Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture

The effects of vitamin K on the morphology and the growth of mouse neuroblastoma (P₂), mouse melanoma (B-16) and rat glioma (C-6) cells in culture were studied. Vitamin K₃ inhibited the growth (due to cell death and partial or complete inhibition of cell division) of all three cell types without causing any morphological differentiation. Vitamin K₃ was more effective than vitamin K₁. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma or glioma cells. Glioma cells did not grow in hormone-supplemented serum-free medium; however, both neuroblastoma and melanoma cells grew to a level 70–80% of that found in serum-supplemented medium. Neuroblastoma cells and melanoma cells cultured in serum-free medium exhibited a 2–3 fold higher sensitivity to vitamin K₃ than those cultured in serum-supplemented medium. This suggests that serum factors attenuate the growth inhibitory effect of vitamin K₃ on tumor cells in culture, probably by reducing the availability of this vitamin to the cells. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma cells even when they were treated in serum-free medium. The fact that micromolar concentrations of vitamin K₃ inhibit the growth of tumor cells in culture suggests that this vitamin may be a potentially useful anticancer agent.     

KINETIC STUDY OF GLUTAMATE TRANSPORT IN RAT BRAIN MITOCHONDRIA

Abstract— The experiments reported here confirm that glutamate can penetrate the inner membrane of isolated rat brain non-synaptosomal mitochondria, either on a glutamate-hydroxyl antiporter or on a glutamate-aspartate antiporter. An inhibition of respiratory activity of mitochondria with glutamate as substrate was obtained in the presence of avenaciolide or N-ethylmaleimide. Swelling of the mitochondria in iso-osmotic NH4⁺-l -glutamate was inhibited in the presence of avenaciolide and N-ethylmaleimide, but mersalyl, kainic acid, glisoxepide and amino-oxyacetic acid had no effect on the glutamate-hydroxyl exchange. Glutamate induced the reduction of intramitochondrial NAD(P), as estimated by double-beam spectrophotometry, and this reduction was inhibited on the one hand by N-ethylmaleimide, avenaciolide or fuscine, on the other hand by aminooxyacetic acid. A direct estimation of the penetration of l -[¹⁴C]glutamate into brain mitochondria was performed by using the centrifugation-stop procedure. This penetration followed saturation kinetics, with a mean apparent K_m of 1.56 M^M at pH 7.4 and at 20°C, the value of Knax was 4.34 nmol per min per mg protein in the same conditions. IV-Ethylmaleimide slowed down the initial rate of glutamate penetration, and this inhibition appeared to be non-competitive with a K_i of 0.7 Mm -at pH 7.4 and at 20°C. The entry of glutamate was pH-dependent and it increased 2-fold in the pH range of 7.4 to 6.4. A temperature-dependence of glutamate transport was also shown between 2 and 25°C; the Arrhenius plot was a straight line, with a calculated E_A of 12.8 kCal per mol of glutamate and a Q₁₀ of 2.16. The activity of γ-glutamyl transpeptidase was practically absent in these rat brain mitochondria. Oxidation of extramitochondrial NADH by the‘malate-aspartate shuttle’reconstituted in vitro was followed in rat brain non-synaptosomal mitochondria. In the absence of extramitochondrial malate or glutamate the ‘shuttle’ did not function, and in the absence of extramitochondrial aspartate the rate of NADH oxidation was low. Glutamine or γ-aminobutyrate did not replace glutamate efficiently. A high inhibition of the‘malate-aspartate shuttle’occurred in the presence of avenaciolide or mersalyl, and a moderate one in the presence of n-ethylmaleimide, glisoxepide or n-butylmalonate. Glutaminase activity in intact brain mitochondria was inhibited in the presence of extramitochondrial glutamate.     

Ba2+ uptake and the inhibition by Ba2+ of K+ flux into rat liver mitochondria

Summary Rapid uptake of Ba²⁺ by respiring rat liver mitochondria is accompanied by a transient stimulation of respiration. Following accumulation of Ba²⁺, e.g. at a concentration of 120 nmol per mg protein, the mitochondria exhibit reduced rates of state 3 and uncoupler-stimulated respiration. ADP-stimulated respiration is inhibited at a lower concentration of Ba²⁺ than is required to affect uncoupler-stimulated respiration, suggesting a distinct effect of Ba²⁺ on mechanisms involved in synthesis of ATP. Ba²⁺, which has an ionic radius similar to that of K⁺, inhibits unidirectional K⁺ flux into respiring rat liver mitochondria. This effect on K⁺ influx is observable at concentrations of Ba²⁺, e.g. 23 to 37 nmol per mg protein, which cause no significant change in state 4 or uncoupler-stimulated respiration. The accumulated Ba²⁺ decreases the measuredV _max of K⁺ influx, while having little effect on the apparentK _m for K⁺. The inhibition of K⁺ influx by Ba²⁺ is seen in the presence and absence of mersalyl, an activator of K⁺ influx. In contrast, under the conditions studied, Ba²⁺ has no apparent effet on the rate of unidirectional K⁺ efflux. These data are consistent with the idea that K⁺ may enter and leave mitochondria via spearate mechanisms.     

Oxidative Metabolism of 4-Aminobutyrate by Rat Brain Mitochondria: Inhibition by Branched-chain Fatty Acid

Abstract: The oxidation of 4-aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched-chain fatty acids 2-methyl-2-ethyl caproate (MEC) and 2, 2-dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by O₂ uptake, was determined in medium containing either 5 or 100 mM-[K⁺]. The apparent K_m for GABA was 1.16 ± 0.19 mM and the V_max in state 3 was 23.8 ± 5.5 ng-atoms O₂. min^?1. mg protein^?1 in 5 mM-[K⁺]. In a medium with 100 mM-[K⁺] the apparent K_m was 1.11 ± 0.17 mM and V_max was 47.4 ± 5.7 ng-atoms O₂. min^?1. mg protein^?1. The K_m for MEC was determined to be 0.58 ± 0.24 or 0.32 ± 0.08 mM, in 5 or 100 mM-[K⁺], respectively. For DMV, the K_i was 0.28 ± 0.05 or 0.34 ± 0.06 mM, in 5 or 100 mM-[K⁺] medium, respectively. The O₂ uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate; the ratio of oxygen uptake to the rate of amino acid formation was close to the theoretical value of 3. Neither the [K²] nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The V_max for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched-chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondria.     

Adenine nucleotide translocase as a site of regulation by ADP of the rat liver mitochondria permeability to H+ and K+ lons

In the presence of oligomycin ADP inhibits the osmotic swelling of the nonenergized rat liver mitochondria in the NH₄NO₃ medium. With the energized mitochondria ADP enhances contraction of the mitochondria swollen in the NH₄NO₃ medium. Carboxyatractyloside and atractyloside abolish or prevent the effects of ADP. The direct measurements of the proton conductance of rat liver mitochondria shows that the inhibitory action of ADP + oligomycin on the H⁺ permeability does not depend on the energization of mitochondria. In these experiments the local anesthetic nupercaine and ADP additively inhibit the inner membrane conductance for protons, but carboxyatractyloside abolishes only the effect of ADP. In the presence of oligomycin ADP also inhibits the osmotic swelling of the nonenergized liver mitochondria in the KNO₃ medium, and the energy-dependent swelling of rat liver mitochondria in the medium with K⁺ ions and P_i. The inhibition by ADP of the membrane passive permeability for K⁺ is also sensitive to carboxyatractyloside. It is concluded that rat liver mitochondria possess an ADP-regulated channel for H⁺ and K⁺. The properties of this pathway for protons and potassium ions favor the idea that ADP regulates the mitochondrial permeability via adenine nucleotide translocase. It is assumed that the adenine nucleotides carrier should operate according to the “gated pore” mechanism.     

On the role of K+ on succinic dehydrogenase activity

The effect of potassium ions on succinic dehydrogenase activity of mitochondria was studied. The results showed that in these organelles K⁺ induces inhibition of the respiratory control; moreover, in submitochondrial particles potassium inhibits the rate of oxidation of succinate. The results showed also that K⁺ does not changes theK _m for succinate but diminishes theV _max. In addition, the data provide evidence that mitochondria oxidizing glutamatemalate in a sucrose medium show a higher activity of succinate dehydrogenase than mitrochondria incubated in KCl.     

The action of two toxic quinones on Anacystis nidulans

Summary A study was made of the factors affecting the toxicity to the blue-green alga, Anacystis nidulans, of two quinones, 2,3-dichloro-1,4-naphthoquinone and 9,10-phenanthraquinone. These two substances, which are known to be far more toxic to blue-green algae than other quinones so far studied, were shown to differ considerably in their properties. For instance, the toxicity of 2,3-dichloro-1,4-naphthoquinone is increased by light, vitamin K₃ and H₂O₂, whilst vitamin K₁ has no effect. On the other hand, the toxicity of 9,10-phenanthraquinone is increased by vitamin K₃, decreased by vitamin K₁, whilst H₂O₂ and light have no effect. The toxicity of both substances is decreased by the extracellular materials of various blue-green algae.     

Modulation of reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by vitamin C-palmitate

An in vitro study of effects of vitamin C-palmitate on the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat microsomes was performed. A sensitive assay method has been developed for the detection of metabolites of NNK in microsomes. Only the reduced metabolite of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-butanol (NNAL), was detected and measured in a time-course study. Vitamin C-palmitate enhanced the reduction of NNK in a concentration-dependent manner. The results indicate a significant increase in V_max and K_m in the presence of vitamin C. However, the rate of formation of NNAL at low substrate concentration varied. The ratio of V_max to K_m decreases. The results suggest that the kinetics are accounted for best by an uncompetitive activator binding model at low concentration of vitamin C. The uncompetitive binding model becomes sketchy at higher concentration of vitamin C. These observations infer that vitamin C loosely binds to the substrate-enzyme complex. Furthermore, the nature of the binding would facilitate the modulation of NNK biotransformation leading to the formation of NNAL. The results also show that vitamin C-palmitate is a potent activator of NNK reduction in rat liver microsomes. Thus, vitamin C-palmitate would mediate the metabolism of NNK through reduction. The resulting NNAL-glucuronide is more readily eliminated in urine.     

Inhibition of electron transfer in the cytochromeb-c 1 segment of the mitochondrial respiratory chain by a synthetic analogue of ubiquinone

A synthetic analogue of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, inhibits oxidation of succinate and NADH-linked substrates by rat liver mitochondria. Inhibition occurs both in the presence (state 3) and absence (state 4) of ADP. With isolated succinate-cytochromec reductase complex from bovine heart mitochondria the quinone analogue inhibits succinate-cytochromec reductase and ubiquinol-cytochromec reductase activities but does not inhibit succinate-ubiquinone reductase activity. Inhibition of cytochromec reductase activities is markedly dependent on pH in the range pH 7–8. At pH 7.0 inhibition occurs with an apparentK _i1×10^–8 M, while at pH 8.0 the apparentK _i is more than an order of magnitude greater than this. Spectrophotometric titrations of 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole show a visibly detectable pK _a at pH 6.5 attributable to ionization of the 6-hydroxy group. These results indicate that this quinone derivative is a highly specific and potent inhibitor of electron transfer in theb-c ₁ segment of the respiratory chain. Because of the structural analogy, it is likely that the mechanism of inhibition involves disruption of normal ubiquinone function. In addition, this inhibition depends on protonation of the ionizable hydroxy group of the inhibitory analogue or on protonation of a functional group in theb-c ₁ segment.     

Production of superoxide and dissipation of mitochondrial transmembrane potential by vitamin K2 trigger apoptosis in human ovarian cancer TYK-nu cells

We reported previously that vitamin K₂ selectively induces apoptosis in human ovary cancer cells (TYK-nu cells) and pancreatic cancer cells (MIA PaCa-2 cells) through a mitochondrion-dependent pathway. In the present study, we examined the details of the mechanism of vitamin K₂-induced apoptosis in TYK-nu cells. We found that superoxide (O₂ ^•−) was produced by TYK-nu cells between 2 and 3 days after the start of treatment with vitamin K₂, whereas it was produced within 30 min after the start of treatment with geranylgeraniol. The vitamin K₂-induced apoptosis was inhibited by anti-oxidants, such as α-tocopherol, Tiron and N-acetyl-L-cysteine (NAC). Furthermore, both the production of superoxide and the induction of apoptosis by vitamin K₂ were inhibited almost completely by cycloheximide, an inhibitor of protein synthesis, suggesting that the synthesis of enzymes for the production of superoxide might be required for these processes. In parallel with the production of superoxide, the mitochondrial transmembrane potential, as measured by staining with Mitotracker Red CMXRos, dissipated during treatment of TYK-nu cells with vitamin K₂ for 3 days. The vitamin K₂-induced depolarization of mitochondrial membranes was completely inhibited by α-tocopherol and, to a lesser extent, by Tiron and NAC. Since α-tocopherol reacts with oxygen radicals, such as superoxide, within the hydrophobic environment of the mitochondrial membrane, we postulate that vitamin K₂-induced oxidative stress in mitochondria might damage mitochondrial membranes, with subsequent release of cytochrome c, the activation of procaspase 3 and, eventually, apoptosis.     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

Species Differences in the Selective Inhibition of Monoamine Oxidase (1-methyl-2-phenylethyl)hydrazine and its Potentiation by Cyanide

The potentiating effects of cyanide on the inhibition of rat liver mitochondrial monoamine oxidase-A & B and of ox liver mitochondrial MAO-B by pheniprazine [(1-methyl-2-phenylethyl)hydrazine] has been studied. Pheniprazine was shown to behave as a mechanism-based MAO inhibitor. For rat liver MAO-B, the initial non-covalent step was characterized by dissociation constant (K _i) of 2450 nM and the first-order rate constant (k ₊₂) for the covalent adduct formation was 0.16 min⁻¹. As a reversible inhibitor it was selective towards rat liver MAO-A (K _i = 420 nM) but the rate of irreversible inhibition of that enzyme was considerably slower (k ₊₂ = 0.06 min⁻¹). MAO-B from ox liver more closely resembled MAO-A from the rat in sensitivity to reversible inhibition by pheniprazine (K _i = 450 nm) but it was closer to rat liver MAO-B in rate of irreversible inhibition (k ₊₂ = 0.29 min⁻¹). The K _i values were significantly decreased in the presence of KCN but there was little effect on the k ₊₂ values. However, sensitivities of the different enzymes to KCN varied widely and considerably higher concentrations of KCN were required for this effect to be apparent with the rat liver mitochondrial MAO-A than with MAO-B from rat and ox liver. The kinetic behaviour of cyanide activation was consistent with partial (non-essential) competitive activation in all cases. Special issue dedicated to Dr. Moussa Youdim.     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

Effect of bedaquiline on the functions of rat liver mitochondria

The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50?μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V₃ and V_DNP. At the same time, at concentrations below 50?μM, BDQ slightly stimulated respiration with substrates of complex I in the state V₂. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II?+?III of the mitochondrial transport chain. It was discovered that at concentrations up to 10?μM, BDQ inhibited H₂O₂ production in mitochondria. BDQ (10–50?μM) suppressed the opening of Ca²⁺-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca²⁺/P_i-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca²⁺ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K⁺ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K⁺ buffer and DNP-induced K⁺ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.     

Specific inhibition by macrocyclic polyethers of mitochondrial electron transport at site I

The macrocyclic polyethers dibenzo-18-crown-6 (XXVIII) and dicyclohexyl-18-crown-6 (XXXI) inhibit the valinomycin-mediated K⁺ accumulation energized by glutamate, -ketoglutarate, malate plus pyruvate or isocitrate but not that promoted by succinate, ascorbate plus TMPD or ATP. The polyethers inhibit the oxidation of the former group of substrates without preventing either the oxidation of succinate or ascorbate plus TMPD or the hydrolysis of ATP.The substrate oxidation inhibited by the macrocyclic polyethers is relieved in intact mitochondria by increasing the concentration of K⁺ in the medium. It is also completely reverted by supplementing the medium with valinomycin, Cs⁺ and phosphate, or else by the addition of vitamin K₃.In submitochondrial sonic particles the macrocyclic polyethers inhibit the oxidation of NADH as well as the ATP-driven reversal of electron flow at the site I of the electron transport chain. They also block the oxidation of NADH in non-phosphorylating Keilin-Hartree particles as well as in Hatefi's NADH-coenzyme Q reductase. The polyethers do not inhibit electron transport in mitochondria from the yeast which lack the first coupling site.The inhibition of electron transport by the polyethers do not require of the addition of alkali metal cations such as K⁺ in intact mitochondria or other membrane preparations.It is established that the macrocyclic polyethers XXVIII and XXXI, already characterized as mobile carrier molecules for K⁺ in model lipid membranes, inhibit electron transport at site I of the electron transport chain from mitochondrial membranes.It is suggested that the ability of the polyethers to coordinate alkali metal cations in aqueous versus lipid environments, but not K⁺ transportper se, is related to their rotenone-like induced inhibition of electron flow in mitochondrial membranes.Supported in part by a Grant from the Research Corporation.     

Effect of potential-dependent potassium uptake on production of reactive oxygen species in rat brain mitochondria

The effect of potential-dependent potassium uptake on reactive oxygen species (ROS) generation in mitochondria of rat brain was studied. It was found that the effect of K⁺ uptake on ROS production in the brain mitochondria under steady-state conditions (state 4) was determined by potassium-dependent changes in the membrane potential of the mitochondria (ΔΨ_m). At K⁺ concentrations within the range of 0–120 mM, an increase in the initial rate of K⁺-uptake into the matrix resulted in a decrease in the steady-state rate of ROS generation due to the K⁺-induced depolarization of the mitochondrial membrane. The selective blockage of the ATP-dependent potassium channel (K _ATP ⁺ -channel) by glibenclamide and 5-hydroxydecanoate resulted in an increase in ROS production due to the membrane repolarization caused by partial inhibition of the potential-dependent K⁺ uptake. The ATP-dependent transport of K⁺ was shown to be ～40% of the potential-dependent K⁺ uptake in the brain mitochondria. Based on the findings of the experiments, the potential-dependent transport of K⁺ was concluded to be a physiologically important regulator of ROS generation in the brain mitochondria and that the functional activity of the native K _ATP ⁺ -channel in these organelles under physiological conditions can be an effective tool for preventing ROS overproduction in brain neurons.     

Short-term vitamin A supplementation at therapeutic doses induces a pro-oxidative state in the hepatic environment and facilitates calcium-ion-induced oxidative stress in rat liver mitochondria independently from permeability transition pore formation

There is a growing body of evidence showing that vitamin A induces toxic effects in several experimental models and in human beings. In the present work, we have investigated the effects of short-term vitamin A supplementation on the adult rat liver redox status. We have found that vitamin A at therapeutic doses induces a hepatic oxidative insult. Furthermore, we have observed increased antioxidant enzyme activity in the liver of vitamin-A-treated rats. Additionally, some mitochondrial dysfunction was found since superoxide anion production was increased in vitamin-A-treated rat liver submitochondrial particles, which may be the result of impaired mitochondrial electron transfer chain activity, as assessed here. We have also isolated rat liver mitochondria and challenged it with 75 μM CaCl₂, a non-oxidant agent that is able to induce mitochondrial oxidative stress indirectly. We have found that mitochondria isolated from vitamin-A-treated rat liver are more sensitive to CaCl₂ than control mitochondria regarding the redox status. Importantly, vitamin A seems to alter mitochondrial redox status independently of the participation of the mitochondrial permeability transition pore, which is activated by Ca²⁺ ions since cyclosporin A did not prevent the oxidative insult elicited by Ca²⁺ addition. Overall, we show here that mitochondria are a target of vitamin-A-associated toxicity also in vivo.     

Inhibition of Acetylcholinesterase by three New Pyridinium Compounds and their Effect on Phosphonylation of the Enzyme

Abstract

Three new mono-pyridinium compounds were prepared: 1-phenacyl-2-methylpyridinium chloride (1), 1-benzoylethylpyridinium chloride (2) and 1-benzoylethylpyridinium-4-aldoxime chloride (3) and assayed in vitro for their inhibitory effect on human blood acetylcholinesterase (EC 3.1.1.7, AChE). All the three compounds inhibited AChE reversibly; their binding affinity for the enzyme was compared with their protective effect (PI) on AChE phosphonylation by soman and VX. Compound 1 was found to bind to both the catalytic and the allosteric (substrate inhibition) sites of the enzyme with estimated dissociation constants of 6.9 μM (K_cat) and 27 μM (K_all), respectively. Compound 2 bound to the catalytic site with K_cat= 59 μM and compound 3 only to the allosteric site with K_all = 328 μM. PI was evaluated from phosphonylation measured in the absence and in presence of the compounds applied in a concentration corresponding to their K_cat or K_all value, and was also calculated from theoretical equations deduced from the reversible inhibition of the enzyme. Compounds 1 and 3 protected the enzyme from phosphonylation by soman and VX, whereas no protection was observed in the presence of compound 2 under the same conditions. Irrespective of the binding sites to AChE, PI for compounds 1 and 3 evaluated from phosphonylation agreed with PI calculated from reversible inhibition. Compound 3 was found to be a weak reactivator of methylphosphonylated AChE with k_r = 1.1 × 10²Lmol^-1 min^-1.     

Modulation of the Permeability Transition Pore by Inhibition of the Mitochondrial KATP Channel in Liver vs. Brain Mitochondria

Inhibition of the mitochondrial K_ATP (mitoK_ATP) channel abrogates the beneficial effects of preconditioning induced by a brief episode of sublethal ischemia. We studied the effect of 5-hydroxydecanoate, a well-known inhibitor of the mitoK_ATP channel, on swelling of isolated liver and brain mitochondria. Volume changes were determined by measurement of light absorbance at 540 nm. Mitochondrial swelling induced by adding Ca²⁺ ions correlated with opening of the permeability transition pore as shown by modulation by 1 μM cyclosporin A. In brain mitochondria, 5-hydroxydecanoate did not significantly affect Ca²⁺-induced swelling. In contrast, 50 or 500 μM 5-hydroxydecanoate increased swelling of liver mitochondria by 9.7 ± 5.1% (n = 6, P = 0.057) and 29.4 ± 1.4% (n = 5, P < 0.0001), respectively. The effect of 5-hydroxydecanoate was blocked by cyclosporin A and was dependent on the presence of potassium in the medium. In medium containing 200 μM ATP to inhibit the mitoK_ATP channel, 5–hydroxydecanoate did not further increase Ca²⁺-induced swelling. We conclude that inhibition of the mitoK_ATP channel exerts its detrimental effect by facilitation of permeability transition pore opening.