

Pyruvate Oxidation and the Permeability of Mitochondria from Blowfly Flight Muscle

The rate of pyruvate oxidation by mitochondria from blowfly flight muscle decreased in the presence of tris (hydroxymethyl) aminomethane (Tris). An increase in the rate of mitochondrial swelling was concomitant with the loss of pyruvate oxidation. These changes were prevented by bovine serum albumin, adenosine triphosphate, and magnesium ions, factors required for mitochondrial contraction. Proline, but not glutamate or malate, restored the rate of pyruvate oxidation to original values. These findings suggest that mitochondrial swelling leads to leakage of intramitochondrial intermediates of the Krebs cycle, accounting for the decrease in the rate of pyruvate oxidation. Exogenous proline penetrates the mitochondrial membrane and is rapidly oxidized, via glutamate, forming intramitochondrial precursors of oxaloacetate. Malate and glutamate were ineffective because of the selective permeability of the mitochondrial membrane.

The effects of neuromuscular blocking agents on mitochondria—II: Effects of d-tubocurarine, pyrrolizidine alkaloids and of complex ions on swelling of mitochondria

The alkaloids, d-tubocurarine, lasiocarpine and heliotrine have been found to inhibit the swelling of isolated rat liver mitochondria in buffered sucrose solutions both in the presence or absence of added substrate. Inhibition of mitochondrial swelling indicated that the increased mitochondrial permeability to NAD caused by these alkaloids was due to a specific permeability change, and not to a general increase in mitochondrial permeability. N-Oxides of the pyrrolizidine alkaloids, lasiocarpine and heliotrine, did not influence the rate of mitochondrial swelling, indicating that the inhibitory effect of the alkaloids on swelling, like their effect on mitochondrial permeability to NAD, NAD status and metabolism, is associated with the positive charge on the ring nitrogen atom. Iron and ruthenium stable complexes affected the swelling of mitochondria in sucrose solutions differently from the alkaloids. Iron complexes were found to accelerate swelling, and ruthenium complexes initially accelerated, then inhibited swelling. Acceleration of swelling by the complexes was largely prevented by the alkaloids or by ATP, but only to a small extent by sodium amytal. Spontaneous swelling of mitochondria in KCl solutions was not influenced by the alkaloids or by the complexes. During the experiments the mitochondrial suspensions were examined microscopically by phase-contrast to interpret correctly the observed optical density changes. The results of these examinations are given and their significance discussed.

The influence of thyroid hormones on normal rat-liver mitochondria II. Thyroxine effects on pretreated mitochondria

1. 1. Dilution of a sucrose suspension of mitochondria with an equal volume of deionized H 2O lowered the respiratory control and permitted a number of thyroxine- induced effects to be observed. 2. 2. The most immediate effect of thyroxine was an increase in the rate of succinate oxidation. This was followed at about 1 min by a further drop in respiratory control and at 2 min by an increase in adenosine-5′-triphosphatase activity. These effects all contributed to a reduction in phosphorylation efficiency in the presence of thyroxine. 3. 3. Although thyroxine increased the rate of mitochondrial swelling, beginning at approx. 2 min after the start of the incubation, this effect was clearly not associated with any of the other thyroxine effects except possibly the stimulation of adenosine-5′-triphosphatase activity. 4. 4. The results support other work in which thyroxine stimulates succinate oxidation under conditions in which it is not limited by the phosphorylative capacity of the mitochondria.

An effect of ethylene on swelling of isolated mitochondria

Mitochondria from rat livers and cauliflower buds were used as model systems to determine if ehtylene treatments applied in vitro could induce changes in properties of lipoprotein membranes leading to increased permeability. The rate of mitochondrial swelling under different treatments was followed by spectrophotometric measurements of changes in the scattering of light by mitochondrial suspensions. Ethylene treatments applied in buffered test solutions of KCl increased the rate of swelling; no increase was observed in sucrose solutions. Calcium ions and carbon tetrachloride caused more rapid swelling than did ethylene. Apparently, ethylene can induce a limited change in membrane permeability. If the mitochondrial membrane is a factor in the regulation of respiration in plant cells, the respiratory response induced in plants by ethylene may be the result of increased membrane permeability.