Changes In Intracellular ATP Levels Research Articles

Ivermectin induces mitophagy in H9c2 cells via activation of the PINK1/Parkin pathway

Abstract Ivermectin (IVM), a remarkable broad-spectrum anthelmintic and insecticides, which also contributes to clinical application. However, IVM induction of cytotoxicity through mitophagy has not been consistently proven in vitro. Here we investigate the cytotoxic effects of ivermectin in mammalian nontarget cells. We have used cck-8 assay to evaluate the cell viability. The Fluorescence labeling was detected the occurrence of autophagy and mitophagy. The ATP bioluminescence detection kit was used to detect changes in intracellular ATP levels. In addition, the western blot assay was applied to reflect the expression of autophagy-related and mitophagy- related proteins. Cellular calcium concentration analysis was captured with a flow cytometer. We also used western blot to reveal expression of the lysosomal membrane protein Lamp2. The expression of lysosomal cathepsin mRNA was evaluated by RT-PCR. The cell viability was significantly diminished in H9c2 cells and the number of autophagosomes increases in a dose-dependent way, at the same time, LC3-II / I ratio was increased. We also found that the H9c2 cellular ATP level was decreased and observed the mPTP opening. The co-localization of mitochondria and lysosomes was detected, also, we found the concentration of Ca2+, the expression of Lamp 2 and mRNA expression levels of Cathepsin B and Cathepsin L significantly increased in a dose-dependent way in H9c2 cells. Finally, the results of expression of PINK1 and Parkin protein increased simultaneously in H9c2 cells. IVM induced mitophagy in H9c2 cells via PINK1/Parkin signaling.

Noise-Induced Loss of Hair Cells and Cochlear Synaptopathy Are Mediated by the Activation of AMPK.

Noise-induced hearing loss (NIHL) is a major unresolved public health problem. Here, we investigate pathomechanisms of sensory hair cell death and suggest a novel target for protective intervention. Cellular survival depends upon maintenance of energy homeostasis, largely by AMP-activated protein kinase (AMPK). In response to a noise exposure in CBA/J mice, the levels of phosphorylated AMPKα increased in hair cells in a noise intensity-dependent manner. Inhibition of AMPK via siRNA or the pharmacological inhibitor compound C attenuated noise-induced loss of outer hair cells (OHCs) and synaptic ribbons, and preserved auditory function. Additionally, noise exposure increased the activity of the upstream AMPK kinase liver kinase B1 (LKB1) in cochlear tissues. The inhibition of LKB1 by siRNA attenuated the noise-increased phosphorylation of AMPKα in OHCs, reduced the loss of inner hair cell synaptic ribbons and OHCs, and protected against NIHL. These results indicate that noise exposure induces hair cell death and synaptopathy by activating AMPK via LKB1-mediated pathways. Targeting these pathways may provide a novel route to prevent NIHL. Our results demonstrate for the first time that the activation of AMP-activated protein kinase (AMPK) α in sensory hair cells is noise intensity dependent and contributes to noise-induced hearing loss by mediating the loss of inner hair cell synaptic ribbons and outer hair cells. Noise induces the phosphorylation of AMPKα1 by liver kinase B1 (LKB1), triggered by changes in intracellular ATP levels. The inhibition of AMPK activation by silencing AMPK or LKB1, or with the pharmacological inhibitor compound C, reduced outer hair cell and synaptic ribbon loss as well as noise-induced hearing loss. This study provides new insights into mechanisms of noise-induced hearing loss and suggests novel interventions for the prevention of the loss of sensory hair cells and cochlear synaptopathy.

Mitochondrial SIRT3 is upregulated by Glucagon-like peptide-1 receptor activation and contributes to reversal of cardiac mitochondrial remodeling induced by type 2 diabetes

Rationale: Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates mitochondrial function and maintains basal ATP yield, whereas the role in myocardium remain uncertain. We reported that the GLP-1 receptor agonist exendin-4 (Ex-4) ameliorated cardiac mitochondrial remodeling in diabetic cardiomyopathy via cyclic AMP axis (ESC 2012, AHA 2012). Changes in intracellular ATP levels are associated with cyclic AMP, which is generated by adenylyl cyclase utilizing ATP, we thus hypothesized that SIRT3 may involve in Ex-4-mediated myocardial reverse remodeling in diabetes. Methods: Type 2 diabetic mice were allocated into Ex-4 (24 nmole/kg/day, DIO/Ex-4) and vehicle (DIO/CON). Heart samples and cultured cardiomyocytes were subjected to mitochondrial fractionation followed by mRNA and protein analyses. Results: Cardiac cyclic AMP concentration and phosphorylation of CREB were elevated in DIO/Ex-4, suggesting successful administration of Ex-4. Electron microscopy revealed that DIO/CON exhibited mitochondria with destroyed cristae structure and defragmented, which was reversed by Ex-4 (Fig. A). The ratio of Mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2), which modulates mitochondria morphology, was consistently suppressed by Ex-4 (Fig. B), suggesting normalization of mitochondrial morphology. DIO-CON exhibited decrease in cardiac SIRT3 level compared to nondiabetic counterpart, which was reversed by Ex-4 (Fig. C). Increased cAMP levels (Isoprotelenol and 8-bromo-cyclic AMP) enhanced SIRT3 mRNA and protein levels in cultured cardiomyocytesm which was blocked by AMP-activated protein kinase (AMPK) inhibitor (compound C). ![Figure][1] Conclusions: Our results indicate that Ex-4 reversed abnormal suppression of SIRT3 in mitochondria via activating the PKA/AMPK-dependent pathway. [1]: pending:yes

Changes in Cytosolic ATP Levels and Intracellular Morphology during Bacteria-Induced Hypersensitive Cell Death as Revealed by Real-Time Fluorescence Microscopy Imaging

Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection.

Dosage effects of resveratrol on ethanol-induced cell death in the human K562 cell line

Previous studies have established that ethanol induces cell apoptosis and necrosis. However, the precise molecular mechanisms are currently unclear. Here, we show that higher concentrations of ethanol (250–400 mM) induced a shift from apoptotic to necrotic cell death in human K562 cells, and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, inhibited or enhanced ethanol-induced apoptosis/necrosis depending on the treatment dosage. Using the cell permeable dye 2′,7′-dichlorofluorescin diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation, we showed that ethanol treatment directly increased intracellular oxidative stress. This intracellular oxidative stress increased in response to high concentrations (100–200 μM) of resveratrol, but remained unchanged following treatment with low concentrations (10–25 μM) of resveratrol. Further studies showed that resveratrol could attenuate or enhance ethanol-induced intracellular oxidative stress generation-dependent on treatment dosage, and that this effect could be correlated with cell apoptosis or necrosis. Importantly, ethanol-induced changes in intracellular ATP levels were also correlated with resveratrol dosage. Taken together, these results indicate that the treatment dosage may determine the effect of resveratrol on ethanol-induced ROS generation, intracellular ATP levels, and cell apoptosis or necrosis. Thus our findings support the possibility that appropriate dosage of resveratrol aids in decreasing the toxic effect of ethanol.

Tumor Necrosis Factor-α Increases ATP Content in Metabolically Inhibited L929 Cells Preceding Cell Death

The effects of tumor necrosis factor-alpha (TNF) on ATP levels were studied in metabolically inhibited L929 cells. Treatment of these cells with TNF in the presence of actinomycin D or cycloheximide induces cyclic changes in the intracellular ATP content preceding cell death. After 3 h of incubation, the intracellular ATP content increased by 48 +/- 6% (p < 0.001), but at 4 h, it decreased to the control level. Two hours later, it increased again by 23 +/- 5% over the control level (p < 0.001). Coinciding with cell death, ATP content decreased progressively until almost complete depletion. These changes in ATP content were associated with parallel alterations in the respiratory coupling and with increased generation of reactive oxygen species. The mechanism by which TNF/actinomycin D or TNF/cycloheximide increased cellular ATP seemed to be dependent on the mitochondrial ATP synthesis and related to the cytotoxic effect of TNF, since blockade of mitochondrial electron transport prevented the increase in cellular ATP, the formation of reactive oxygen species, and the apoptotic cell death caused by TNF. We suggest that the TNF/actinomycin D- or TNF/cycloheximide-induced changes in intracellular ATP levels may be involved in the cytotoxic effect of TNF in metabolically inhibited L929 cells.

ATP is a coupling modulator of parallel Na,K-ATPase-K-channel activity in the renal proximal tubule.

A fundamental and essential property of nearly all salt-transporting epithelia is the tight parallel coupling between the magnitude of the K-conductive pathway at the basolateral membrane and the activity of the Na,K-dependent ATPase (Na,K-ATPase). In the present study, we demonstrate that the coupling response in the renal proximal tubule is governed, at least in part, through the interaction between ATP-sensitive K channels and Na,K-ATPase-mediated changes in intracellular ATP levels. First, we identified a K-selective channel at the basolateral membrane, which is inhibited by the cytosolic addition of ATP. Second, conventional microelectrode analysis in the isolated perfused proximal straight tubule revealed that these channels are the major determinant of the macroscopic K conductance so that ATP-mediated changes in the open probability of the K channel could alter the extent of K recycling. Indeed, the increase in the macroscopic K conductance upon stimulation of transcellular Na transport and pump activity was found to be paralleled by a decrease in intracellular ATP. Finally, a causal link between parallel Na,K-ATPase-K-channel activity and ATP was established by the finding that intracellular ATP loading uncoupled the response. With our recent observations that similar ATP-sensitive K channels are expressed abundantly in other epithelia, we postulate that ATP may act as a universal coupling modulator of parallel Na,K-ATPase-K-channel activity.

Reversible inactivation of the Ah receptor associated with changes in intracellular ATP levels.

We have previously demonstrated that incubation of Hepa-1 cells in the presence of cytochalasin B (CB) results in a time- and temperature-dependent loss of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity (Gudas et al., 1986). We show here that this loss of binding activity is probably attributable to a CB induced inhibition of glucose transport, as incubation of cells in the presence of glycolytic inhibitors or in glucose free medium caused a similar effect. All conditions leading to loss of binding also caused a marked reduction in cellular ATP concentration, suggesting that ATP (or perhaps another energy-dependent molecule) is required for maintaining the receptor in the active state. Inactivation of the Ah receptor occurred in the cytosol but not when it had translocated to the nucleus. Reactivation of receptor binding activity occurred readily in vivo and did not require de novo protein synthesis. However, attempts to restore receptor binding activity in vitro were not successful. To our knowledge this is the first reported evidence indicating that the TCDD binding Ah receptor can exist in both an inactive and an active form, with the amount present in the active ligand binding form being coupled to the energy state of the cells.

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis.

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.

Microcalorimetric studies of tissue cells and bacteria

AbstractAnalytical microcalorimetric studies of cultures of Enterobacter aerogenes and Clostridium pasteurianum revealed a complex pattern of heat production. While both organisms gave thermograms which followed the exponential growth curves in the early stages, the later events in the heat effect appeared to reflect deficiencies in the growth medium and induction of “secondary” nutrients. Synchronously dividing Schizosaccharomyces pombe is known to give maxima in oxygen uptake during the cell cycle. These peaks are enhanced by the uncoupling agent, 2‐oxomalononitrile m‐chlorophenylhydrazone (CCCP). The rate of heat production in control cultures pumped through the LKB microcalorimeter increased at a linear rate, doubling over each complete cell cycle. Introduction of CCCP to cultures gave an altered thermogram in which the maxima of heat evolution which occurred corresponded in time to the peaks in oxygen uptake.When suspensions of muscle fibroblasts from 9 day old chick embryos were pumped through the aeration vessel, they produced heat at a rate which was proportional to cell concentration. Inhibitors of respiration decreased heat evolution by similar percentages to their effect on oxygen uptake. Cells of the same type in monolayer gave slightly lower levels of heat production. The thermogram was characterised by maxima over 4 h, which may be a reflection of temporal changes in intracellular ATP levels. When muscle fibroblasts were mixed with excess (zero order kinetics) ATP and pumped through the gold vessel of the microcalorimeter, the rate of heat production was proportional to cell concentration, the latter representing the hydrolysing enzyme. 4,4′‐Dithiodi(nicotinic acid), a sulphydryl inhibitor which does not enter cells, depressed the calorimetric response vs cell concentration curve by 78%, indicating that a sulphydryl enzyme was present at the cell surface. Batch calorimetric experiments to determine the heat of interaction of antibodies with cells gave thermograms which differed in form and value, the latter changing in sign and magnitude. The results illustrate the need for careful model experiments to disect this complex system. The present results underline the value of microcalorimetry in giving analytical data on growth and respiration in cells and organisms and illustrates the potential of the technique in studies of surface‐localised reactions.