Home About us Contact
|
Home About us Contact | ||
|
|
||
Mitochondrial Potential (mitochondrial + potential)
Selected AbstractsCharacterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivationFEBS JOURNAL, Issue 7 2005Angela M. Falchi Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were analyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochondrial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase). Succinic acid (mitochondrial complex II substrate, given in the permeable form of dimethyl ester) abolished the rotenone-induced inhibition of R phases. Taken together, these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions, resulting in a progressive depolarization of mitochondria accompanied by NAD(P)H oxidation and Ca2+ influx. D phases were not blocked by cyclosporin A (inhibitor of the permeability transition pore) or o -phenyl-EGTA (a Ca2+ chelator), suggesting that the permeability transition pore was not involved in mitochondrial potential fluctuations. [source] Effectiveness of extracellular lactate/pyruvate for sustaining synaptic vesicle proton gradient generation and vesicular accumulation of GABAJOURNAL OF NEUROCHEMISTRY, Issue 3 2006A. S. Tarasenko Abstract The effects of extracellular monocarboxylates pyruvate and lactate on membrane potentials, acidification and neurotransmitter filling of synaptic vesicles were investigated in experiments with rat brain synaptosomes using [3H]GABA and fluorescent dyes, potential-sensitive rhodamine 6G and pH-sensitive acridine orange. In experiments investigating accumulation of acridine orange in synaptic vesicles within the synaptosomes, monocarboxylates, similarly to glucose, ensured generation of the vesicle proton gradient by available and recycled vesicles, and pyruvate demonstrated the highest efficacy. An increase in the level of proton gradient correlated with enhanced accumulation of [3H]GABA in synaptic vesicles and resulted in enlarged exocytosis and attenuated the transporter-mediated [3H]GABA release. Pyruvate added to glucose-contained medium caused more active binding of rhodamine 6G by synaptosomes that reflected mitochondrial membrane hyperpolarization, and this intensification of nerve terminal energy metabolism resulted in an increase in total ATP content by ,25%. Pyruvate also prolonged the state of metabolic competence of nerve terminal preparations, keeping the mitochondrial potential and synaptic vesicle proton gradient at steady levels over a long period of time. Thus, besides glucose, the extracellular monocarboxylates pyruvate and lactate can provide sufficient support of energy-dependent processes in isolated nerve terminals, allowing effective functioning of neurotransmitter release and reuptake systems. [source] Oxidative stress promotes proliferation and dedifferentiation of retina glial cells in vitroJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2009Carolina E. Abrahan Abstract Oxidative damage is involved in triggering neuronal death in several retinal neurodegenerative diseases. The recent finding of stem cells in the retina suggests that both preventing neuronal death and replacing lost neurons might be useful strategies for treating these diseases. We have previously shown that oxidative stress induces apoptosis in cultured retinal neurons. We now investigated the response of Müller cells, proposed as retina stem cells, to this damage. Treatment of glial cell cultures prepared from rat retinas with the oxidant paraquat (PQ) did not induce glial cell apoptosis. Instead, PQ promoted their rapid dedifferentiation and proliferation. PQ decreased expression of a marker of differentiated glial cells, simultaneously increasing the expression of smooth muscle actin, shown to increase with glial dedifferentiation, the levels of cell-cycle markers, and the number of glial cells in the cultures. In addition, glial cells protected neurons in coculture from apoptosis induced by PQ and H2O2. In pure neuronal cultures, PQ induced apoptosis of photoreceptors and amacrine neurons, simultaneously decreasing the percentage of neurons preserving mitochondrial membrane potential; coculturing neurons with glial cells completely prevented PQ-induced apoptosis and preserved mitochondrial potential in both neuronal types. These results demonstrate that oxidative damage activated different responses in Müller glial cells; they rapidly dedifferentiated and enhanced their proliferation, concurrently preventing neuronal apoptosis. Glial cells might not only preserve neuronal survival but also activate their cell cycle in order to provide a pool of new progenitor cells that might eventually be manipulated to preserve retinal functionality. © 2008 Wiley-Liss, Inc. [source] P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca2+ rise and change in mitochondrial potentialJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2007Stefan Kahlert Abstract With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca2+ level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y1/2/4 receptor agonist in rat, caused a large cytosolic Ca2+ increase in astrocytes and a considerably smaller Ca2+ response in neighboring neurons. The P2Y1 receptor antagonist MRS2179 completely blocked the ATP-induced Ca2+ response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca2+ level. Stimulation with UDP, a P2Y6 receptor agonist; UTP, a P2Y2/4 receptor agonist; 2MeSATP, a P2Y1 receptor agonist; or 2MeSADP, a P2Y1/12/13 receptor agonist, evoked significant Ca2+ responses in astrocytes but small Ca2+ responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca2+ peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca2+ level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain. © 2007 Wiley-Liss, Inc. [source] Glutamate-mediated influx of extracellular Ca2+ is coupled with reactive oxygen species generation in cultured hippocampal neurons but not in astrocytesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Stefan Kahlert Abstract Generation of reactive oxygen species (ROS) in brain tissue leads to neurodegeneration. The major source of ROS is the mitochondrial respiratory chain. We studied regulation of Ca2+ level, mitochondrial potential, and ROS generation in defined mixed hippocampal cell cultures exposed to glutamate (100 ,M). Recordings were made from individually identified astrocytes and neurons to compare the physiologic responses in both cell types. Neurons identified by synaptotagmin immunoreactivity were characterized functionally by the fast Ca2+ increase with K+ (50 mM) stimulation, and the astrocytes identified by glial fibrillary acidic protein (GFAP) staining had the functional characteristic of a transient Ca2+ peak in response to ATP (10 ,M) stimulation. We found that the glutamate-mediated Ca2+ response in neurons is due largely to influx of extracellular Ca2+. This is consistent with our finding that in cultured hippocampal neurons, stores depending on the activity of the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) pump had a low Ca2+ content, regardless of whether the neurons were challenged or not with K+ before applying the SERCA inhibitor cyclopiazonic acid (CPA). Astrocytes displayed a large CPA-mediated Ca2+ response, indicating a high level of Ca2+ load in the stores in astrocytes. Importantly, the rise in ROS generation due to glutamate application was cell-type specific. In neurons, glutamate induced a marked rise in generation of ROS, but not in astrocytes. In both astrocytes and neurons, the mitochondrial potential was increased in response to glutamate challenge. We conclude that in neurons, Ca2+ influx accounts for the increased ROS generation in response to glutamate. This might explain the high vulnerability of neurons to glutamate challenge compared to the vulnerability of astrocytes. The high resistance of astrocytes is accompanied by an efficient downregulation of cytosolic Ca2+, which is not found in neurons. © 2004 Wiley-Liss, Inc. [source] Alsterpaullone, a novel cyclin-dependent kinase inhibitor, induces apoptosis by activation of caspase-9 due to perturbation in mitochondrial membrane potential,MOLECULAR CARCINOGENESIS, Issue 4 2003Tyler Lahusen Abstract The majority of human neoplasms have aberrations in the retinoblastoma pathway due to hyperactivation of cyclin-dependent kinases (CDK). Based on this observation, novel small molecules, such as flavopiridol and UCN-01, are being developed and are currently being tested in the clinic. Efforts to develop CDK modulators led us to the discovery of a novel class of CDK inhibitors, the paullones [Cancer Res 1999;59:2566]. Initial studies demonstrated that paullones inhibit CDKs in vitro, thereby blocking cell-cycle progression. However, the exact mechanism for the antiproliferative effects of paullones was never explored. In this report, we demonstrate for the first time that the most potent paullone, alsterpaullone (Alp), induced apoptosis and promoted loss in clonogenicity in the Jurkat cell line. Alp caused early activation of both caspase-8 and -9, leading to cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, apoptosis by Alp was not associated with loss in anti-apoptotic proteins such as XIAP or BCL-XL. Pre-incubation with cell-permeable inhibitors z-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethylketone and benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (ZVAD) blocked Alp-induced apoptosis. Moreover, the general caspase inhibitor ZVAD blocked the cleavage and activation of most caspases tested except caspase-9. Studies of mitochondrial membrane potential also demonstrated that Alp is able to disrupt mitochondrial potential in the presence of ZVAD, suggesting that the activation of caspase-9 by Alp follows mitochondrial perturbation. Pre-incubation of Jurkat cells with ZVAD did not prevent the depletion of cyclin D3, loss of CDK, or cell-cycle arrest by Alp. In summary, these experiments suggest that Alp activates caspase-9 via mitochondrial perturbation. Active caspase-9 cleaves and activates caspase-8 and caspase-3, leading to apoptosis. In the presence of the general caspase inhibitor ZVAD, the cell-cycle effects of Alp are unaltered while apoptosis is blocked, suggesting that the CDK effects of Alp are not sufficient for Alp-induced apoptosis. Additional studies with paullones are warranted to further characterize their preclinical effects and to explore their potential use in the clinical setting. Published 2003 Wiley-Liss, Inc. [source] Activity of nitric oxide synthase in mature and immature human spermatozoaANDROLOGIA, Issue 2 2010C. Roessner Summary Nitric oxide (NO) is known to be involved in multiple signal transduction pathways of male germ cells, including sperm capacitation. In somatic cells, NO production was found to be part of apoptosis signalling. The aim of our study was to further clarify the role of NO in spermatozoa by investigation of NO synthase activity with regard to sperm maturity and sperm apoptosis signalling. Semen specimens from 19 healthy donors were subjected to density gradient centrifugation to separate the predominantly mature and immature sperm fraction. NO synthase activity was evaluated using diaminofluoresceine-2-diacetate by FACS. Apoptosis signalling was monitored by flowcytometric analyses of caspase-3 (CP3) and integrity of the transmembrane mitochondrial potential (TMP). TUNEL assay was used to detect DNA fragmentations. Maturity of human spermatozoa was associated with increased NO synthase activity and inactivated apoptosis signalling (lower levels of disrupted TMP, active CP3 and DNA fragmentations, P < 0.05). Activation of apoptosis signalling was significantly negatively correlated to NO production, indicating a rather anti-apoptotic effect of NO. This might underline the recently proposed role of NO in physiological sperm signal transduction, e.g. during capacitation. [source] Overproduction of BCR-ABL induces apoptosis in imatinib mesylate-resistant cell linesCANCER, Issue 1 2005Vanessa Desplat Ph.D. Abstract BACKGROUND Imatinib mesylate, a BCR-ABL tyrosine kinase inhibitor, induces apoptosis in chronic myeloid leukemia cells. Resistance to imatinib is currently the most important concern of this treatment. One of the main mechanisms of this resistance is overexpression of BCR-ABL. METHODS In the current study, the authors investigated the correlation between BCR-ABL overexpression and apoptosis in BaF/BCR - ABL and LAMA84 cell lines resistant to imatinib suddenly deprived of the inhibitor, and compared with their sensitive counterpart. RESULTS Removal of imatinib from culture medium led to a decrease in Bcr-Abl protein expression by Day 5, which was sustained for , 3 weeks of imatinib deprivation. Apoptosis was observed after 3 days of imatinib deprivation in resistant lines accompanied by caspase activation, loss of membrane asymmetry (annexin V staining), and alteration of mitochondrial potential (dihexyloxacarbocyanine iodide [DiOC6]). Transient activation of the STAT5/Bcl-xL pathway and Akt kinase activity preceded these responses. CONCLUSIONS Thus, imatinib removal led to apoptosis of BCR-ABL,overexpressing leukemic cells, a phenomenon that could be exploited to sensitize imatinib-resistant cells to the cytotoxic effect of other drugs. Cancer 2005. © 2004 American Cancer Society. [source] Activity of triptolide against human mast cells harboring the kinase domain mutant KITCANCER SCIENCE, Issue 7 2009Yanli Jin Gain-of-function mutations of the receptor tyrosine kinase KIT can cause systemic mastocytosis (SM) and gastrointestinal stromal tumors. Most of the constitutively active KIT can be inhibited by imatinib; D816V KIT cannot. In this study, we investigated the activity of triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook. f., in cells expressing mutant KIT, including D816V KIT. Imatinib-sensitive HMC-1.1 cells harboring the mutation V560G in the juxtamembrane domain of KIT, imatinib-resistant HMC-1.2 cells harboring both V560G and D816V mutations, and murine P815 cells, were treated with triptolide, and analyzed in terms of growth, apoptosis, and signal transduction. The in vivo antitumor activity was evaluated by using the nude mouse xenograft model. Our results demonstrated that triptolide potently inhibits the growth of both human and murine mast cells harboring not only imatinib-sensitive KIT mutation but also imatinib-resistant D816V KIT. Triptolide markedly inhibited KIT mRNA levels and strikingly reduced the levels of phosphorylated and total Stat3, Akt, and Erk1/2, downstream targets of KIT. Triptolide triggered apoptosis by inducing depolarization of mitochondrial potential and release of cytochrome c, downregulation of Mcl-1 and XIAP. Furthermore, triptolide significantly abrogated the growth of imatinib-resistant HMC-1.2 cell xenografts in nude mice and decreased KIT expression in xenografts. Our data demonstrate that triptolide inhibits imatinib-resistant mast cells harboring D816V KIT. Further investigation of triptolide for treatment of human neoplasms driven by gain-of-function KIT mutations is warranted. (Cancer Sci 2009; 100: 1335,1343) [source] | |||||||||||||