Effect of In Vivo Administration of Naloxone on ATP-ase's Enzyme Systems of Synaptic Plasma Membranes from Rat Cerebral Cortex

Naloxone is a specific competitive antagonist of morphine, acting on opiate receptors, located on neuronal membranes. The effects of in vivo administration of naloxone on energy-consuming non-mitochondrial ATP-ases were studied in two different types of synaptic plasma membranes from rat cerebral cortex, known to contain a high density of opiate receptors. The enzyme activities of Na⁺, K⁺-ATP-ase, Ca²⁺, Mg²⁺-ATP-ase and Mg²⁺-ATP-ase and of acetylcholinesterase (AChE) were evaluated on synaptic plasma membranes obtained from control and treated animals with effective dose of naloxone (12g · kg^–1 i.m. 30 minutes). In control (vehicle-treated) animals specific enzyme activities assayed on these two types of synaptic plasma membranes are different, being higher on synaptic plasma membranes of II type than of I type, because the first fraction is more enriched in synaptic plasma membranes. The acute treatment with naloxone produced a significant decrease in Ca²⁺,Mg²⁺-ATP-ase activity and an increase in AChE activity, only in synaptic plasma membranes of II type. The decrease of Ca²⁺,Mg²⁺-ATP-ase enzymatic activity and the increased AChE activity are related to the interference of the drug on Ca²⁺ homeostasis in synaptosoplasm, that leads to the activation of calcium-dependent processes, i.e. the extrusion of neurotransmitter. These findings give further evidence that pharmacodynamic characteristics of naloxone are also related to increase [Ca²⁺] _i , interfering with enzyme systems (Ca²⁺,Mg²⁺-ATP-ase) and that this drug increases acetylcholine catabolism in synaptic plasma membranes of cerebral cortex.     

Effect of In Vivo <Emphasis Type="SmallCaps">l</Emphasis>-Acetylcarnitine Administration on ATP-ases Enzyme Systems of Synaptic Plasma Membranes from Rat Cerebral Cortex

The maximum rates (V _max) of some enzymatic activities related to energy consumption (ATP-ases) were evaluated in two types of synaptic plasma membranes (SPM) isolated from cerebral cortex of rats subjected to in vivo treatment with l-acetylcarnitine at two different doses (30 and 60 mg kg⁻¹ i.p., 28 days, 5 days/week). The following enzyme activities were evaluated: acetylcholinesterase (AChE); Na⁺, K⁺, Mg²⁺-ATP-ase; ouabain insensitive Mg²⁺-ATP-ase; Na⁺, K⁺-ATP-ase; direct Mg²⁺-ATP-ase; Ca²⁺, Mg²⁺-ATP-ase; Low- and High-affinity Ca²⁺-ATP-ase. Sub-chronic treatment with l-acetylcarnitine increased Na⁺, K⁺-ATP-ase activity on SPM 2 and Ca²⁺, Mg²⁺-ATP-ase activity on both SPM fractions. These results suggest (1) that the sensitivity to drug treatment is different between the two populations of SPM, confirming the micro-heterogeneity of these sub-fractions, probably originating from different types of synapses, (2) the specificity of the molecular site of action of the drug on SPM and (3) its interference on ion homeostasis at synaptic level.     

Variance analysis of Cd2+, Zn2+, and Mn2+ effect on membrane Na+,K(+)-ATPase activity of loach embryos

The evaluation of influences Cd2+, Zn2+, Mn2+ in concentrations 10(-6), 10(-5), 10(-4) M (factor of dose) on the Na+, K(+)-ATP-ase activity in the early period of development (60-330 min.) of loach embryos (time factor) using one- and two-factor analysis of variance has been performed. It has been detected, that the changes of enzyme activity are mainly caused by action of the explored cations and do not depend on time of embryos development. The most influence on activity in the indicated period of embryos development of loach renders Cd2+ in concentration 10(-4) M, relative value of its influence being 95.7% (p = 0.01). Substantial concentration dependence of the Na+, K(+)-ATP-ase activity is exposed to the action of each of cations. The values of the influence of their concentration changes during the studied period of development differ insignificantly for all cations and make 76.2-77.5% (p < 0.01).     

Effect of chronic ethanol consumption on emotional stress in the white rat

Chronic pain emotional stress (PES), paired action of the white noise and electric skin stimulation and chronic (during 7 months) ethanol consumption in white rats were shown to act in the same direction. Hypertension, decrease of respiratory rate and increase of Hildebrandt index were observed as a result of PES, ethanol consumption, and especially under PES during ethanol consumption. Ethanol consumption by the animals led to their growth retardation and increase of the spleen and heart mass. Accidental thymus involution was noted both under ethanol consumption and PES. Activation of lipid peroxidation and decrease of superoxide dismutase activity (of its mitochondrial form especially) as well as of Na+,K+-ATP-ase activity were observed in brain homogenates of the rats after PES, while the general ATP-ase activity remained unchanged. An increase of triiodothyronine level and the tendency to thyroxine level increase as well as a decrease of superoxide dismutase activity were observed in the blood serum of these animals. A tendency towards lipid peroxidation level decrease and to brain superoxide dismutase activity increase, as well as blood antioxidation activity increase (evaluated by transferrin and coeruloplasmin contents and by serum superoxide dismutase activity) and a decrease of thyroxine level were observed as a result of ethanol consumption. The mechanisms are discussed of the "anti-stress" action of short-term ethanol consumption and of the action of its chronic consumption, additive to PES.     

Effect of in vivo treatment of clonidine on ATP-ase's enzyme systems of synaptic plasma membranes from rat cerebral cortex

The effects on energy-consuming ATP-ases were studied in two types of synaptic plasma membranes from rat cerebral cortex after in vivo injection of clonidine. To study the mechanism of action of clonidine at subcellular level, the enzyme activities of Na⁺, K⁺-ATP-ase, Ca²⁺, Mg²⁺-ATP-ase, Low- and High-affinity Ca²⁺-ATP-ase, and Mg²⁺-ATP-ase were evaluated on synaptic plasma membranes of control and treated animals with clonidine (5 g · kg^–1; i.p. 30 minutes). Acute treatment with clonidine decreased the catalytic activity of Ca²⁺, Mg²⁺-ATP-ase and of low-affinity Ca²⁺-ATP-ase only in synaptic plasma membranes of II type, that is the fraction enriched in synaptic plasma membranes. The decreases of these enzymatic activities are related to the interference of the drug on Ca²⁺ homeostasis in synaptoplasm. The reductions of these enzyme-consuming ATP-ases give further evidence that clonidine has not only neuroreceptorial effects, but that the drug also affects the energy metabolism of cerebral tissue, improving the knowledges about the pharmacology of clonidine. Because the elevation of [Ca²⁺]_i, during ischemia/hypoxia contributes to cellular injury, these findings may suggest that the prevention of calcium overload may be the key mechanism of protection by ₂-agonist.     

Activity of Ca(+2)Mg(+2) --ATPase in erythrocyte membranes of women with diabetes mellitus type I]

The activity of Ca(++)-Mg++ ATP-ase present in erythrocyte membranes was determined in basal conditions and following stimulation with calmodulin in 8 women with insulin-dependent diabetes and in 9 healthy women. The isolation of erythrocyte membranes and the determination of activity of Ca(++)-Mg(++)-ATP-ase were carried out according to the method of Gietzen et al. A decrease in the activity of Ca(++)-Mg(++)-ATP-ase in basal conditions was found in fractions with the highest erythrocyte content obtained from diabetic patients. After stimulation with calmodulin the activity of Ca(++)-Mg(++)-ATP-ase in all the fractions was lower in diabetic patients than in the controls. Low activities of the enzyme were accompanied by high values of HbA1c. The results suggest that glycosylation of the ATP-ase or/and calmodulin may be the main cause of the observed fall in the enzyme activity in diabetes. Also the disturbances concerning the cumulation of intracellular calcium may be related to the changes caused by glycosylation of Ca(++)-Mg(++)-ATP-ase or/and calmodulin.     

The effect of (+) cyanidanol-3 on the Na+-K+-ATP-ase and Mg++-ATP-ase activities of the rat brain in the presence and absence of ascorbic acid

The authors investigated the effects of (+) cyanidanol-3-(Catergen) in vitro on the activities of rat brain plasma membrane and microsomal Na+-K+-ATP-ase and Mg++-ATP-ase, in the presence and absence of ascorbic acid. Due to lipid peroxidation induced by low concentration of ascorbid acid, activity of both ATP-ase decreased. (+) cyanidanol-3 proved to be an effective antioxidant in this system. It inhibited the decrease of ATP-ase activity which occurred as a result of lipid peroxidation promoted by ascorbic acid.     

The role of Ca++ in regulation of oxoglutarate dehydrogenase complex activity from bovine adrenal cortex

Binding of Ca2+ ions by EGTA is established to increase the oxoglutarate dehydrogenase complex K'm for 2-oxoglutarate up to 25 mM in spite of the Mg2+ ions presence in the medium. The maximum reaction rate remains unchanged. Addition, besides EGTA of an equimolar Ca2+ amount to the medium induces a more than 100-fold decrease in K'm. A positive effect of Ca2+ ions is intensified by ADP. Ca2+ counteracts the inhibition of the oxoglutarate dehydrogenase complex activity by NADH. When chelating Ca2+ by EGTA with NADH available, a nonhyperbolic dependence of the reaction rate on the 2-oxoglutarate concentration is observed. When Ca2+ is absent, signs of a positive cooperative interaction of the enzyme with ADP and NADH are observed under conditions when 2-oxoglutarate concentration is much lower than the saturating one.     

The effect of antibodies against Ca2(+)-ATPase and its hydrophobic fragment on the activity of this enzyme and on the passive transport of Ca2(+)]

It is shown that both Ca(2+)-ATPase and its hydrophobic fragment are immunogenic factors. A region between hydrophobic and hydrophilic parts of Ca(2+)-ATP-ase is immunogenic. These antibodies clearly inhibit inflow and outflow of Ca2+ through a hydrophobic fragment reconstructed into liposomes and do not influence Ca(2+)-ATPase activity.     

The study of Ca2+ influx in human erythrocytes in isotonic polyethylene (glycol) 1500 (PEG-1500) and sucrose media

Effects of isotonic solutions of polyethylene (glycol) 1500 (PEG-1500) and sucrose on Ca2+ influx into ATP-depleted red blood cells were studied using the Ca2+ -sensitive fluorescent dye fura-2AM. When incubated in isotonic low ionic strength media (containing 2 mM CaCl2 in addition to sucrose and PEG-1500), the initial rate of Ca2+ influx was higher than that for the cells in physiological (normal ionic strength) medium. After 20 minutes of incubation in the PEG-1500-containing solution, a 10-fold increase of Ca2+ influx was observed, whereas in the sucrose medium the rate of Ca2+ influx decreased compared to that in physiological medium. 1H-NMR data provided no evidence of direct interaction between PEG-1500 and the erythrocyte membrane. Moreover, PEG-1500 did not affect lipid peroxidation (LPO) induction in erythrocyte membranes. We propose that a change in the hydrogen environment of Ca2+ -ATPase of the erythrocytes suspended in the PEG-1500 solution is the primary cause of altered Ca2+ homeostasis in these cells. The activation of the Ca2+ -ATP-ase in sucrose medium may result in an incomplete suppression of the Ca2+-pump activity in ATP-depleted cells, which is accelerated when calmodulin binds with the Ca2+-ATP-ase under the conditions of rapid Ca2+ accumulation.     

Characterization of an endogenous inhibitor of lung 15-hydroxyprostaglandin dehydrogenase

An endogenous inhibitor of the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase was isolated from the 105,000 X g supernatant fraction of lungs of pregnant rabbits following DEAE chromatography. The material was heat stable and was resistant to pronase treatment. The inhibitor contained a mixture of saturated and mono-unsaturated fatty acids and cholesterol with palmitate and oleate representing the major fatty acids in the inhibitory factor. The factor inhibited prostaglandin dehydrogenase activity but had only minor effects on the activity of NAD+-dependent alcohol and lactate dehydrogenases or the NADP+-dependent isocitrate dehydrogenase. In an attempt to develop a greater understanding of the inhibitory action of fatty acids on prostaglandin dehydrogenase activity, a variety of standard fatty acids were examined for their ability to decrease enzymic activity. Oleate and palmitate inhibited enzymic activity by 70% at 10 microM, whereas arachidonate and myristate were only 30% inhibitory at this concentration. A comparison among the 18-carbon-containing fatty acids demonstrated that oleate was more potent than linoleate and linolenate in inhibiting prostaglandin dehydrogenase activity. The coenzyme A derivatives of oleate, linoleate and linolenate were less inhibitory than the free fatty acids.     

Activation of V1-receptors by vasopressin stimulates inositol phospholipid hydrolysis and arachidonate metabolism in human platelets.

The effects of Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within intact mitochondria prepared from control and insulin-treated rat epididymal adipose tissue was explored by incubating the mitochondria in medium containing the ionophore A23187. The apparent Ka for Mg2+ was approximately halved in the mitochondria derived from insulin-treated tissue in both the absence and the presence of Ca2+. In this system, the major effect of Ca2+ was also to decrease the apparent Ka for Mg2+, rather than to change the Vmax. of the phosphatase. Damuni, Humphreys & Reed [(1984) Biochem. Biophys. Res. Commun. 124, 95-99] have reported that spermine activates ox kidney pyruvate dehydrogenase phosphate phosphatase. Studies were carried out on phosphatase from pig heart and rat epididymal adipose tissue which confirm and extend this observation. The major effect of spermine is shown to be a decrease in the Ka for Mg2+, which is apparent in both the presence and the absence of Ca2+. Spermine did not affect the sensitivity of the phosphatase to Ca2+ at saturating concentrations of Mg2+. Other polyamines tested were not as effective as spermine. No alteration in the maximum activity or Mg2+-sensitivity of pyruvate dehydrogenase phosphate phosphatase was apparent in extracts of mitochondria from insulin-treated tissue. The close similarity of the effects of spermine and the changes in kinetic properties of pyruvate dehydrogenase phosphate phosphatase within mitochondria from insulin-treated adipose tissue suggests that insulin may activate pyruvate dehydrogenase by increasing the concentration of spermine within the mitochondria. However, it is concluded that insulin is more likely to alter the interaction of the pyruvate dehydrogenase system with some other polybasic intramitochondrial component whose action can be mimicked by spermine.     

Effect of fluorine on enzyme activity in the small intestine mucosa during absorption of sodium, potassium, monosaccharides and amino acids

Effect of NaF on ATP-ase, creatine kinase, acid and basic phosphatases activity of small intestine mucosa in white rats during enteral administration of NaCl, KCl solutions has been studied under conditions of acute experiment. Inhibition of the Mg2+-ATP-ase activity by 13%, general ATP-ase activity by 25%, creatine kinase by 22% and a 2.2-3 fold inhibition of Na, K+-ATP-ase activity is observed. Acid and basic phosphatase activity does not change, swelling of mucosa is observed. The acid phosphatase activity in the intestinal contents increases 1.5 times, basic phosphatase activity--1.7 times, creatine amount--1.7 times, Pi amount 1.8 times. In experiments in vitro F- produces 20% activation of the basic phosphatase of mucosa and a 2.6-fold inhibition of the acid phosphatase. Rate of fructose absorption falls by 34%, that of methionine--by 29%, glucose--by 24%, glutaminic acid--by 10%. Activity of general ATP-ase in this case decreases by 22, 15, 27, 20% respectively. It is supposed that the F- effect results in destabilization of the membrane structures of the intestine mucosa.     

Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes.

The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.     

Effect of halothane on lung carcinoma cells A 549

The halogenated hydrocarbons, such as halothane, are widely used as anesthetics in clinical practice; however their application is often accompanied with metabolic, cardiovascular and respiratory complications. One of the possible factors for this negative outcome might be the severe toxicity of these agents. In this paper, we investigate in vitro effects of halothane on human lung carcinoma A 549 cells, namely on their cytotoxicity, adhesive properties and metabolic activity. The cytotoxicity response of lung carcinoma A 549 cells to halothane was determined by lactate dehydrogenase (LDH) assay (for cytotoxicity), by detachment assay after adhesion to type IV collagen (for cell adhesive properties) and by surface tension measurements of culture medium (for cell metabolic activity). Regarding the cytotoxicity, the determined maximal non-toxic concentration of halothane on A 549 cells, given here as volume percentages (vol.%) was 0.7 vol.% expressed as aqueous concentration in the culture medium. Direct measurement of the actual halothane concentration in the culture medium showed that 0.7 vol.% corresponds to 1.05 mM and 5.25 aqueous-phase minimum alveolar concentration (MAC). Concentrations equal or higher than 1.4 vol.% (2.1 mM; 10.5 MAC) of halothane provoked complete detachment (cell death), or reduction of initial adhesion to collagen IV in half of the cell population. Surfactant production of A 549 cells, registered up to 48 h after halothane treatment, was inhibited by halothane concentrations as low as 0.6 vol.% (0.9 mM; 4.5 MAC). Our results demonstrate that sub toxic halothane concentrations of 0.6 vol.% inhibits surfactant production; concentrations in the range 0.8-1.4 vol.% induce membrane damages and concentrations equal and higher than 1.4 vol.%--cell death of approximately 50% of the cells.     

Aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12 (lambda). Activation by monovalent cations and an analysis of the effect of the adenosine triphosphate-magnesium ion complex on this activation process.

The dehydrogenase activity of the aspartokinase I-homoserine dehydrogenase I complex isolated from Escherichia coli K12 is subject to a cooperative activation by K+ or Rb+, which is characterized by a Hill coefficient of approximately 2. Ionic strength has little effect on the Hill coefficient for this activation process; however, high ionic strength appears to increase the enzyme's affinity for K+ and decrease its affinity for Rb+. The Vmax of the K+-activated dehydrogenase is greater than that of the Rb+-activated dehydrogenase. The results of a study of the competition between K+ and Rb+ in the activation process suggest the presence of an activated species containing both K+ and Rb+. The cooperative activation by K+ is antagonized by Na+ via a process that is noncooperative with respect to Na+. The MgATP-2- complex, a substrate for the kinase activity of aspartokinase I-homoserine dehydrogenase I, has a marked effect on the K+ activation of the dehydrogenase activity. Kinetic studies of this effect of MgATP-2- on the K+ requirement of the dehydrogenase at pH 8.9 indicate that: (a) activation by a monovalent cation is essential in the presence as well as in the absence of MgATP-2-; (b) the concentration of K+ required to activate fully the dehydrogenase is reduced in the presence of MgATP-2-; (c) activation of the dehydrogenase by K+ is noncooperative in the presence of MgATP-2-; and (d) the maximum velocity for the dehydrogenase catalyzed oxidation of homoserine is greater in the presence of MgATP-2- than in its absence. Based on these results, a simple model consistent with these data is proposed. Destruction of the kinase activity and the threonine sensitivity of the aspartokinase-homoserine dehydrogenase complex by treatment with 5,5'-dithiobis(2-nitrobenzoic acid) or by incubation at pH 9 also converts the K+ activation of the dehydrogenase from a cooperative to a noncooperative process. Marked protection of the enzyme against loss of threonine sensitivity at pH 9 is afforded by MgATP-2- plus K+ and homoserine. The apparent molecular radius of the enzyme complex as determined by gel filtration at pH 8.85 in the presence of threonine or MgATP-2- plus K+ and homoserine is dependent on the enzyme concentration. The observed apparent molecular radii of 70 A at high enzyme concentrations and 61 A at low enzyme concentrations are consistent with the enzyme's undergoing a concentration-dependent dissociation from a tetrameric to a dimeri     

重金属离子对猪红细胞膜Ca~(2+)-ATP酶的作用

猪红细胞膜Ca~(2+)-ATP酶是一种钙调蛋白(CaM)依赖酶,其活力又依赖巯基的完整性。实验应用Ca~(2+)-ATP酶这一模型体系观察到重金属离子,Pb~(2+)、Cd~(2+)和Hg~(2+)都能替代Ca~(2+),激活CaM,从而激活Ca~(2+)-ATP酶;其最大刺激活力分别为85％、80％和30％,半刺激浓度分别为32、27和0.7μmol/L。当三种重金属离子的浓度增加时,则与Ca~(2+)-ATP酶的巯基结合,抑制酶的活力,Pb2~(2+)、Cd~(2+)和Hg~(2+)的半抑制浓度分别为370、440和2μmol/L。抑制作用为渐进性过程,而刺激作用为即时效应。抑制作用可为巯基化物,特别是二巯基化物所逆转。研究结果提示,CaM可能是重金属中毒最初作用的靶分子,而重金属中毒不仅使CaM“开关”失灵,还可能导致细胞内Ca~(2+)的调节全面失控。     

In vitro study of AMP-deaminase from fish (Cyprinus carpio) treated with hydrolyzable tannins

AMP-deaminase (EC 3.5.4.6) is an enzyme responsible for stabilising adenylate energy changes. The properties of this enzyme are controlled by various ligands of hydrophobic nature. An investigation of enzyme activity alterations under the influence of natural phenolic acids (tannic, ellagic and gallic) which are soluble in water, could evidence the biological toxicity of these compounds. In our study purified AMP-deaminase isolated from white muscle of Cyprinus carpio was exposed to phenolic acids in the concentration range of 1 to 50 microM as well as to tannic acid in the presence of Cu2+ ions (5 microM). On the basis of the obtained results we can conclude that among the tested acids, gallic acid did not contribute to the change in AMP-deaminase activity, whereas ellagic acid diminished its activity at the highest concentration (50 microM). Tannic acid caused a significant decrease in the enzyme activity in comparison to control for all used concentrations. Cu2+ ions alone reduced the activity of AMP-deaminase for all studied concentrations. A combined action of a chosen Cu2+ ions concentration (5 microM) with tannic acid at the concentration higher than 2 microM resulted in a decrease in the enzyme activity, but for lower tannic acid concentration of 1 microM the activity of AMP-deaminase was stimulated. These experiments showed that tannic acid may stop free radical chain reactions only at low concentrations (1 microM) in the presence of Cu2+ ions (5 microM).     

The Na+,K+-ATP-ase Activity and Lipids of Trout and Rat Brain Cell Membranes at the Pressure of 101 Atmospheres

The effect of elevated heliox pressure (101 ATA) on activity of Na⁺,K⁺-ATP-ase and some characteristics of fatty acid composition was studied in membrane phospholipids of trout and rat brain synaptosomal and mitochondrial fractions. The Na⁺,K⁺-ATP-ase activity was shown to decrease by 25% in both fractions of the rat brain; in mitochondrial fraction of the trout brain it decreased by 47%, while in synaptosomal fraction, only by 11%. It has also been established that under experimental conditions, the unsaturation index of fatty acids of phosphatidylcholine and phosphatidylethanolamine of trout synaptosomes decreased, with no changes in these lipids in mitochondrial fraction. The phosphatidylcholine unsaturation index in rats did not practically change in both fractions, while in rat phosphatidylethanolamine it increased in mitochondrial fraction and slightly decreased in synaptosomal fraction. Thus, under conditions of high pressure the reduction of the enzyme activity is also determined, specifically, by peculiarities of the phospholipid fatty acid composition in the subcellular fractions studied. A possibility of changes of the enzyme activity as a result of transition of its lipid component from the liquid crystalline to the gel state under effect of an enhancement of lipid peroxidation under conditions of elevated pressure is discussed.     

Effect of magnesium ions on Ca2+, Mg2+-ATPase on plasma membranes of thymocytes]

Ca2+, Mg(2+)-ATP-ase of plasmatic membranes of thymocites were studied as affected by Mg ions. A complex character of the reaction rate dependence on Mg2+ concentration in the range of 0.002-25. OmM was revealed. The data obtained indicate the existence of Mg(2+)-binding centre of ATP-ase which regulates the enzyme catalytic activity.