Protective and Antioxidative Effects of GM1 Ganglioside in PC12 Cells Exposed to Hydrogen Peroxide are Mediated by Trk Tyrosine Kinase

GM1 ganglioside was found to increase the survival of PC12 cells exposed to H₂O₂, its action was blocked by Trk tyrosine kinase inhibitor K-252a. Thus, the inhibition of H₂O₂ cytotoxic action by GM1 constituted 52.8 ± 4.3%, but in the presence of 1.0 μM K-252a it was only 11.7 ± 10.8%, i.e. the effect of GM1 became insignificant. Exposure to GM1 markedly reduced the increased accumulation of reactive oxygen species (ROS) and diminished the inactivation of Na⁺,K⁺-ATPase induced in PC12 cells by H₂O₂, but in the presence of K-252a GM1 did not change these metabolic parameters. The inhibitors of extracellular signal-regulated protein kinase, phosphatidyl inositol 3-kinase and protein kinase C decreased the effects of GM1. A combination of these protein kinase inhibitors reduced inhibition of H₂O₂ cytotoxic action by GM1 to the larger extent than each of the inhibitors and practically abolished the ability of GM1 to decrease H₂O₂-induced ROS accumulation. The protective and antioxidative effects of GM1 in PC12 cells exposed to H₂O₂ appear to be mediated by activation of Trk receptor tyrosine kinase and the protein kinases downstream from this enzyme.     

A Decrease of neuroprotective effect of ganglioside GM1 on PC12 cells under conditions of oxidative stress in the presence of inhibitor of tyrosine kinase of Trk-receptors

Effects of inhibitors of tyrosine kinases (K-252a, genistein) and of phospholipase A₂ (bromophenacyl bromide) on viability of PC12 cells are studied in the presence of hydrogen peroxide and ganglioside GM1. The degree of inhibition of hydrogen peroxide cytotoxic effects by ganglioside GM1 amounted to 52.8 ± 4.2%. However, in the presence in the medium of 0.1 and 1 μM inhibitors of tyrosine kinase of Trk-receptors (K-252a) it was as low as 32.7 ± 6.5% and 11.7 ± 9.8%, respectively. GM1 prevented Na⁺,K⁺-ATPase oxidative inactivation produced by H₂O₂, but in the presence of 1 μM K-252a this effect was practically not pronounced. In the presence of another inhibitor of tyrosine kinases-genistein, a tendency for a decrease of the GM1 protective effect was observed at its concentrations 0.1 and 1 μM, whereas at a higher concentration 10 μM, genistein depressed statistically significantly the GM1 neuroprotective effect. It was found that inhibitor of phospholipase A₂ bromophenacyl bromide did not affect the action of GM1 aimed at increasing the viability of cells under action of hydrogen peroxide on them. It seems that this enzyme is not involved in the cascade of reactions participating in realization of the ganglioside protective effect. Thus, inhibitor of tyrosine kinase of Trk-receptors K-252a decreases or practically prevents the ganglioside GM1 neuroprotective effect on PC12 cells under stress conditions; the same ability is characteristic of genistein—an inhibitor of tyrosine kinases of the wider spectrum of action.     

Neuroprotective Effect of Ganglioside GM1 on the Cytotoxic Action of Hydrogen Peroxide and Amyloid β-peptide in PC12 cells

Ganglioside GM1 was shown to increase the viability of PC12 cells exposed to hydrogen peroxide or amyloid β-peptide (Aβ_25–35). The PC12 cells transfected with mutant gene (expressing APP_SW) were found to be more sensitive to oxidative stress than the cells transfected with wild type gene (expressing APP_WT) or vector-transfected cells, GM1 being effective in enhancing the viability of the cells transfected with mutant gene. The exposure to hydrogen peroxide or Aβ_25–35 results in a partial inactivation of Na⁺,K⁺-ATPase in PC12 cells, H₂O₂ increases MDA accumulation in these cells. But these effects could be partially prevented or practically abolished by GM1 ganglioside. In the presence of the inhibitor of tyrosine kinase of Trk receptors (K-252a) the protective and metabolic effects of GM1 on PC12 cells in conditions of oxidative stress caused by hydrogen peroxide are not observed or are markedly diminished.     

Metabolic effects of ganglioside GM1 on PC12 cells in oxidative stress depend on modulation of activity of tyrosine kinase Trk of receptors

There have been obtained evidences that not only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of cells of the neuronal line PC12 under action of H₂O₂. By the example of GM1 and GD1a, gangliosides have been shown to produce a protective effect on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. For the first time, GM1 at nanomolar concentrations was shown to decrease the H₂O₂-induced formation of reactive oxygen species (ROS). It was found that in the presence of inhibitor of tyrosine kinase Trk of receptors K-252a, GM1 at concentrations of 10 μM and 10 nM lost its ability to produce such metabolic effects as a decrease of ROS accumulation and of the degree of oxidative inactivation of Na⁺,K⁺-ATPase in PC12 cells, as well as ceased to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of gangliosides GM1 in PC12 cells treated with H₂O₂ on modulation of activity of activity of tyrosine kinase Trk receptors (i.e., from the same signal system) agrees with concept about the essential role of oxidant effect of GM1 in its increase of cell viability.     

Effects of oxidative stress inducers, neurotoxins, and ganglioside GM1 on Na+, K+-ATPase in PC12 and brain synaptosomes

To elucidate mechanism of ganglioside neuroprotection, it is important to study their metabolic effects, specifically of action on Na+, K+ -ATPase. It has been shown that under effect of oxidative stress inductors and neurotoxins an oxidative inactivation of this enzyme takes place in PC12 cells and brain cortex synaptosomes, this inactivation being able to be prevented or decreased by ganglioside GM1. Thus, for instance, 24 h after action of 1 mM H2O2, activity of Na+, K+ -ATPase in PC12 cells decreased more than twice. However, in the case of preincubation of the cells with ganglioside GM1 prior to the H2O2 action this enzyme activity did not differ statistically significantly from control. Ganglioside GM1 also was able to increase significantly the enzyme activity decreased by action on the PC12 cells of amyloid beta-peptide (AP) causing lesion of neurons in Alzheimer's disease and at low H202 concentrations. Experiments on brain cortex synaptosomes have established that not only antioxidants--alpha-tocopherol and superoxide dismutase--but also ganglioside GM1 prevent the glutamateproduced Na+, K+ -ATPase oxidative inactivation. The obtained data agree with a suggestion that the ganglioside neuroprotective effect at action on nerve cells of such toxins as Abeta, glutamate or reactive oxygen species is due to their ability to inhibit the free-radical reactions.     

Different Metabolic Effects of Ganglioside GM1 in Brain Synaptosomes and Phagocytic Cells

The metabolic effects of ganglioside GM1 were found to be quite different in brain synaptosomes and phagocytic cells. Incubation of rat brain cortex synaptosomes with GM1 was shown to decrease the production of reactive oxygen species induced by Fe²⁺-H₂O₂ system and measured by chemiluminometric method in the presence of luminol. Gangliosides GM1, GD1a, and GT1b significantly diminished the induced accumulation of lipid peroxidation product in brain synaptosomes, but protein kinase inhibitor (polymyxin B) abolished this effect. Incubation with antioxidants or GM1 significantly diminished the increase of ⁴⁵Ca²⁺ influx and oxidative inactivation of Na⁺,K⁺-ATPase in brain synaptosomes exposed to glutamate, the effect of GM1 was concentration-dependent in the range 10^–11–10^–8 M. But the incubation of human neutrophils and mouse peritoneal macrophages with 10^–11–10^–10 M GM1, on the contrary, increased several times the luminol-dependent chemiluminescence response of these cells to activation by low concentrations of 12-myristate-13-acetate phorbol ester. The opposite effects of GM1 in the nerve endings and phagocytic cells seem to be protective in both cases as the inhibition of reactive oxygen species production in the nerve cells may enhance their viability in damaged brain, while the intensification of their production in phagocytic cells may promote the resistance of organism to infection.     

Quinone and oxyradical scavenging properties of N-acetylcysteine prevent dopamine mediated inhibition of Na+, K+-ATPase and mitochondrial electron transport chain activity in rat brain: Implications in the neuroprotective therapy of Parkinson's disease

Dopamine oxidation products such as H₂O₂ and reactive quinones have been held responsible for various toxic actions of dopamine, which have implications in the aetiopathogenesis of Parkinson's disease. This study has shown that N-acetylcysteine (0.25–1 mm) is a potent scavenger of both H₂O₂ and toxic quinones derived from dopamine and it further prevents dopamine mediated inhibition of Na⁺,K⁺-ATPase activity and mitochondrial respiratory chain function. The quinone scavenging ability of N-acetylcysteine is presumably related to its protective effect against dopamine mediated inhibition of mitochondrial respiratory chain activity. However, both H₂O₂ scavenging and quinone scavenging properties of N-acetylcysteine probably account for its protective effect against Na⁺,K⁺-ATPase inhibition induced by dopamine. The results have important implications in the neuroprotective therapy of sporadic Parkinson's disease since inactivation of mitochondrial respiratory activity and Na⁺,K⁺-ATPase may trigger intracellular damage pathways leading to the death of nigral dopaminergic neurons.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

Alterations on Na+,K+-ATPase and Acetylcholinesterase Activities Induced by Amyloid-β Peptide in Rat Brain and GM1 Ganglioside Neuroprotective Action

Alzheimer’s disease (AD) is a neurodegenerative disorder whose pathogenesis involves production and aggregation of amyloid-β peptide (Aβ). Aβ-induced toxicity is believed to involve alterations on as Na⁺,K⁺-ATPase and acetylcholinesterase (AChE) activities, prior to neuronal death. Drugs able to prevent or to reverse these biochemical changes promote neuroprotection. GM1 is a ganglioside proposed to have neuroprotective roles in AD models, through mechanisms not yet fully understood. Therefore, this study aimed to investigate the effect of Aβ1-42 infusion and GM1 treatment on recognition memory and on Na⁺,K⁺-ATPase and AChE activities, as well as, on antioxidant defense in the brain cortex and the hippocampus. For these purposes, Wistar rats received i.c.v. infusion of fibrilar Aβ1-42 (2 nmol) and/or GM1 (0.30 mg/kg). Behavioral and biochemical analyses were conducted 1 month after the infusion procedures. Our results showed that GM1 treatment prevented Aβ-induced cognitive deficit, corroborating its neuroprotective function. Aβ impaired Na⁺,K⁺-ATPase and increase AChE activities in hippocampus and cortex, respectively. GM1, in turn, has partially prevented Aβ-induced alteration on Na⁺,K⁺-ATPase, though with no impact on AChE activity. Aβ caused a decrease in antioxidant defense, specifically in hippocampus, an effect that was prevented by GM1 treatment. GM1, both in cortex and hippocampus, was able to increase antioxidant scavenge capacity. Our results suggest that Aβ-triggered cognitive deficit involves region-specific alterations on Na⁺,K⁺-ATPase and AChE activities, and that GM1 neuroprotection involves modulation of Na⁺,K⁺-ATPase, maybe by its antioxidant properties. Although extrapolation from animal findings is difficult, it is conceivable that GM1 could play an important role in AD treatment.     

Natural products inspired synthesis of neuroprotective agents against H2O2-induced cell death

Stroke is a debilitating disease and the third leading cause of death in the USA, where over 2000 new stroke cases are diagnosed every day. Treatment options for stroke-related brain damage are very limited and there is an urgent need for effective neuroprotective agents to treat these conditions. Comparison of the structures of several classes of neuroprotective natural products such as limonoids and cardiac glycosides revealed the presence of a common structural motif which may account for their observed neuroprotective activity. Several natural product mimics that incorporate this shared structural motif were synthesized and were found to possess significant neuroprotective activity. These compounds enhanced cell viability against H₂O₂ induced oxidative stress or cell death in PC12 neuronal cells. The compounds were also found to enhance and modulate Na⁺/K⁺-ATPase activity of PC12 cells, which may suggest that the observed neuroprotective activity is mediated, at least partly, through interaction with Na⁺/K⁺-ATPase.     

Overexpression of Na+/K+-ATPase Parallels the Increase in Sodium Transport and Potassium Recycling in an In Vitro Model of Proximal Tubule Cellular Ageing

Na⁺/K⁺-ATPase plays a key role in the transport of Na⁺ throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na⁺ and K⁺ ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na⁺/K⁺-ATPase activity and K⁺ conductance were evaluated using electrophysiological methods. Na⁺K⁺-ATPase α₁– and β₁-subunit expression was quantified by Western blot techniques. Na⁺/H⁺ exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na⁺ transport, which results from an increase in the number of Na⁺/K⁺-ATPase α₁- and β₁-subunits, in the membrane. Increases in Na⁺/K⁺-ATPase activity were accompanied by increases in K⁺ conductance as a result of functional coupling between Na⁺/K⁺-ATPase and basolateral K⁺ channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na⁺/H⁺ exchanger activity were observed. H₂O₂ production was increased in aged cells, but exposure for 5 days to 1 and 10 μM of H₂O₂ had no effect on Na⁺/K⁺-ATPase expression. Ouabain (100 nM) increased α₁-subunit, but not β₁-subunit, Na⁺/K⁺-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.     

Dopamine Oxidation Products Inhibit Na+, K+-ATPase Activity in Crude Synaptosomal–mitochondrial Fraction from Rat Brain

The diverse damaging effects of dopamine (DA) oxidation products on brain subcellular components including mitochondrial electron transport chain have been implicated in dopaminergic neuronal death in Parkinson's disease. It has been shown in this study that DA (50–200?μM) causes dose-dependent inhibition of Na⁺, K⁺-ATPase activity of rat brain crude synaptosomal–mitochondrial fraction during in vitro incubation up to 2?h. The enzyme inactivation is prevented by catalase and the metal-chelator (diethylenetriamine penta-acetic acid) but not by superoxide dismutase or hydroxyl-radical scavengers like mannitol and dimethylsulphoxide (DMSO). Further, reduced glutathione and cysteine, markedly prevent DA-mediated inactivation of Na⁺, K⁺-ATPase. Under similar conditions of incubation, DA (200?μM) leads to the formation of quinoprotein adducts (protein-cysteinyl catechol) with synaptosomal–mitochondrial proteins and the phenomenon is also prevented by glutathione (5?mM) or cysteine (5?mM).

The available data imply that the inactivation of Na⁺, K⁺-ATPase in this system involves both H²O² and metal ions. The reactive quinones by forming adducts with protein thiols also probably contribute to the process, since reduced glutathione and cysteine which scavenge quinones from the system protect Na⁺, K⁺-ATPase from DA-mediated damage. The inactivation of neuronal Na⁺, K⁺-ATPase by DA may give rise to various toxic sequelae with potential implications for dopaminergic cell death in Parkinson's disease.     

Role of tyrosine kinase of Trk-Receptors in realization of antioxidant effect of ganglioside GM1 in PC12 cells

Ganglioside GM1 has been shown to increase viability of PC12 cells at their induction of oxidative stress by hydrogen peroxide. However, in the presence of inhibitor of tyrosine kinase Trkreceptors K-252a this GM1 effect decreases or virtually disappears. To understand mechanism of the protective effect, there was studied action of H₂O₂, GM1, and inhibitor K-252a on formation of reactive oxygen species (ROS). It has been shown that ganglioside GM1 decreases significantly the H₂O₂-induced ROS accumulation in PC12 cells; however, in the presence of inhibitor of tyrosine kinase of Trk-receptors, this GM1 effect is not revealed. It has been found that inhibitors of each of protein kinases present at the signal realization stages following the stages of activation of tyrosine kinase Trk-receptors—Erk 1/2, PI3-kinases, and PKC, decreased the GM1 ability to reduce the H₂O₂-induced ROS accumulation, while at the combined use of inhibitors of these three protein kinases, the GM1 effect was completely absent. Thus, the ganglioside GM1 antioxidant effect on PC12 is mediated by activation of tyrosine kinase Trk-receptors and protein kinases perceiving signal from this enzyme.     

C-peptide,Na+,K+-ATPase,and Diabetes

Na⁺,K⁺-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na⁺,K⁺-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na⁺,K⁺-ATPase activity was strongly related to blood C-peptide levels in non–insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene.Apolymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na⁺,K⁺-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na⁺,K⁺-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na⁺,K⁺-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na⁺,K⁺-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na⁺,K⁺-ATPase activity. This impairment in Na⁺,K⁺-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetesinduced decrease in Na⁺,K⁺-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na⁺,K⁺-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na⁺,K⁺-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na⁺,K⁺-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

GM1 Ganglioside Activates ERK1/2 and Akt Downstream of Trk Tyrosine Kinase and Protects PC12 Cells Against Hydrogen Peroxide Toxicity

Ganglioside GM1 at micro- and nanomolar concentrations was shown to increase the viability of pheochromocytoma PC12 cells exposed to hydrogen peroxide and diminish the accumulation of reactive oxygen species and oxidative inactivation of Na⁺,K⁺-ATPase, the effects of micromolar GM1 being more pronounced than those of nanomolar GM1. These effects of GM1 were abolished by Trk receptor tyrosine kinase inhibitor and diminished by MEK1/2, phosphoinositide 3-kinase and protein kinase C inhibitors. Hydrogen peroxide activates Trk tyrosine kinase; Akt and ERK1/2 are activated downstream of this protein kinase. GM1 was found to activate Trk receptor tyrosine kinase in PC12 cells. GM1 (100 nM and 10 µM) increased the basal activity of Akt, but did not change Akt activity in cells exposed to hydrogen peroxide. Basal ERK1/2 activity in PC12 cells was increased by GM1 at a concentration of 10 µM, but not at nanomolar concentrations. Activation of ERK1/2 by hydrogen peroxide was enhanced by GM1 at a concentration of 10 µM and to a lesser extent at a concentration of 100 nM. Thus, the protective and metabolic effects of GM1 ganglioside on PC12 cells exposed to hydrogen peroxide appear to depend on the activation of Trk receptor tyrosine kinase and downstream activation of Akt and ERK1/2.     

Regulation of Na,K-ATPase Transport Activity by Protein Kinase C

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the α-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC. Received: 10 July 1996/Revised: 19 September 1996     

Iron-Induced Inhibition of Na+, K+-ATPase and Na+/Ca2+ Exchanger in Synaptosomes: Protection by the Pyridoindole Stobadine

The effect of oxidative stress, induced by Fe²⁺-EDTA system, on Na⁺,K⁺-ATPase, Na⁺/Ca²⁺ exchanger and membrane fluidity of synaptosomes was investigated. Synaptosomes isolated from gerbil whole forebrain were incubated in the presence of 200 M FeSO₄-EDTA per mg of protein at 37°C for 30 min. The oxidative insult reduced Na⁺,K⁺-ATPase activity by 50.7 ± 5.0 % and Na⁺/Ca²⁺ exchanger activity measured in potassium and choline media by 47.1 ± 7.2 % and 46.7 ± 8.6 %, respectively. Membrane fluidity was also significantly reduced as observed with the 1,6-diphenyl-1,3,5-hexatriene probe. Stobadine, a pyridoindole derivative, prevented the decrease in membrane fluidity and in Na⁺/Ca²⁺ exchanger activity. The Na⁺,K⁺-ATPase activity was only partially protected by this lipid antioxidant, indicating a more complex mechanism of inhibition of this protein. The results of the present study suggest that the Na⁺/Ca²⁺ exchanger and the Na⁺,K⁺-ATPase are involved in oxidation stress-mediated disturbances of intracellular ion homeostasis and may contribute to cell injury.     

Relations Between Intracellular Ions and Energy Metabolism Under Acidotic Conditions: A Study with Nigericin in Synaptosomes, Neurons, and C6 Glioma Cells

Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na⁺]_i., [K⁺]_i, and [Ca²⁺]_i, and pH under conditions when [H⁺]_i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K⁺ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca²⁺], and [Na⁺]., The increase in [Na⁺], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K⁺ and subsequent depolarization lead to an increase in Na⁺ influx and stimulation of the Na⁺/K⁺ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H⁺] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K⁺], depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Characterization,quantitation, similarity evaluation and combination with Na+,K+-ATPase of cardiac glycosides from Streblus asper

Streblus asper Lour. (Moraceae) is a medicinal plant in Asian countries including India and Thailand, possessing activities of anti-tumor, anti-allergy, anti-parasitic and anti-bacterial. In this paper, characterization, quantitation and similarity evaluation of cardiac glycosides in different parts of S. asper were investigated by HPLC-Q-TOF-MS and chemometric methods. Then, the inhibition of Na⁺,K⁺-ATPase activity by the compounds isolated from S. asper was measured. Meanwhile, enzyme kinetics and molecular docking were determined to exhibit the combination modes between cardiac glycosides and Na⁺,K⁺-ATPase. As a result, twenty peaks of cardiac glycosides were assigned. Strophanthidin-3-O-α-l-rhamnopyranosyl-(1 → 4)-6-deoxy-β-d-allopyranoside (1), glucostrebloside (2), strebloside (4) and mansonin (8) with a significant activity of inhibiting Na⁺,K⁺-ATPase (IC₅₀ 7.55–13.60 μM) were chosen for the determination of enzyme kinetics, exhibiting anticompetitive inhibitory characteristics towards Na⁺,K⁺-ATPase. Compound 4 could reasonably bind to the active sites of Na⁺,K⁺-ATPase, proved by molecular docking. Furthermore, the contents of the major compounds in four different parts of S. asper were extremely different, analyzed by chemometric methods, similarity analysis and principle compounds analysis. All these findings indicated that the contents of major compounds in different parts of S. asper were extremely different with a significant activity of inhibiting Na⁺,K⁺-ATPase, providing a reference for determination of effective part and administered dosage. The combination modes between cardiac glycosides and Na⁺,K⁺-ATPase were also revealed by enzyme kinetics and molecular docking, which provided a basis for further study of pharmacological activity.