Cytosolic ATP Levels Research Articles

Abstract 1046: Miro1 Promotes Mitochondria ER Contact Sites Formation In Vascular Smooth Muscle Cells During G1/s

Vascular smooth muscle cell (VSMC) proliferation plays a key role in vascular disorders including atherosclerosis. Mitochondria promote VSMC proliferation by mitochondrial Ca 2+ regulation, ROS and ATP production. Mitochondria-ER contact sites (MERCS) promote signal transduction to mitochondria. MIRO1, an outer mitochondrial membrane GTPase, drives intracellular mitochondrial mobility. In drosophila, MIRO1 controls MERCS formation. However, it is unknown whether this interaction promotes key cellular eukaryotic phenotypes, such as proliferation. Here, we tested whether MIRO1 promotes VSMC proliferation by modulating MERCS and mitochondrial Ca 2+ entry. MIRO1 deletion decreased neointima formation in vivo, and abolished VSMC proliferation and G1/S transition in vitro. In WT VSMCs, proximity ligation assays (PLA) and split-GFP imaging revealed that MERCS formation increased in G1/S phase along with recruitment of MIRO1. MIRO1 deletion abolished cell cycle-dependent regulation of MERCS while increasing the expression of MERCS proteins GRP75 and IP3R. MIRO1 deletion also blocked increases in baseline mitochondrial [Ca 2+ ] by mitoPericam imaging and decreases in ER [Ca 2+ ] by CEPIA-ER in G1/S phase. Additionally, the mitochondrial membrane potential (TMRM) and mitochondrial respiration significantly increased at G1/S in WT VSMCs but not in MIRO1-/- cells. In WT VSMCs, pyruvate dehydrogenase activity increased G1/S phase while cytosolic ATP levels (ATP sensor) were increased. In MIRO1-/- cells, both parameters did not change. These data demonstrate that MERCS formation and mitochondrial [Ca 2+ ] increases during the cell cycle and is controlled by MIRO1. Furthermore, MIRO1 deletion decreases cellular ATP levels in VSMCs. In conclusion, we propose that MIRO1 controls MERCS and increased mitochondrial [Ca 2+ ] that drives metabolic activity and proliferation.

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Response to Letter

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Response to Letter

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Letter

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Letter

Mild excitotoxic effect on mitochondrial membrane potential, Ca2+ homeostasis and energy production in a mouse model of Alzheimer’s disease

AbstractBackgroundNeurons are bound to maintain a tight control over their cellular homeostasis in order to preserve their many functions, and specifically to precisely regulate ion fluxes for the generation of action potential and neurotransmission. In particular, the energetic balance has to be finely adjusted since its alteration may hamper cellular functionality with consequent cell suffering and death. Mitochondria play a central role in the homeostatic regulation within the cells due to the many vital tasks they perform in terms of energy production, participation in the regulation of Ca2+ concentration, modulation of intracellular signaling that allow the cell to respond to external solicitationMethodPrimary hippocampal neuronal cultures of 14‐17 DIV from WT and AD mouse model were challenged with μM concentration of glutamate to induce a mild excitotoxic effect; mitochondrial membrane potential, mitochondrial an cytosolic Ca2+ transients and cytosolic ATP levels were evaluated with fluorescent probes. Cells were treated also with inhibitors of the permeability transition pore to evaluate its effect on cell functionalityResultWe observed that in a model of Familial Alzheimer’s disease, hippocampal neurons are sensitive to a modest excitotoxic signal that produces a transient cytosolic and mitochondrial matrix Ca2+ with consequent partial loss of mitochondrial membrane potential and delayed recovery of mitochondrial matrix [Ca2+] which might contribute in the activation of the mitochondria permeability transition pore and consequent impairment of mitochondrial and cellular functions. This is accompanied by a reduced cytosolic ATP content that can result in an energy deficit for the cell. A brief treatment with inhibitors of the permeability transition pore is able to reduce these effects and might have protective roles against exogenous insults to the cells and help mitigate the neuronal loss characteristic of Alzheimer’s disease.ConclusionAD hippocampal neurons are more sensitive to a mild excitotoxic insult that affect mitochondrial functionality and in particular the activation of the permeability transition pore (PTP). Treatment with μM concentrations of two different inhibitors of the PTP prevent mitochondrial impairment.

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management

Sodium pyruvate improves the plasma amino acid profile in rats with L-arginine-induced acute pancreatitis.

Plasma amino acid levels are altered upon many pathological conditions including acute pancreatitis. It is unclear whether amino acids can be used as specific biomarker of acute pancreatitis severity or recovery. Development of acute pancreatitis is associated with mitochondrial dysfunction and decreased cytosolic ATP level. Sodium pyruvate is considered as a potential treatment of pancreatitis due to its ability to sustain mitochondrial oxidative and ATP-productive capacity in vitro. This study investigated the effect of sodium pyruvate on pancreatic morphology and plasma amino acid levels in rats with acute pancreatitis. Acute pancreatitis in rats was induced by administration of L-arginine (5g/kg) Experimental treatment group received sodium pyruvate (1g/kg) for 4days. On day 8 of the experiment, animals were killed, blood was collected and plasma amino acid concentration was determined with high-performance liquid chromatography. Histological examination showed large areas of fibrosis in the pancreas of animals treated with L-arginine irrespectively of sodium pyruvate administration. Sodium pyruvate improved the plasma amino acid levels. Rats with acute pancreatitis had significantly lower levels of most essential and non-essential amino acids and increased glutamate and aspartate in plasma. Administration of sodium pyruvate completely or partially restored the levels of methionine, phenylalanine, tryptophan, leucine, isoleucine, aspartate, asparagine and ornithine levels, while increasing glutamine and serine to levels significantly higher than control. Plasma lysine, alanine, arginine and taurine remained unaffected in all experimental groups. Sodium pyruvate may be considered for use as a maintenance therapy in acute pancreatitis.

Pulsed Radiofrequency Decreases pERK and Affects Intracellular Ca2+ Influx, Cytosolic ATP Level, and Mitochondrial Membrane Potential in the Sensitized Dorsal Root Ganglion Neuron Induced by N-Methyl D-Aspartate.

The molecular mechanism of pulsed radiofrequency (PRF) in chronic pain management is not fully understood. Chronic pain involves the activation of specific N-Methyl D-Aspartate receptors (NMDAR) to induce central sensitization. This study aims to determine the effect of PRF on central sensitization biomarker phosphorylated extracellular signal-regulated kinase (pERK), Ca2+ influx, cytosolic ATP level, and mitochondrial membrane potential (Δψm) of the sensitized dorsal root ganglion (DRG) neuron following NMDAR activation. This study is a true experimental in-vitro study on a sensitized DRG neuron induced with 80 µM NMDA. There are six treatment groups including control, NMDA 80 µM, Ketamine 100 µM, PRF 2Hz, NMDA 80 µM + PRF 2 Hz, and NMDA 80 µM + PRF 2 Hz + ketamine 100 µM. We use PRF 2 Hz 20 ms for 360 seconds. Statistical analysis was performed using the One-Way ANOVA and the Pearson correlation test with α=5%. In the sensitized DRG neuron, there is a significant elevation of pERK. There is a strong correlation between Ca2+, cytosolic ATP level, and Δψm with pERK intensity (p<0.05). PRF treatment decreases pERK intensity from 108.48 ± 16.95 AU to 38.57 ± 5.20 AU (p<0.05). PRF exposure to sensitized neurons also exhibits a Ca2+ influx, but still lower than in the unexposed neuron. PRF exposure in sensitized neurons has a higher cytosolic ATP level (0.0458 ± 0.0010 mM) than in the unexposed sensitized neuron (0.0198 ± 0.0004 mM) (p<0.05). PRF also decreases Δψm in the sensitized neuron from 109.24 ± 6.43 AU to 33.21 ± 1.769 AU (p<0.05). PRF mechanisms related to DRG neuron sensitization are by decreasing pERK, altering Ca2+ influx, increasing cytosolic ATP level, and decreasing Δψm which is associated with neuron sensitization following NMDAR activation.

The SGLT2 inhibitor empagliflozin improves cardiac energy status via mitochondrial ATP production in diabetic mice

Empagliflozin, a sodium-glucose co-transporter 2 inhibitor developed, has been shown to reduce cardiovascular events in patients with type 2 diabetes and established cardiovascular disease. Several studies have suggested that empagliflozin improves the cardiac energy state which is a partial cause of its potency. However, the detailed mechanism remains unclear. To address this issue, we used a mouse model that enabled direct measurement of cytosolic and mitochondrial ATP levels. Empagliflozin treatment significantly increased cytosolic and mitochondrial ATP levels in the hearts of db/db mice. Empagliflozin also enhanced cardiac robustness by maintaining intracellular ATP levels and the recovery capacity in the infarcted area during ischemic-reperfusion. Our findings suggest that empagliflozin enters cardiac mitochondria and directly causes these effects by increasing mitochondrial ATP via inhibition of NHE1 and Nav1.5 or their common downstream sites. These cardioprotective effects may be involved in the beneficial effects on heart failure seen in clinical trials.

Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.

ER-to-Golgi Transport in HeLa Cells Displays High Resilience to Ca2+ and Energy Stresses.

One third of all human proteins are either transmembrane or soluble secretory proteins that first target the endoplasmic reticulum (ER). These proteins subsequently leave the ER and enter the Golgi apparatus via ER-Golgi intermediate vesicular structures. Live-cell imaging of cargos fused to fluorescent proteins (FPs) enables the high-resolution visualization and characterization of secretory transport processes. Here, we performed fluorescence time-lapse imaging to assess the Ca2+ and energy dependency of ER-to-Golgi transport in living HeLa cells, a cancer cell model which has been well investigated. Our data revealed that ER-to-Golgi transport remained highly efficient in the absence of ATP-generating substrates, despite clear reductions in cytosolic and mitochondrial ATP levels under these energy stress conditions. However, cell treatment with 2-deoxy-D-glucose (2-DG), which severely diminished subcellular ATP levels, abolished ER-to-Golgi transport. Interestingly, while 2-DG elevated cytosolic Ca2+ levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca2+ mobilizations, which strongly affected the motility of these vesicles, did not considerably impair ER-to-Golgi transport. In summary, we highlight that ER-to-Golgi transport in HeLa cells remains functional despite high energy and Ca2+ stress levels.

Single-cell dynamics of pannexin-1-facilitated programmed ATP loss during apoptosis.

ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hr. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a 'programmed' process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP.

Metabolic regulation of calcium pumps in pancreatic cancer: role of phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3)

BackgroundHigh glycolytic rate is a hallmark of cancer (Warburg effect). Glycolytic ATP is required for fuelling plasma membrane calcium ATPases (PMCAs), responsible for extrusion of cytosolic calcium, in pancreatic ductal adenocarcinoma (PDAC). Phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3) is a glycolytic driver that activates key rate-limiting enzyme Phosphofructokinase-1; we investigated whether PFKFB3 is required for PMCA function in PDAC cells.MethodsPDAC cell-lines, MIA PaCa-2, BxPC-3, PANC1 and non-cancerous human pancreatic stellate cells (HPSCs) were used. Cell growth, death and metabolism were assessed using sulforhodamine-B/tetrazolium-based assays, poly-ADP-ribose-polymerase (PARP1) cleavage and seahorse XF analysis, respectively. ATP was measured using a luciferase-based assay, membrane proteins were isolated using a kit and intracellular calcium concentration and PMCA activity were measured using Fura-2 fluorescence imaging.ResultsPFKFB3 was highly expressed in PDAC cells but not HPSCs. In MIA PaCa-2, a pool of PFKFB3 was identified at the plasma membrane. PFKFB3 inhibitor, PFK15, caused reduced cell growth and PMCA activity, leading to calcium overload and apoptosis in PDAC cells. PFK15 reduced glycolysis but had no effect on steady-state ATP concentration in MIA PaCa-2.ConclusionsPFKFB3 is important for maintaining PMCA function in PDAC, independently of cytosolic ATP levels and may be involved in providing a localised ATP supply at the plasma membrane.

Carnosic Acid, a Natural Diterpene, Attenuates Arsenic-Induced Hepatotoxicity via Reducing Oxidative Stress, MAPK Activation, and Apoptotic Cell Death Pathway.

The present studies have been executed to explore the protective mechanism of carnosic acid (CA) against NaAsO2-induced hepatic injury. CA exhibited a concentration dependent (1–4 μM) increase in cell viability against NaAsO2 (12 μM) in murine hepatocytes. NaAsO2 treatment significantly enhanced the ROS-mediated oxidative stress in the hepatic cells both in in vitro and in vivo systems. Significant activation of MAPK, NF-κB, p53, and intrinsic and extrinsic apoptotic signaling was observed in NaAsO2-exposed hepatic cells. CA could significantly counteract with redox stress and ROS-mediated signaling and thereby attenuated NaAsO2-mediated hepatotoxicity. NaAsO2 (10 mg/kg) treatment caused significant increment in the As bioaccumulation, cytosolic ATP level, DNA fragmentation, and oxidation in the liver of experimental mice (n = 6). The serum biochemical and haematological parameters were significantly altered in the NaAsO2-exposed mice (n = 6). Simultaneous treatment with CA (10 and 20 mg/kg) could significantly reinstate the NaAsO2-mediated toxicological effects in the liver. Molecular docking and dynamics predicted the possible interaction patterns and the stability of interactions between CA and signal proteins. ADME prediction anticipated the drug-likeness characteristics of CA. Hence, there would be an option to employ CA as a new therapeutic agent against As-mediated toxic manifestations in future.

General Anesthetics Regulate Autophagy via Modulating the Inositol 1,4,5-Trisphosphate Receptor: Implications for Dual Effects of Cytoprotection and Cytotoxicity

General anesthetics are both neuroprotective and neurotoxic with unclear mechanisms. General anesthetics may control cell survival via their effects on autophagy by activation of type 1 inositol triphosphate receptor (InsP3R-1). DT40 or SH-SY5Y cells with only or over 99% expression of InsP3R-1 were treated with isoflurane or propofol. Cell viability was determined by MTT reduction or LDH release assays. Apoptosis was determined by measuring Caspase-3 or by TUNEL assay. Autophagy activity was determined by measuring LC3 II and P62. We evaluated mitochondrial integrity using MitoTracker Green and cytosolic ATP levels. Fura2-AM was used to measure the concentrations of cytosolic calcium ([Ca2+]c). Propofol significantly increased peak and integrated calcium response (P < 0.001) in cells with InsP3R-1 but not in cells with triple knockout of InsP3R. Both propofol and isoflurane increased autophagy induction (P < 0.05) in an mTOR- and InsP3R- activity dependent manner. Short exposure to propofol adequately activated InsP3-1 to provide sufficient autophagy for cytoprotection, while prolonged exposure to propofol induced cell apoptosis via impairment of autophagy flux through over activation of InsP3-1. Propofol damaged mitochondria and decreased cytosolic ATP. The effects of general anesthetics on apoptosis and autophagy are closely integrated; both are caused by differential activation of the type 1 InsP3R.

BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells.

ATP levels may represent fundamental health conditions of cells. However, precise measurement of intracellular ATP levels in living cells is hindered by the lack of suitable methodologies. Here, we developed a novel ATP biosensor termed “BTeam”. BTeam comprises a yellow fluorescent protein (YFP), the ATP binding domain of the ε subunit of the bacterial ATP synthase, and an ATP-nonconsuming luciferase (NLuc). To attain emission, BTeam simply required NLuc substrate. BTeam showed elevated bioluminescence resonance energy transfer efficiency upon ATP binding, resulted in the emission spectra changes correlating with ATP concentrations. By using values of YFP/NLuc emission ratio to represent ATP levels, BTeam achieved steady signal outputs even though emission intensities were altered. With this biosensor, we succeeded in the accurate quantification of intracellular ATP concentrations of a population of living cells, as demonstrated by detecting the slight distribution in the cytosol (3.7–4.1 mM) and mitochondrial matrix (2.4–2.7 mM) within some cultured cell lines. Furthermore, BTeam allowed continuous tracing of cytosolic ATP levels of the same cells, as well as bioluminescent imaging of cytosolic ATP dynamics within individual cells. This simple and accurate technique should be an effective method for quantitative measurement of intracellular ATP concentrations.

The effects of metformin and simvastatin on the growth of LNCaP and RWPE-1 prostate epithelial cell lines

The anti-diabetic drug metformin and cholesterol-lowering statins inhibit prostate cancer cell growth in vitro and have been linked with lowered risk of prostate cancer in epidemiological studies. We evaluated the effects of these drugs on cancerous and non-cancerous prostate epithelial cell lines. Cancer (LNCaP) and normal (RWPE-1) prostate epithelial cell lines were treated with pharmacologic concentrations of metformin and simvastatin alone and in combinations. Relative changes in cell number were measured with crystal violet staining method. Drug effects on apoptosis and cell cycle were measured with flow cytometry. We also measured changes in the activation and expression of a set of reported target proteins of metformin and statins with Western blotting. Metformin decreased the relative cell number of LNCaP cells by inducing G1 cell cycle block, autophagy and apoptosis, and slightly increased cytosolic ATP levels, whereas RWPE-1 cells were resistant to metformin. However, RWPE-1 cells were sensitive to simvastatin, which induced G2 cell cycle block, autophagy and apoptosis, and increased cytosolic ATP levels in these cells. Combination of metformin and simvastatin synergistically decreased cytosolic ATP levels, increased autophagy and instead of apoptosis, induced necrosis in LNCaP cells. Synergistic effects were not observed in RWPE-1 cells. These results suggest, that prostate cancer cells may be more vulnerable to combined growth-inhibiting effects of metformin and simvastatin compared to normal cells. The data presented here provide evidence for the potency of combined metformin and statin, also at pharmacologic concentrations, as a chemotherapeutic option for prostate cancer.

Restrictions in ATP diffusion within sarcomeres can provoke ATP-depleted zones impairing exercise capacity in chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by the inability of patients to sustain a high level of ventilation resulting in perceived exertional discomfort and limited exercise capacity of leg muscles at average intracellular ATP levels sufficient to support contractility. MethodsMyosin ATPase activity in biopsy samples from healthy and COPD individuals was implemented as a local nucleotide sensor to determine ATP diffusion coefficients within myofibrils. Ergometric parameters clinically measured during maximal exercise tests in both groups were used to define the rates of myosin ATPase reaction and aerobic ATP re-synthesis. The obtained parameters in combination with AK- and CK-catalyzed reactions were implemented to compute the kinetic and steady-state spatial ATP distributions within control and COPD sarcomeres. ResultsThe developed reaction–diffusion model of two-dimensional sarcomeric space identified similar, yet extremely low nucleotide diffusion in normal and COPD myofibrils. The corresponding spatio-temporal ATP distributions, constructed during imposed exercise, predicted in COPD sarcomeres a depletion of ATP in the zones of overlap between actin and myosin filaments along the center axis at average cytosolic ATP levels similar to healthy muscles. ConclusionsATP-depleted zones can induce rigor tension foci impairing muscle contraction and increase a risk for sarcomere damages. Thus, intra-sarcomeric diffusion restrictions at limited aerobic ATP re-synthesis can be an additional risk factor contributing to the muscle contractile deficiency experienced by COPD patients. General significanceThis study demonstrates how restricted substrate mobility within a cellular organelle can provoke an energy imbalance state paradoxically occurring at abounding average metabolic resources.

Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria

Background/Aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca²⁺ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca²⁺ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca²⁺ uptake upon intracellular Ca²⁺ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. Conclusions: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca²⁺ overload and subsequent cancer cell death.

Stimulation of Eryptosis by Combretastatin A4 Phosphate Disodium (CA4P)

Background/Aims: Combretastatin A4 phosphate disodium (CA4P) is utilized for the treatment of malignancy. The substance has previously been shown to trigger suicidal cell death or apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), ceramide, oxidative stress and ATP depletion. The present study explored, whether CA4P induces eryptosis and, if so, to gain insight into mechanisms involved. Methods: Flow cytometry has been employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCF fluorescence, glutathione (GSH) abundance from CMF fluorescence and ceramide abundance from fluorescent antibodies. In addition cytosolic ATP levels were quantified utilizing a luciferin-luciferase-based assay and hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to CA4P (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. CA4P did not appreciably increase hemolysis. Hundred µM CA4P significantly increased Fluo3-fluorescence. The effect of CA4P (100 µM) on annexin-V-binding was significantly blunted, but not abolished, by removal of extracellular Ca²⁺. CA4P (≥ 50 µM) significantly decreased GSH abundance and ATP levels but did not significantly increase ROS or ceramide. Conclusions: CA4P triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to entry of extracellular Ca²⁺ and energy depletion.

Dynamic Changes in Cytosolic ATP Levels in Cultured Glutamatergic Neurons During NMDA-Induced Synaptic Activity Supported by Glucose or Lactate.

We have previously shown that synaptic transmission fails in cultured neurons in the presence of lactate as the sole substrate. Thus, to test the hypothesis that the failure of synaptic transmission is a consequence of insufficient energy supply, ATP levels were monitored employing the ATP biosensor Ateam1.03YEMK. While inducing synaptic activity by subjecting cultured neurons to two 30s pulses of NMDA (30µM) with a 4min interval, changes in relative ATP levels were measured in the presence of lactate (1mM), glucose (2.5mM) or the combination of the two. ATP levels reversibly declined following NMDA-induced neurotransmission activity, as indicated by a reversible 10-20% decrease in the response of the biosensor. The responses were absent when the NMDA receptor antagonist memantine was present. In the presence of lactate alone, the ATP response dropped significantly more than in the presence of glucose following the 2nd pulse of NMDA (approx. 10 vs. 20%). Further, cytosolic Ca(2+) homeostasis during NMDA-induced synaptic transmission is partially inhibited by verapamil indicating that voltage-gated Ca(2+) channels are activated. Lastly, we showed that cytosolic Ca(2+) homeostasis is supported equally well by both glucose and lactate, and that a pulse of NMDA causes accumulation of Ca(2+) in the mitochondrial matrix. In summary, we have shown that ATP homeostasis during neurotransmission activity in cultured neurons is supported by both glucose and lactate. However, ATP homeostasis seems to be negatively affected by the presence of lactate alone, suggesting that glucose is needed to support neuronal energy metabolism during activation.