Academic literature on the topic 'EGTA-AM'

We have investigated the requirement for Ca2+ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca2+ reduced the amount of fusion. The requirement for Ca2+ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca2+ plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

Activity-dependent alterations of synaptic transmission important for learning and memory are often induced by Ca2+ signals generated by depolarization. While it is widely assumed that Ca2+ is the essential transducer of depolarization into cellular plasticity, little effort has been made to test whether Ca2+-independent responses to depolarization might also induce memory-like alterations. It was recently discovered that peripheral axons of nociceptive sensory neurons in Aplysia display long-lasting hyperexcitability triggered by conditioning depolarization in the absence of Ca2+ entry (using nominally Ca2+-free solutions containing EGTA, “0Ca/EGTA”) or the absence of detectable Ca2+ transients (adding BAPTA-AM, “0Ca/EGTA/BAPTA-AM”). The current study reports that depolarization of central ganglia to ∼0 mV for 2 min in these same solutions induced hyperexcitability lasting >1 h in sensory neuron processes near their synapses onto motor neurons. Furthermore, conditioning depolarization in these solutions produced a 2.5-fold increase in excitatory postsynaptic potential (EPSP) amplitude 1–3 h afterward despite a drop in motor neuron input resistance. Depolarization in 0 Ca/EGTA produced long-term potentiation (LTP) of the EPSP lasting ≥1 days without changing postsynaptic input resistance. When re-exposed to extracellular Ca2+ during synaptic tests, prior exposure to 0Ca/EGTA or to 0Ca/EGTA/BAPTA-AM decreased sensory neuron survival. However, differential effects on neuronal health are unlikely to explain the observed potentiation because conditioning depolarization in these solutions did not alter survival rates. These findings suggest that unrecognized Ca2+-independent signals can transduce depolarization into long-lasting synaptic potentiation, perhaps contributing to persistent synaptic alterations following large, sustained depolarizations that occur during learning, neural injury, or seizures.

A pot experiment was made to study effects of ethylene glycol tetraacetic acid (EGTA, an inhibitor of Ca2+) and trifluoperazine (TFP, an inhibitor of calmodulin (CaM) on mycorrhizal colonization, growth performance, and chlorophyll, sucrose and glucose concentrations of four-month-old trifoliate orange (Poncirus trifoliata) seedlings under mycorrhization with Rhizophagus intraradices. Exogenous EGTA and TFP notably inhibited root mycorrhizal colonization, and the addition of EGTA also decreased soil hyphal length. In general, EGTA treatment decreased but TFP increased easily extractable glomalin-related soil protein (EE-GRSP) and total GRSP (T-GRSP) concentrations. In addition, EGTA and TFP applications generally significantly inhibited growth performance (height, stem diameter, leaf number, and shoot and root biomass), root traits (total length, surface area, volume, and number of 1st, 2nd and 3rd order lateral root), and chlorophyll a,b and a+b concentrations, the mycorrhizal inoculation generally reversed the negative effects and markedly increased these variables, irrespective of whether the seedlings were applied by inhibitors or not. EGTA and TFP treatments generally inhibited sucrose and glucose levels of leaf and root, except that TFP application notably increased root glucose in AM and non-AM seedlings. AMF inoculation resulted in carbohydrate modification: decrease in leaf sucrose, increase in root sucrose and leaf glucose, as well increase in root glucose under no-inhibitor and EGTA conditions and decrease in root glucose under TFP. It suggests that Ca2+ and CaM were involved in mycorrhizal and root development in trifoliate orange.

The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1−/−) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 μM). In WT and Panx1−/− mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1−/− mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 μM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1−/− mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1−/− NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 μM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1−/− mice to above the control level.

Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was used as an intracellular Ca2+ chelator to shorten the duration of the Ca2+ transient and to determine the rate-limiting steps in relaxation of frog skeletal muscle at 10 and 20 degrees C. Incubation with the acetoxymethyl ester of EGTA (EGTA-AM) produced a linear approximately twofold increase in the rate of fall in twitch Ca2+ concentration ([Ca2+]) over 0-70 min at 10 degrees C. The rate of relaxation initially increased from 5 to 9/s over 0-13 min and then leveled, as the rate of fall in [Ca2+] continued to increase and peak force decreased. Increasing the rate of fall in [Ca2+] increased the rate of relaxation, until a rate-limiting step was reached at approximately 9/s at 10 degrees C. At 20 degrees C, incubation with EGTA-AM produced a linear approximately twofold increase in the rate of fall in twitch [Ca2+] without affecting the rate of relaxation (27/s). Ca2+ dissociated from the regulatory sites of purified troponin (Tn) at approximately 8/s at 10 degrees C and at 23/s at 20 degrees C. When the duration of the Ca2+ transient was decreased by EGTA or increased by partial inhibition of the sarcoplasmic reticulum Ca(2+)-ATPase, twitch force increased linearly with Ca2+ transient duration. Thus the duration of the Ca2+ transient is a primary determinant of twitch force and, whereas the rate of fall in [Ca2+] is rate limiting for relaxation at 10 degrees C, Ca2+ dissociation from Tn may be rate limiting at 20 degrees C.

Cells prepared from frog esophageal peptic glands by dispersal in low-Ca2+ medium (peptic acini) or 1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-containing medium ("EGTA cells") were compared. EGTA cells were characterized by decreased secretory responses to agonists [bombesin (BB), acetylcholine, and isoproterenol] and intracellular messenger activators (forskolin, 12-O-tetradecanoyl-phorbol-13-acetate), decreased relative intrinsic efficacies of muscarinic agonists, and somatostatin insensitivity. Decreased BB and muscarinic receptor responses were not associated with changes in receptor number or characteristics. The time course of BB- and acetylcholine-stimulated pepsinogen secretion indicated that the marked reduction was confined largely to the late secretory phase (2–30 min), dependent on extracellular Ca2+, rather than early phase (2 min) secretion, which is related to release of intracellular Ca2+. The defect could be reversed by the Ca2+ ionophore A23187. BB-stimulated intracellular Ca2+ mobilization measured with fura-2/AM was similar in the two cell preparations, whereas BB-stimulated 45Ca2+ uptake was reduced threefold in EGTA cells, and this defect was also reversed by A23187. Somatostatin inhibited both BB-stimulated secretion and 45Ca uptake by peptic acini, but it had no significant effect on these parameters in EGTA cells. Cytochalasin B inhibited BB stimulation in peptic acini but not EGTA cells. These findings suggest that peptic cells isolated with EGTA exhibit decreased secretory responses that are due at least in part to impairment of a mechanism for uptake of extracellular Ca2+.

We investigated regulation of intracellular Ca2+ induced by photodynamic therapy (PDT) with a new chlorin-based photosensitizer, DH-II-24, in human gastric adenocarcinoma cells. DH-II-24-mediated PDT induced necrotic cell death according to post-irradiation time, and produced intracellular reactive oxygen species (ROS) in an irradiation time-dependent manner. PDT also increased intracellular Ca2+ , and this Ca2+ elevation was largely inhibited by BAPTA-AM but not by EGTA. BAPTA-AM inhibited the ROS production by PDT, whereas NAC and Trolox had no effect on the PDT-induced Ca2+ response. In the presence of EGTA, pre-incubation with thapsigargin, Gly-Phe-β-naphthylamide or brefeldin A had no significant effect on the PDT-induced elevation in intracellular Ca2+ . However, ruthenium red affected the initial and late Ca2+ responses to PDT. Thus, DH-II-24-mediated PDT produces intracellular ROS via elevation in intracellular Ca2+ , contributed, at least in part, by mitochondria, which results in necrotic death of the human gastric adenocarcinoma cells.

In rat liver epithelial (WB) cells, Ca2+ pool depletion induced by two independent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method, Ca2+ pool depletion by thapsigargin increased the activity of ERK, even when rise in cytosolic Ca2+ was blocked with the Ca2+ chelator BAPTA-AM. For the second method, addition of extracellular EGTA at a concentration shown to deplete intracellular Ca2+pools also increased ERK activity. In each instance, ERK activation, as measured by an immunocomplex kinase assay, was greatly reduced by the tyrosine kinase inhibitor genistein, suggesting that Ca2+ store depletion increased ERK activity through a tyrosine kinase pathway. The intracellular Ca2+-releasing agent thapsigargin increased Fyn activity, which was unaffected by BAPTA-AM pretreatment, suggesting that Fyn activity was unaffected by increased cytosolic free Ca2+. Furthermore, depletion of intracellular Ca2+ with EGTA caused inactivation of protein phosphatase 2A and protein tyrosine phosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK were augmented in cells pretreated with BAPTA-AM, but ANG II-induced expression of the dual-specificity phosphatase mitogen-activated protein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment. Together these results indicate that ERK activity is regulated by the balance of phosphorylation vs. dephosphorylation reactions in intact cells and that the amount of Ca2+stored in intracellular pools plays an important role in this regulation.

Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether interalveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca2+ uncaging in intact alveoli of the isolated, blood-perfused rat lung. We loaded alveolar cells with the Ca2+ cage o-nitrophenyl EGTA-AM (NP-EGTA-AM) together with the fluorophores, fluo 4, or LysoTracker green (LTG) to determine, respectively, the cytosolic Ca2+ concentration ([Ca2+]cyt) or type 2 cell secretion. To uncage Ca2+ from NP-EGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased, whereas LTG fluorescence decreased, in the photo-targeted region, indicating that uncaging both increased [Ca2+]cyt and induced secretion. Concomitantly, [Ca2+]cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of interalveolar communication. These conducted responses were inhibited by pretreating alveoli with the connexin43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted [Ca2+]cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, interalveolar Ca2+ signals regulate type 2 cell secretion in adjacent alveoli. Such interalveolar communication might facilitate acinar coordination of alveolar function.

1. We characterized the kinetics of presynaptic Ca2+ ion concentration in optic nerve fibers and terminals of the optic tectum in Rana pipiens with the use of microfluorimetry. Isolated frog brains were incubated with the membrane-permeant tetraacetoxymethyl ester (AM) of the Ca2+ indicator fura-2. An optic nerve shock caused a transient decrease in the 380-nm excited fluorescence in the optic tectum with a rise time of <15 ms and a recovery to prestimulus levels on a time scale of seconds. 2. In normal saline, the amplitude of the fluorescence transients was dependent on stimulus intensity and at all levels it was directly correlated with the amplitude of postsynaptic field potentials produced by activation of unmyelinated optic nerve fibers. In the presence of the non-N-methyl-D-aspartate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, the amplitude and time course of fluorescence transients remained essentially unchanged while postsynaptic field potential amplitude was greatly reduced. Replacing extracellular Ca2+ with Ba2+ blocked unfacilitated postsynaptic field potentials while fluorescence transients remained significant. In reduced-Ca2+ salines (<1 mM), the amplitude of fluorescence transients increased approximately linearly with extracellular [Ca2+], whereas the amplitude the corresponding field potential was nonlinearly related to the fluorescent transient amplitude (approximately 2.5 power). In thin sections of labeled tecta, fluorescence labeling was localized to 1-micron puncta in the termination zone of optic nerve fibers in the superficial layers. Taken together, these results provide strong evidence that the fluorescence transients correspond to an increase in Ca2+ in presynaptic terminals of unmyelinated optic nerve fibers. 3. During trains of optic nerve stimulation, the amplitude of fluorescence transients to succeeding action potentials became smaller. The decrement of the amplitudes was not observed in mag-fura-5-labeled tecta, when the intracellular Ca2+ buffering capacity of fura-2-labeled terminals was increased by incubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-AM, or in low-Ca2+ saline. We conclude that the Ca2+ influx per action potential is constant during the train and that the reduced response was produced by saturation of the fura-2. We provide a mathematical analysis of this saturation effect and use it to estimate the Ca2+ change per action potential. 4. Both BAPTA-AM and EGTA-AM reduced the overall amplitude of fura-2-measured Ca2+ transients and reduced the saturation effect in action potential trains. However, there was a qualitative difference in their effects on the shape of the transient. Incubation with the fast buffer BAPTA prolonged the decay to baseline. In contrast, the slow buffer EGTA (or EDTA) produced an initial decay faster than the control condition while also producing the slower subsequent phase observed with BAPTA. We demonstrate that these results are consistent with numerical simulations of Ca2+ dynamics in a single-compartment model where the fast initial decay is produced by the forward rate of Ca2+ binding to EGTA. 5. Ca2+ influx into tectal presynaptic structures, and also into unmyelinated axons in the isolated optic nerve, was diminished (60-70%) in the presence of the voltage-activated Ca2+ channel blocker omega-conotoxin GVIA, but was only weakly affected (approximately 10%) by omega-agatoxin IVA. 6. After 10- to 50-Hz stimulus trains, synaptic enhancement of unmyelinated fibers decayed with a characteristic time similar to fura-2 fluorescence decays. Incubation with EDTA-AM or EGTA-AM produced little effect on evoked release but reduced both the amplitude of the fura-2-measured Ca2+ transient and the amplitude of short-term synaptic enhancement.

2011/2012
During the development of spinal cord, the maturation of neuronal circuits is a complex process, involving genetic and epigenetic mechanisms cooperating for the maturation of motor control (Jessell, 2000; Kiehn, 2006). Variations in the concentration of intracellular Ca2+ are crucial signals in this process of maturation; in fact, Ca2+ signals may lead to the emergence of specific neuronal phenotypes or guide the formation of cellular connectivity. The organotypic cultures of embryonic mouse spinal cord represent an ideal experimental approach to study the maturation and physiology of the individual neurons and spinal networks. In fact, this experimental model reproduces in vitro the heterogeneous populations of cells, the three dimensional connections between these cells and the basic cytoarchitecture of the spinal cord observed in in vivo development (Avossa et al., 2003). In this experimental model, three different types of Ca2+ activity have been identified and characterized: waves, bursts and oscillations (Fabbro et al., 2007; Sibilla et al., 2009). These Ca2+ signals are all generated by ventral interneurons, but each of them shows a specific pattern of expression during development and has different underlying mechanisms. In this thesis, I focused my attention on the most peculiar of these Ca2+ signals: the electrical activity-independent Ca2+ oscillations. The main aim of my thesis was to better clarify the mechanisms underlying the generation and role of Ca2+ oscillations in spinal neurons, by investigating the effects of pharmacological manipulation of intracellular Ca2+ buffering on Ca2+ oscillations behavior, neuronal biophysical properties and neuronal network activity in organotypic spinal cultures. To this aim, I treated spinal cord slices with two different intracellular Ca2+ buffers, BAPTA-AM and EGTA-AM, and I monitored their impact using both Ca2+-imaging and single cell patch-clamp techniques. Initially, I investigated the effects on Ca2+ oscillations induced by both chronic and acute treatment with BAPTA-AM. For the first time I described a change in the activity of oscillating neurons. In particular, after chronic incubation with BAPTA-AM, I reported a significant increase in the number of neurons recruited to generate Ca2+ oscillations, which was accompanied by a modulation of oscillations kinetic. Ca2+ oscillations recorded after chronic incubation with BAPTA-AM maintained their peculiar features (Fabbro et al., 2007; Sibilla et al., 2009), in particular their Ca2+-dependence, thus supporting the idea that the BAPTA-induced oscillations represent an amplification of the true oscillations phenomena, amplified by a prolonged intracellular Ca2+ buffering. Despite a potentiating effect of chronic BAPTA-AM treatment on Ca2+ oscillations, its acute application completely blocked Ca2+ oscillations in all neurons. The next step was to verify whether the observed effects could be related to changes in the biophysical properties of neurons or in neuronal network electrical activity. By patch clamp experiments I showed that the chronic BAPTA-AM treatment induces a significant enhancement in the frequency of heterogeneous (GABA-glycine and AMPA mediated) spontaneous post-synaptic currents (PSCs) when compared to untreated cultures. Neuronal membrane capacitance and input resistance were comparable to those of control neurons, thus confirming neuronal health. As the reported results pointed to an increased excitability at the level of single neuron, I analyzed the impact of BAPTA treatment on the functional expression of a family of channels extremely important in the regulation of neuronal excitability: voltage-gated K+ channels. I observed the presence of a significant increase in the amplitude of K+ currents (IK) in slices chronically treated with BAPTA-AM. To analyze the type of IK involved I separated the different IK components (Ca2+-dependent -IK(Ca)- , transient -IK(A)- and delayed-rectifier -IK(DR)-), demonstrating that, in BAPTA-AM treated cultures, the IK(Ca) and IK(A) components were similar to control cultures. Conversely, I found a potentiation of IK(DR), i.e. an increase in its maximal current amplitude. Furthermore, I found that acute application of BAPTA-AM partially reduces the magnitude of total IK. Action potentials are other critical players reflecting neuronal excitability. Chronic BAPTA-AM treatment did not affect action potential kinetic; however, I found that BAPTA-treated neurons show a different distribution profile of excitability, with a widening of the population of ventral spinal interneurons displaying a tonic firing pattern and a decrease in the one showing an adapting firing behavior. To explore the specificity of BAPTA-AM effects I employed another intracellular Ca2+ chelator: EGTA-AM. I reported, as a consequence of a chronic EGTA-AM treatment of spinal neurons, an increase in the population of oscillating neurons (similarly to BAPTA treatment), but, without changes in oscillations kinetic. However, the study of synaptic activity in EGTA-AM treated slices did not reveal any change in the frequency or kinetic of spontaneous or miniature PSCs. Interestingly, in contrast to BAPTA treatment, EGTA-AM had no effect on IK. Overall, the results reported in this thesis show, on one hand, a specific effect of BAPTA-AM on K+; most importantly, on the other hand, they support the needing of a correct intracellular Ca2+ homeostasis for the genesis of Ca2+ oscillations and indicate the presence of a homeostatic adaptation as a rebound effect of chronic manipulation of intracellular Ca2+.
XXV Ciclo
1982

Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are detected with indo 1. They are extremely rapid and are maximimal by 1015 seconds. Suspension studies, which reflect an average value over 3 × 107 cells/ml, indicate a thrombin dose-dependent increase in cytoplasmic calcium. However, flow cytometry indicates that subpopulations of cells are responding differently. The responses of these populations seem to be dependent on thrombin concentration. A maximal overall response is found when more than 50 % of the cells respond. This may explain multiple stimulations of the same suspension of cells at low thrombin dosesElevated extracellular Ca++ increases the maximal cytoplasmic response and significantly retards its subsequent decrease. When extracellular Ca++ is removed via addition of EGTA just before stimulation, the initial response is halved and the subsequent slow decrease is more marked and returns to lower (near-basal) levels. Intracellular Ca++ can be chelated with s - (0-am inophenoxy) - e thane-N, N, N',N'-tetraacet ic acid (BAPTA), which has a twofold greater affinity far Ca++ than does indo, but which does not fluoresce (at indo sensitive wavelengths) upon binding of Ca++. Cells loaded with both indo and BAPTA exhibit no rise in cytosolic Ca++ or altered membrane potential when stimulated with αthrombin, unlike cells loaded with indo alone. When Ca++ (2 mM) is added back to the extracellular buffer, these cells will take up the Ca++ at a constant rate as seen by a rise in indo fluorescence. When cytosolic Ca levels reach those of the resting platelets loaded with indo alone, these cells recover the αthrombin-indue ed Ca response of control platelets. However, they no longer depolarize partially under these same Ca++ replenished conditions. This implies that some of the Ca++ required for the platelet thrombin response comes from non-replenishable internal stores.