Decrease In Cellular ATP Concentration Research Articles

Ochratoxin A causes mitochondrial dysfunction, apoptotic and autophagic cell death and also induces mitochondrial biogenesis in human gastric epithelium cells.

Ochratoxin A (OTA) is a common natural contaminant found in human and animal food worldwide. Our previous work has shown that OTA can cause oxidative DNA damage, G2 arrest and malignant transformation of human gastric epithelium (GES-1) cells. Mitochondria are considered to be target for the action of many cytotoxic agents. However, the role of mitochondria in the cytotoxicity of OTA remains unknown. The aim of this study is to explore the putative role of mitochondria on OTA cytotoxicity by analyzing mitochondrial changes in GES-1 cells. The results showed that OTA treatment (5, 10, 20µM) for different times caused increases in the production of reactive oxygen species, and induced mitochondrial damage, shown by loss of mitochondrial membrane potential (ΔΨM), and decrease in cellular ATP concentration. Subsequently, the mitochondrial apoptotic pathway was activated, presented by increase of apoptotic rate and activation of apoptotic proteins. Autophagic cell death was also triggered, demonstrated by the conversion of light chain 3B (LC3B)-I to LC3B-II and elevated levels of green fluorescent protein-LC3 (GFP-LC3) puncta. Moreover, Parkin-dependent mitophagy was also activated presented by the colocalization of MitoTracker with LysoTracker or GFP-LC3 puncta. The inhibition of autophagy and mitophagy by inhibitors or siRNA attenuated the toxic effect of OTA on cell growth. Interestingly, OTA treatment also enhanced mitochondrial biogenesis confirmed by activation of AMPK/PGC-1α/TFAM pathway and promoted cell survival. Collectively, the effects of OTA on mitochondria of GES-1 cells are complex. OTA could cause mitochondrial function disturbance, apoptotic and autophagic cell death and also induce mitochondrial biogenesis.

Nelfinavir and other protease inhibitors in cancer: mechanisms involved in anticancer activity.

To review the mechanisms of anti-cancer activity of nelfinavir and other protease inhibitors (PIs) based on evidences reported in the published literature. We extensively reviewed the literature concerning nelfinavir (NFV) as an off target anti-cancer drug and other PIs. A classification of PIs based on anti-cancer mode of action was proposed. Controversies regarding nelfinavir mode of action were also addressed. The two main mechanisms involved in anti-cancer activity are endoplasmic reticulum stress-unfolded protein response pathway and Akt inhibition. However there are many other effects, partially dependent and independent of those mentioned, that may be useful in cancer treatment, including MMP-9 and MMP-2 inhibition, down-regulation of CDK-2, VEGF, bFGF, NF-kB, STAT-3, HIF-1 alfa, IGF, EGFR, survivin, BCRP, androgen receptor, proteasome, fatty acid synthase (FAS), decrease in cellular ATP concentration and upregulation of TRAIL receptor DR5, Bax, increased radiosensitivity, and autophagy. The end result of all these effects is slower growth, decreased angiogenesis, decreased invasion and increased apoptosis, which means reduced proliferation and increased cancer cells death. PIs may be classified according to their anticancer activity at clinically achievable doses, in AKT inhibitors, ER stressors and Akt inhibitors/ER stressors. Beyond the phase I trials that have been recently completed, adequately powered and well-designed clinical trials are needed in the various cancer type settings, and specific trials where NFV is tested in association with other known anti-cancer pharmaceuticals should be sought, in order to find an appropriate place for NFV in cancer treatment. The analysis of controversies on the molecular mechanisms of NFV hints to the possibility that NFV works in a different way in tumor cells and in hepatocytes and adipocytes.

Nelfinavir and other protease inhibitors in cancer: mechanisms involved in anticancer activity

To review the mechanisms of anti-cancer activity of nelfinavir and other protein inhibitors (PIs) based on evidences reported in the published literature. We extensively reviewed the literature concerning nelfinavir (NFV) as an off target anti-cancer drug and other PIs. A classification of PIs based on anti-cancer mode of action was proposed. Controversies regarding nelfinavir mode of action were also addressed. The two main mechanisms involved in anti-cancer activity are endoplasmic reticulum stress-unfolded protein response pathway and Akt inhibition. However there are many other effects, partially dependent and independent of those mentioned, that may be useful in cancer treatment, including MMP-9 and MMP-2 inhibition, down-regulation of CDK-2, VEGF, bFGF, NF-kB, STAT-3, HIF-1 alfa, IGF, EGFR, survivin, BCRP, androgen receptor, proteasome, fatty acid synthase (FAS), decrease in cellular ATP concentration and upregulation of TRAIL receptor DR5, Bax, increased radiosensitivity, and autophagy. The end result of all these effects is slower growth, decreased angiogenesis, decreased invasion and increased apoptosis, which means reduced proliferation and increased cancer cells death. PIs may be classified according to their anticancer activity at clinically achievable doses, in AKT inhibitors, ER stressors and Akt inhibitors/ER stressors. Beyond the phase I trials that have been recently completed, adequately powered and well-designed clinical trials are needed in the various cancer type settings, and specific trials where NFV is tested in association with other known anti-cancer pharmaceuticals should be sought, in order to find an appropriate place for NFV in cancer treatment. The analysis of controversies on the molecular mechanisms of NFV hints to the possibility that NFV works in a different way in tumor cells and in hepatocytes and adipocytes.

Extracts of Edible and Medicinal Plants Damage Membranes of Vibrio cholerae

The use of natural compounds from plants can provide an alternative approach against food-borne pathogens. The mechanisms of action of most plant extracts with antimicrobial activity have been poorly studied. In this work, changes in membrane integrity, membrane potential, internal pH (pH(in)), and ATP synthesis were measured in Vibrio cholerae cells after exposure to extracts of edible and medicinal plants. A preliminary screen of methanolic, ethanolic, and aqueous extracts of medicinal and edible plants was performed. Minimal bactericidal concentrations (MBCs) were measured for extracts showing high antimicrobial activity. Our results indicate that methanolic extracts of basil (Ocimum basilicum L.), nopal cactus (Opuntia ficus-indica var. Villanueva L.), sweet acacia (Acacia farnesiana L.), and white sagebrush (Artemisia ludoviciana Nutt.) are the most active against V. cholera, with MBCs ranging from 0.5 to 3.0 mg/ml. Using four fluorogenic techniques, we studied the membrane integrity of V. cholerae cells after exposure to these four extracts. Extracts from these plants were able to disrupt the cell membranes of V. cholerae cells, causing increased membrane permeability, a clear decrease in cytoplasmic pH, cell membrane hyperpolarization, and a decrease in cellular ATP concentration in all strains tested. These four plant extracts could be studied as future alternatives to control V. cholerae contamination in foods and the diseases associated with this microorganism.

Abstract 1440: Endothelial And Cardiomyocyte Toxicity Of Endogenously Produced Nucleoside: 4-pyridone-3-carboxamide-1-β-d-riboside

Our recent studies identified hitherto unknown, naturally occurring nucleotide: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PYTP). This nucleotide was found to accumulate in the erythrocytes of patients with chronic renal failure, a condition that precipitates heart failure and atherosclerosis. We found also increase in plasma concentration of nucleoside precursor of 4PYTP: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) in patients with renal failure. This study was aimed to evaluate metabolism and potential toxic effect of this nucleoside in cardiomyocytes and endothelial cells. We used human erythrocytes isolated from peripheral blood, human endothelial cell line HMEC-1 cell line and cardiomyocytes isolated from rat hearts. Cells were incubated with chemically synthesized 4PYR. Metabolic assessments were performed with HPLC or liquid chromatography/mass spectrometry. We demonstrated formation of 4PYTP in human erythrocytes during incubation with 4PYR. We noted however, preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP. Concentration of 4PYMP increased in the erythrocytes from below 5 μM to 76.9±7.1, 254.7±13.9 and 674.3±34.3 μM after 6h incubation with 0.1, 0.3 and 1 mM 4PYR. 4PYMP progressively accumulated in the cultured endothelial cells during incubation with 4PYR up to 72 h. Cardiomyocytes were also shown to accumulate 4PYMP. In all cell types formation of 4PYR nucleotides was accompanied by decrease in cellular ATP concentration. Furthermore, treatment with 4PYR and formation of its nucleotides in endothelial cells reduced NO synthase pathway while in cardiac myocytes resulted in prolongation of action potential. We conclude that endogenously formed 4PYR is effectively metabolized to nucleotide derivatives in the erythrocytes, cardiomyocytes and endothelium. Depletion of ATP in these cells accompanying 4PYR metabolism and deleterious effects on cell function suggests that accumulation of 4PYR and its enhanced metabolism to nucleotides in endothelium and cardiomyocyted may contribute to cardiovascular pathology observed in chronic renal failure.

Impairment of the mitochondrial respiratory enzyme activity triggers sequential activation of apoptosis‐inducing factor‐dependent and caspase‐dependent signaling pathways to induce apoptosis after spinal cord injury

The mitochondrion participates in caspase-independent or caspase-dependent apoptotic pathways through the release of apoptosis-inducing factor or cytochrome c. Whether both mitochondrial apoptotic cascades are triggered in the injured spinal cord remains unknown. Here, we demonstrated that neurons, astrocytes and microglia in spinal segments proximal to a complete spinal cord transection underwent two phases of apoptotic cell death. The early phase of high-molecular weight (HMW) DNA fragmentation was associated with nuclear translocation of apoptosis-inducing factor, reduction in mitochondrial respiratory chain enzyme activity and decrease in cellular ATP concentration. The delayed phase of low-molecular weight (LMW) DNA fragmentation was accompanied by cytosolic release of cytochrome c, activation of caspases 9 and 3, and resumption of mitochondrial respiratory functions and ATP contents. Microinfusion of coenzyme Q(10), an electron carrier in mitochondrial respiratory chain, into the epicenter of the transected spinal cord attenuated both phases of induced apoptosis, and reversed the elicited mitochondrial dysfunction, bioenergetic failure, and activation of apoptosis-inducing factor, cytochrome c, or caspases 9 and 3. We conclude that mitochondrial dysfunction after spinal cord transection represents the initiating cellular events that trigger the sequential activation of apoptosis-inducing factor-dependent and caspase-dependent signaling cascades, leading to apoptotic cell death in the injured spinal cord.

Reduced iC3b-mediated phagocytotic capacity of pulmonary neutrophils in cystic fibrosis

Cystic fibrosis (CF) is characterized by a neutrophil-dominated chronic inflammation of the airways with persistent infections. In order to investigate whether neutrophils contribute to an inadequacy in the pulmonary defence mechanism, the phagocytic activity of pulmonary and peripheral blood neutrophils from CF and non-CF respiratory patients were compared. Neutrophils were isolated from both the blood and bronchoalveolar lavage fluid of 21 patients with CF (12 male, 9 female; mean age 7.5 years, range 0.25-16.4 years) and 17 non-CF subjects (9 male, 8 female; mean age 5.4 years, range 0.2-13.1 years). The ex vivo phagocytic rate of normal pulmonary neutrophils to internalize zymosan particles opsonized with iC3b was faster than that of circulating neutrophils (P < 0.05), but the maximum capacity (9 particles/cell) was similar. In contrast, pulmonary neutrophils from patients with CF had a lower phagocytic capacity than circulating neutrophils either from the same patients or from normal subjects. This deficiency could not be attributed to (i) the cell surface density of CR3 (CD18/CD11b) receptors, which were not significantly different between the other groups (ii) the signalling ability of the CR3 receptors, using cytosolic free Ca(2+) signalling as the receptor activity read-out or (iii) a decrease in cellular ATP concentration. As CFTR was not detectable on neutrophils from any source by either histochemistry or Western blotting, it was concluded that the reduced phagocytic capacity was not the direct result of a CFTR mutation, but was attributed to a failure of neutrophil phagocytic priming during translocation into the CF lung.

The effect of adenosine metabolism inhibition and nucleotide precursor supply on adenosine production in human heart endothelial cells.

Supply of adenine together with inhibitors of adenosine metabolism allowed to protect ATP in human endothelial cells, but unlike in rat endothelial cells no further increase in adenosine production was observed. Inhibition of adenosine kinase and adenosine deaminase with supply of adenine is an effective procedure to persistently increase endogenous adenosine production in the endothelium of the human heart without causing decrease in cellular ATP concentration.

Anoxia-tolerant hepatocytes: model system for study of reversible metabolic suppression.

Chrysemys picta bellii is well known for its ability to survive extended anoxic periods and has been widely used as a model system to study anoxic metabolism. Described here is a method for the isolation of anoxia-tolerant hepatocytes from this species. Freshly isolated hepatocytes were determined to be viable based on trypan blue exclusion, gluconeogenic capacity from [14C]lactate, responsiveness to epinephrine and glucagon, and maintenance of cellular adenylate concentrations. Under anoxic conditions for 10 h there was no significant increase in cell staining and no decrease in cellular ATP concentration. Furthermore, the addition of cyanide at the 5-h mark did not result in any significant differences in these parameters; however, iodoacetate added at this time caused trypan blue staining to increase and ATP concentrations to fall. The rate of glucose production from the cells was threefold greater under anoxic than normoxic conditions, underscoring the important role of the liver in supplying substrate during anoxia. From the rate of O2 consumption and rate of lactate production under anaerobic conditions, ATP turnover rates were calculated to be 68.4 +/- 7.2 and 6.5 +/- 0.43 mumol ATP.g-1.h-1, respectively; this corresponds to a 90% decrease in metabolic rate during anoxia. Within a cellular system such as this the more complex regulatory mechanisms involved in a large coordinated reduction in metabolism can be probed.

Tumor necrosis factor induced oxidative stress in isolated mouse hepatocytes

Tumor necrosis factor α (TNFα) is a macrophage-derived cytokine which participates in homeostatic tissue repair. It is also a potentially useful antitumor agent. Liver toxicity, however, limits TNFα's clinical utility and suggests that it may play a role in liver toxicity of various etiologies. To determine the direct effects of TNFα on hepatocytes, in the absence of infiltrating leukocytes and other inflammatory mediators, an isolated mouse hepatocyte model has been used in the present study. Hepatocytes exposed to recombinant human TNFα (1–10 μg/ ml) exhibited intracellular GSH depletion and GSSG efflux during the first 2 hr of exposure, but no cytotoxicity was observed. However, TNFα was toxic to hepatocytes pretreated with 1,3-bis(chloroethyl)-1-nitrosourea to inhibit GSSG-reductase activity. Furthermore, these cells exhibited a greater efflux of GSSG upon exposure to TNFα. TNFα also caused a marked decrease in cellular ATP concentrations, which occurred after initiation of effects on the glutathione pool. These findings indicate that high concentrations of TNFα induce an oxidant stress in isolated hepatocytes. The antioxidants mannitol and benzoate, as well as the iron chelator deferoxamine, reduced the extent of TNFα-induced oxidant effects in hepatocytes, which indicates that the oxidant stress may involve hydroxyl radical generation. Hepatocytes treated with ruthenium red or fructose were less susceptible to TNFα-induced ATP depletion, which suggests that mitochondrial calcium cycling may be involved in disruption to energy metabolism.

Transport of 4-hydroxyphenylacetic acid in Klebsiella pneumoniae

Klebsiella pneumoniae M5a1 has been shown to possess an inducible transport system for 4-hydroxyphenylacetate (4-HPA). This transport system has a K t of 16.3 μ m and a maximal velocity of 31.2 nmol/min (milligrams dry weight). The transport system has been inhibited by inhibitors of energy metabolism with a concomitant decrease in cellular ATP concentrations, and the 4-HPA binding activity has been detected in the crude shock extracts. All these observations indicate that 4-HPA uptake is an active transport which involves a periplasmic binding protein and it seems to be energized by phosphate bond energy.

Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na +/K +/Cl − cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 μM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener 86Rb + to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of 86Rb + influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359–367) as being mediated by a Na +/K +/Cl − cotransporter. Inhibition by 10 μM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of 86Rb + influx, mediated by Na +/K +-ATPase. After 3 h of incubation with 10 μM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [ 3H]TPMP +, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K + influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumour activity, did not inhibit 86Rb + influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 ± 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na +/K +/Cl − cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.

Dependence of fluid-phase pinocytosis in arterial smooth-muscle cells on temperature, cellular ATP concentration and the cytoskeletal system.

125I-labelled poly(vinylpyrrolidone) was used as a marker of fluid-phase pinocytosis in cultured pig arterial smooth-muscle cells. The rate of pinocytosis was temperature-dependent. A decrease in cellular ATP concentrations as a result of inhibition of either glycolysis or oxidative phosphorylation was associated with a similar decrease in pinocytosis. A microfibrillar-disruptive agent, cytochalasin B, caused a concentration-dependent stimulation of pinocytosis, whereas the microtubular-disruptive agents colchicine and vinblastine decreased pinocytosis to approximately half of control values at all concentrations used. These results indicate that fluid-phase pinocytosis in smooth-muscle cells is dependent on a continuing supply of energy and the integrity of the microtubules. Furthermore, microfilaments appear to exert a certain degree of constraint on pinocytosis, possibly by restricting invagination of the plasma membrane.

Adrenergic regulation of haemoglobin oxygen affinity in rainbow trout red cells

Adrenaline (initial concentration 5×10−6 M) caused an increase in haemoglobin oxygen affinity of rainbow trout red cells incubated in vitro in physiological saline. The increase was more pronounced at low than at high extracellular pH values. Adrenaline also induced an increase in intracellular pH, swelling of red cells and a metabolic decrease in cellular ATP concentration. The most important reason for increased oxygen affinity was the increase in intracellular pH resulting, to a great extent, from decreased concentration of impermeable polyanions, ATP and haemoglobin, in the cell. In addition, the decrease in cellular ATP concentration as such increases oxygen affinity of haemoglobin.

ATP concentration in Escherichia coli during oxygen toxicity

Escherichia coli, strain E-26, grown in defined salts medium with glucose as the sole carbon and energy source, contained 1.50 ± 0.16 · 10 6 molecules of ATP/cell. ATP was extracted with HClO 4 and assayed with a Dupont Luminescence Biometer using the luciferin-luciferase assay. Exposure during exponential growth at 37°C to 4.2 atm of oxygen resulted in complete growth cessation within 5 min, and to cyclic changes in cellular ATP concentration over a 2 h period. However, significant decrease in cellular ATP concentration occurred after growth inhibition in hyperbaric oxygen; hence, lack of ATP was not the cause of growth inhibition from oxygen toxicity.

Isolated rat hepatocytes as an experimental tool in the study of cell injury. Effect of anoxia

Rat hepatocytes were isolated by liver perfusion in the presence of collagenase and hyaluronidase and incubated in the absence or presence of oxygen. As a result of anoxia, there was a gradual increase in plasma membrane permeability, noted as an increase in succinate-stimulated oxygen uptake, a decrease in trypan blue exclusion frequency, a leakage of cytosolic lactate dehydrogenase activity and an increased proportion of swollen and disrupted cells. After anaerobic incubation for 30 minutes — but not for 60 minutes — there were signs of recovery from anoxic cell injury upon re-oxygenation. The changes in plasma membrane permeability properties in anoxia seemed to be preceded by a marked decrease in cellular ATP level; aerobic incubation of hepatocytes in the presence of an uncoupler of phosphorylation from respiration led to a similar decrease in cellular ATP concentration followed by similar disturbances in plasma membrane permeability properties. It is suggested that a disturbed plasma membrane function caused by a decreased energy level is of primary importance for the initiation of cell death in anoxia.