VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release.
AuthorsMadesh M, Hajnóczky G
JournalJ Cell Biol
PubMed ID11739410
'Enhanced formation of reactive oxygen species (ROS), superoxide (O2*-), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2*- elicited rapid and massive cytochrome c ... More
Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic beta-cells.
AuthorsBraun M, Wendt A, Birnir B, Broman J, Eliasson L, Galvanovskis J, Gromada J, Mulder H, Rorsman P
JournalJ Gen Physiol
PubMed ID14769845
'We have explored whether gamma-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic beta-cells. To this end, beta-cells were engineered to express GABA(A)-receptor Cl(-)-channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 ... More
'Synaptically released Zn2+ can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn2+-sensitive probe, Newport green, examined levels of [Zn2+]i attained in cultured cortical neurons on exposure to N-methyl-D-asparte, kainate, or high K+ (to activate voltage-sensitive Ca2+ channels) in the presence of 300 microM Zn2+. Indicating particularly ... More
Cellular activation by Ca2+ release from stores in the endoplasmic reticulum but not by increased free Ca2+ in the cytosol.
AuthorsStrayer DS, Hoek JB, Thomas AP, White MK
JournalBiochem J
PubMed ID10548531
'Ca(2+) release from intracellular stores and/or transmembrane influx can increase the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). Such changes in [Ca(2+)](i) might transduce signals regulating transcription, motility, secretion, and so on. Surfactant secretagogues such as ATP and ionomycin stimulate the release and transmembrane influx of Ca(2+), both of which increase [Ca(2+)](i). ... More
Presynaptic strontium dynamics and synaptic transmission.
AuthorsXu-Friedman MA, Regehr WG
JournalBiophys J
PubMed ID10096899
'Strontium can replace calcium in triggering neurotransmitter release, although peak release is reduced and the duration of release is prolonged. Strontium has therefore become useful in probing release, but its mechanism of action is not well understood. Here we study the action of strontium at the granule cell to Purkinje ... More
Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC.
AuthorsHyrc KL, Bownik JM, Goldberg MP
JournalCell Calcium
PubMed ID10756974
'Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ ... More
Calcium sensitivity of glutamate release in a calyx-type terminal.
AuthorsBollmann JH, Sakmann B, Borst JG
JournalScience
PubMed ID10937999
'Synaptic efficacy critically depends on the presynaptic intracellular calcium concentration ([Ca2+]i). We measured the calcium sensitivity of glutamate release in a rat auditory brainstem synapse by laser photolysis of caged calcium. A rise in [Ca2+]i to 1 micromolar readily evoked release. An increase to >30 micromolar depleted the releasable vesicle ... More
Rapid Ca2+-dependent increase in oxygen consumption by mitochondria in single mammalian central neurons.
AuthorsHayakawa Y, Nemoto T, Iino M, Kasai H
JournalCell Calcium
PubMed ID15755497
'Oxygen consumption increases within a fraction of a second after the onset of neuronal activity, a phenomenon referred to as the "initial dip" in functional imaging studies of the living brain. The cellular mechanism that underlies this rapid increase in oxygen consumption has remained unclear, however. We have now used ... More
Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca2+ deregulation.
AuthorsBolshakov AP, Mikhailova MM, Szabadkai G, Pinelis VG, Brustovetsky N, Rizzuto R, Khodorov BI,
JournalCell Calcium
PubMed ID18037484
'To clarify the role of the mitochondrial permeability transition pore (MPT) in the mechanism of the glutamate-induced delayed calcium deregulation (DCD) and mitochondrial depolarization (MD), we studied changes in cytosolic (pH(c)) and mitochondrial pH (pH(m)) induced by glutamate in cultured cortical neurons expressing pH-sensitive fluorescent proteins. We found that DCD ... More
Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes.
Authors Patel Sandip; Gaspers Lawrence D; Boucherie Sylviane; Memin Elisabeth; Stellato Kerri Anne; Guillon Gilles; Combettes Laurent; Thomas Andrew P;
JournalJ Biol Chem
PubMed ID12097323
'Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the ... More
Quantification of calcium signal transmission from sarco-endoplasmic reticulum to the mitochondria.
AuthorsPacher P, Csordás P, Schneider T, Hajnóczky G
JournalJ Physiol
PubMed ID11118489
'Recent studies have shown that ryanodine and IP3 receptor (RyR/IP3R)-mediated cytosolic Ca2+ signals propagate to the mitochondria, initiating chains of events vital in the regulation of different cellular functions. However, the fraction of released Ca2+ utilized by the mitochondria during these processes has not been quantified. To measure the amount ... More
Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3.
AuthorsDevinney MJ, Reynolds IJ, Dineley KE
JournalCell Calcium
PubMed ID15670869
'Elevation of intracellular free zinc ([Zn2+]i) probably contributes to cell death in injury paradigms involving calcium deregulation and oxidative stress such as glutamate excitotoxicity. However, it is difficult to monitor both ions simultaneously in live cells. Here we present a new method using fluorescence microscopy and the ion sensitive indicators ... More
Calcium requirements for exocytosis do not delimit the releasable neuropeptide pool.
AuthorsLu X, Ellis-Davies GC, Levitan ES
JournalCell Calcium
PubMed ID12618147
'Recently, it was proposed that secretory vesicles have widely varying Ca(2+) thresholds for exocytosis. This model can explain adaptation of secretory responses and predicts that incomplete release is a consequence of insufficient Ca(2+). However, membrane capacitance-based measurements have not supported varying Ca(2+) thresholds. Here, Green Fluorescent Protein (GFP) imaging is ... More
Glucose regulation of free Ca(2+) in the endoplasmic reticulum of mouse pancreatic beta cells.
AuthorsTengholm A, Hellman B, Gylfe E
JournalJ Biol Chem
PubMed ID10601240
'Free Ca(2+) was measured in organelles of individual mouse pancreatic beta cells loaded with the low affinity indicator furaptra. After removal of cytoplasmic indicator by controlled digitonin permeabilization the organelle Ca(2+) was located essentially in the endoplasmic reticulum (ER), >90% being sensitive to inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPases. The Ca(2+) ... More
Measurement of free [Ca2+] changes in agonist-sensitive internal stores using compartmentalized fluorescent indicators.
AuthorsHofer AM,
JournalMethods Mol Biol
PubMed ID16422202
This chapter provides a guide for the use of compartmentalized fluorescentdyes to measure free [Ca2+] in the agonist-sensitive internal store of single cells. The basic steps involve incubating cells with the AM-ester derivative of the appropriate dye (resulting in partitioning of the probe in both the cytoplasm and organelles), and ... More
Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum.
AuthorsGolovina VA, Blaustein MP
JournalScience
PubMed ID9054358
The organization of calcium (Ca2+) stores in the sarcoplasmic and endoplasmic reticulum (S-ER) is poorly understood. The dynamics of the storage and release of calcium in the S-ER of intact, cultured astrocytes and arterial myocytes were studied with high-resolution imaging methods. The S-ER appeared to be a continuous tubular network; ... More
Zn(2+): a novel ionic mediator of neural injury in brain disease.
AuthorsWeiss JH, Sensi SL, Koh JY
JournalTrends Pharmacol Sci
PubMed ID11050320
Zn(2+) is the second most prevalent trace element in the body and is present in particularly large concentrations in the mammalian brain. Although Zn(2+) is a cofactor for many enzymes in all tissues, a unique feature of brain Zn(2+) is its vesicular localization in presynaptic terminals, where its release is ... More
Intracellular calcium dependence of transmitter release rates at a fast central synapse.
AuthorsSchneggenburger R, Neher E
JournalNature
PubMed ID10972290
Calcium-triggered fusion of synaptic vesicles and neurotransmitter release are fundamental signalling steps in the central nervous system. It is generally assumed that fast transmitter release is triggered by elevations in intracellular calcium concentration ([Ca2+]i) to at least 100 microM near the sites of vesicle fusion. For synapses in the central ... More
NMR-sensitive fluorinated and fluorescent intracellular calcium ion indicators with high dissociation constants.
AuthorsLondon RE, Rhee CK, Murphy E, Gabel S, Levy LA
JournalAm J Physiol
PubMed ID8203496
A new series of high-dissociation constant (KD) Ca2+ indicators has been developed to reduce perturbations due to buffering of transients, to carry out measurements in cells and organelles with high basal Ca2+ concentrations, and to measure cytosolic Ca2+ levels in the presence of perturbations that may significantly increase these levels. ... More
Glucose deprivation produces a prolonged increase in sensitivity to glutamate in cultured rat cortical neurons.
AuthorsVergun O, Han YY, Reynolds IJ
JournalExp Neurol
PubMed ID14552910
In this study we investigated whether the link between mitochondrial dysfunction and deregulation of Ca(2+) homeostasis preceding excitotoxic cell death is mediated by cellular deenergization. Glycolytic and/or mitochondrial ATP synthesis was inhibited with 2-deoxy-D-glucose (2DG) and oligomycin, respectively. Changes in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) and mitochondrial membrane potential were simultaneously ... More
Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons.
AuthorsLomax RB, Camello C, Van Coppenolle F, Petersen OH, Tepikin AV
JournalJ Biol Chem
PubMed ID11994289
We have studied the Ca(2+) leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca(2+) in the endoplasmic reticulum ([Ca(2+)](ER)). Cytosolic Ca(2+) ([Ca(2+)](C)) was clamped to the resting level by a BAPTA-Ca(2+) mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of ... More
Polycarboxylate fluorescent indicators as ion concentration probes in biological systems.
AuthorsKaterinopoulos HE, Foukaraki E
JournalCurr Med Chem
PubMed ID11860359
A large number of techniques have been applied to monitor the function of free metal ions in biological systems. Fluorescent ion probes have evolved into an extremely useful tool for contemporary experimentalists. Polycarboxylate indicators are widely used in the determination of metal ion concentrations, especially due to their cell membrane ... More
Ca(2+)- and voltage-dependent gating of Ca(2+)- and ATP-sensitive cationic channels in brain capillary endothelium.
AuthorsCsanády L, Adam-Vizi V
JournalBiophys J
PubMed ID12829486
Biophysical properties of the Ca(2+)-activated nonselective cation channel expressed in brain capillaries were studied in inside-out patches from primary cultures of rat brain microvascular endothelial cells. At -40 mV membrane potential, open probability (P(o)) was activated by cytosolic [Ca(2+)] > 1 micro M and was half-maximal at approximately 20 micro ... More
Cytosolic-free calcium increases to greater than 100 micromolar in ATP-depleted proximal tubules.
AuthorsWeinberg JM, Davis JA, Venkatachalam MA
JournalJ Clin Invest
PubMed ID9239420
Previous studies have shown that cytosolic-free Ca2+ (Caf) increases to at least low micromolar concentrations during ATP depletion of isolated kidney proximal tubules. However, peak levels could not be determined precisely with the Ca2+-sensitive fluorophore, fura-2, because of its high affinity for Ca2+. Now, we have used two low affinity ... More
Characterization of a range of fura dyes with two-photon excitation.
AuthorsWokosin DL, Loughrey CM, Smith GL
JournalBiophys J
PubMed ID14990500
Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of ... More
Mitochondria suppress local feedback activation of inositol 1,4, 5-trisphosphate receptors by Ca2+.
AuthorsHajnóczky G, Hager R, Thomas AP
JournalJ Biol Chem
PubMed ID10318833
The concerted action of inositol 1,4,5-trisphosphate (IP3) and Ca2+ on the IP3 receptor Ca2+ release channel (IP3R) is a fundamental step in the generation of cytosolic Ca2+ oscillations and waves, which underlie Ca2+ signaling in many cells. Mitochondria appear in close association with regions of endoplasmic reticulum (ER) enriched in ... More
Minimal requirements for calcium oscillations driven by the IP3 receptor.
AuthorsHajnóczky G, Thomas AP
JournalEMBO J
PubMed ID9218795
Hormones and neurotransmitters that act through inositol 1,4,5-trisphosphate (IP3) can induce oscillations of cytosolic Ca2+ ([Ca2+]c), which render dynamic regulation of intracellular targets. Imaging of fluorescent Ca2+ indicators located within intracellular Ca2+ stores was used to monitor IP3 receptor channel (IP3R) function and to demonstrate that IP3-dependent oscillations of Ca2+ ... More
Intracellular Ca2+ thresholds that determine survival or death of energy-deprived cells.
AuthorsDong Z, Saikumar P, Griess GA, Weinberg JM, Venkatachalam MA
JournalAm J Pathol
PubMed ID9422540
Increase of intracellular ionized or free Ca2+ is thought to play a central role in cell death due to ATP depletion. However, concurrently operative mechanisms of injury that do not require intracellular Ca2+ increases have made it difficult to test this hypothesis or to determine the concentrations at which intracellular ... More
Presynaptic mitochondrial calcium sequestration influences transmission at mammalian central synapses.
AuthorsBillups B, Forsythe ID
JournalJ Neurosci
PubMed ID12122046
Beyond their role in generating ATP, mitochondria have a high capacity to sequester calcium. The interdependence of these functions and limited access to presynaptic compartments makes it difficult to assess the role of sequestration in synaptic transmission. We addressed this important question using the calyx of Held as a model ... More
Rapid Ca2+ entry through Ca2+-permeable AMPA/Kainate channels triggers marked intracellular Ca2+ rises and consequent oxygen radical production.
AuthorsCarriedo SG, Yin HZ, Sensi SL, Weiss JH
JournalJ Neurosci
PubMed ID9742143
The widespread neuronal injury that results after brief activation of highly Ca2+-permeable NMDA channels may, in large part, reflect mitochondrial Ca2+ overload and the consequent production of injurious oxygen radicals. In contrast, AMPA/kainate receptor activation generally causes slower toxicity, and most studies have not found evidence of comparable oxygen radical ... More
Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones.
AuthorsVergun O, Keelan J, Khodorov BI, Duchen MR
JournalJ Physiol
PubMed ID10457062
1. The objective of this study was to clarify the relationships between loss of mitochondrial potential and the perturbation of neuronal Ca2+ homeostasis induced by a toxic glutamate challenge. Digital fluorescence imaging techniques were employed to monitor simultaneously changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and mitochondrial potential (DeltaPsim) in individual ... More
Calcium influx constitutes the ionic basis for the maintenance of glutamate-induced extended neuronal depolarization associated with hippocampal neuronal death.
AuthorsLimbrick DD, Sombati S, DeLorenzo RJ
JournalCell Calcium
PubMed ID12531183
Excessive activation of neuronal glutamate receptors has been implicated in the pathophysiology of stroke, epilepsy, and traumatic brain injury. Previously, it has been demonstrated that excitotoxic glutamate exposure results in the induction of an extended neuronal depolarization (END), as well as protracted elevations in free intracellular calcium ([Ca(2+)](i)). Both END ... More
Ca2+-dependent protein kinase--a modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells.
AuthorsBruce JI, Yule DI, Shuttleworth TJ
JournalJ Biol Chem
PubMed ID12368283
Cross-talk between cAMP and [Ca(2+)](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca(2+) release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type ... More
The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria.
AuthorsChalmers S, Nicholls DG
JournalJ Biol Chem
PubMed ID12660243
Three sequential phases of mitochondrial calcium accumulation can be distinguished: matrix dehydrogenase regulation, buffering of extramitochondrial free calcium, and finally activation of the permeability transition. Relationships between these phases, free and total matrix calcium concentration, and phosphate concentration are investigated in rat liver and brain mitochondria. Slow, continuous calcium infusion ... More
Dual responses of CNS mitochondria to elevated calcium.
AuthorsBrustovetsky N, Dubinsky JM
JournalJ Neurosci
PubMed ID10627586
Isolated brain mitochondria were examined for their responses to calcium challenges under varying conditions. Mitochondrial membrane potential was monitored by following the distribution of tetraphenylphosphonium ions in the mitochondrial suspension, mitochondrial swelling by observing absorbance changes, calcium accumulation by an external calcium electrode, and oxygen consumption with an oxygen electrode. ... More
Blockade of mitochondrial Ca2+ uptake by mitochondrial inhibitors amplifies the glutamate-induced calcium response in cultured cerebellar granule cells.
AuthorsKhodorov B, Pinelis V, Storozhevykh T, Yuravichus A, Khaspekhov L
JournalFEBS Lett
PubMed ID10481057
The objective of this study was to evaluate the role of mitochondrial Ca2+ uptake (MCU) in modulation (shaping) of the glutamate (Glu)-induced changes in neuronal cytoplasmic Ca2+ ([Ca2+]i). In order to block MCU, nerve cells were treated with mitochondrial inhibitors (MI) inducing collapse of the mitochondrial potential (Delta Psim). Measurements ... More
[Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release.
AuthorsMironov SL, Ivannikov MV, Johansson M
JournalJ Biol Chem
PubMed ID15516333
The positioning and dynamics of organelles depend on membrane-cytoskeleton interactions. Mitochondria relocate along microtubules (MT), but it is not clear whether MT have direct effects on mitochondrial function. Using two-photon microscopy and the mitochondrial fluorescent dyes rhodamine 123 and Rhod-2, we showed that Taxol and nocodazole, which correspondingly stabilize and ... More
Endoplasmic reticulum D-myo-inositol 1,4,5-trisphosphate-sensitive stores regulate nuclear factor-kappaB binding activity in a calcium-independent manner.
The transcription factor nuclear factor-kappaB (NF-kappaB) plays critical roles in neuronal survival and plasticity and in activation of immune responses. The activation of NF-kappaB has been closely associated with changes in intracellular calcium levels, but the relationship between the two remains unclear. Here we report that inhibition of endoplasmic reticulum ... More
Excitotoxic mitochondrial depolarisation requires both calcium and nitric oxide in rat hippocampal neurons.
AuthorsKeelan J, Vergun O, Duchen MR
JournalJ Physiol
PubMed ID10545145
1. Glutamate neurotoxicity has been attributed to cellular Ca2+ overload. As mitochondrial depolarisation may represent a pivotal step in the progression to cell death, we have used digital imaging techniques to examine the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial potential (DeltaPsim) during glutamate toxicity, and to define the ... More
AMPA exposures induce mitochondrial Ca(2+) overload and ROS generation in spinal motor neurons in vitro.
AuthorsCarriedo SG, Sensi SL, Yin HZ, Weiss JH
JournalJ Neurosci
PubMed ID10627601
The reason for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is primarily unknown. A possible factor is the expression by motor neurons of Ca(2+)-permeable AMPA/kainate channels, which may permit rapid Ca(2+) influx in response to synaptic receptor activation. However, other subpopulations of central neurons, most notably ... More