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1.
Methods Mol Biol ; 1942: 123-129, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900180

RESUMO

Calcium is a major regulator of neuronal activity and calcium signaling is critically important for normal neuronal function. Ca imaging is a well-established tool for studying neuronal function and ongoing spontaneous Ca2+ transients are a good indicator of neuronal maturity. There are various indicators available today, differing by their sensitivity, spectra, and loading method. Here we present a method for measurement of Ca2+ transients in neurons using two different Ca2+ indicators, Oregon Green BAPTA-1 and GCaMP6.


Assuntos
Potenciais de Ação , Cálcio/metabolismo , Diferenciação Celular , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Neurônios/metabolismo , Sinalização do Cálcio , Células Cultivadas , Quelantes/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Neurônios/citologia , Compostos Orgânicos/metabolismo
2.
Biochem Biophys Res Commun ; 507(1-4): 211-216, 2018 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-30415775

RESUMO

Ciliary beating frequency (CBF) was investigated in ciliated nasal epithelial cells (cMNECs) isolated from mice using video microscopy equipped with a high-speed camera. In cMNECs, a spontaneous CBF oscillation was observed. The CBF oscillation was abolished by BAPTA-AM but not by Ca2+-free solution. The addition of thapsigargin, which depletes Ca2+ from internal stores, also abolished CBF oscillation. Moreover, the intracellular Ca2+ concentration [Ca2+]i, spontaneously oscillated even with the Ca2+-free solution. Moreover, 2APB (an inhibitor of the IP3 receptor) abolished CBF oscillation in cMNECs. Overall, these findings suggest that the CBF oscillation in cMNECs is triggered by the release of Ca2+ from the IP3-sensitive internal stores. Moreover, IBMX, an inhibitor of phosphodiesterase, did not affect CBF oscillation in cMNECs, although it slightly increased CBF. These results suggest that CBF oscillations were induced by [Ca2+]i oscillation controlled via the release of Ca2+ from IP3-sensitive stores, rather than via cAMP accumulation. CBF oscillation possibly plays a crucial role in maintaining an efficient mucociliary clearance in the nasal epithelia.


Assuntos
Cálcio/metabolismo , Cílios/metabolismo , Espaço Intracelular/metabolismo , Mucosa Nasal/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Compostos de Boro/farmacologia , Cílios/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Camundongos Endogâmicos C57BL , Mucosa Nasal/efeitos dos fármacos , Tapsigargina/farmacologia
3.
Cell Calcium ; 73: 82-87, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29689523

RESUMO

The study of cellular Ca2+ signalling is indebted to Roger Tsien for the invention of fluorescent indicators that can be readily loaded into living cells and provide the means to measure cellular Ca2+ changes over long periods of time with sub-second resolution and microscopic precision. However, a recent study [1] reminds us that as useful as these tools are they need to be employed with caution as there can be off-target effects. This article summarises these recent findings within the wider context of confounding issues that can be encountered when using chemical and genetically-encoded Ca2+ indicators, and briefly discusses some approaches that may mitigate against misleading outcomes.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Indicadores e Reagentes/metabolismo , Animais , Cálcio/análise , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/análise , Ácido Egtázico/metabolismo , Ácido Egtázico/farmacologia , Humanos , Indicadores e Reagentes/análise , Indicadores e Reagentes/farmacologia
4.
J Cell Biochem ; 118(11): 3722-3729, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28374913

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the newest discovered intracellular second messengers, which is able to release Ca2+ stored within endolysosomal (EL) vesicles. NAADP-induced Ca2+ signals mediate a growing number of cellular functions, ranging from proliferation to muscle contraction and differentiation. Recently, NAADP has recently been shown to regulate angiogenesis by promoting endothelial cell growth. It is, however, still unknown whether NAADP stimulates proliferation also in endothelial progenitor cells, which are mobilized in circulation after an ischemic insult to induce tissue revascularization. Herein, we described a novel approach to prepare NAADP-containing liposomes, which are highly cell membrane permeable and are therefore amenable for stimulating cell activity. Accordingly, NAADP-containing liposomes evoked an increase in intracellular Ca2+ concentration, which was inhibited by NED-19, a selective inhibitor of NAADP-induced Ca2+ release. Furthermore, NAADP-containing liposomes promoted EPC proliferation, a process which was inhibited by NED-19 and BAPTA, a membrane permeable intracellular Ca2+ buffer. Therefore, NAADP-containing liposomes stand out as a promising tool to promote revascularization of hypoxic/ischemic tissues by favoring EPC proliferation. J. Cell. Biochem. 118: 3722-3729, 2017. © 2017 Wiley Periodicals, Inc.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , NADP/análogos & derivados , Neovascularização Fisiológica/efeitos dos fármacos , Adulto , Carbolinas/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Células Endoteliais/citologia , Feminino , Humanos , Lipossomos , Masculino , NADP/farmacologia , Piperazinas/metabolismo
5.
Mol Biochem Parasitol ; 213: 26-29, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28274857

RESUMO

To investigate the role of Ca2+ signaling in starvation-induced autophagy in Trypanosoma brucei, the causative agent of human African trypanosomiasis, we used cell-permeant Ca2+ chelator BAPTA-AM and cell impermeant chelator EGTA, and examined the potential involvement of several intracellular Ca2+ signaling pathways in T. brucei autophagy. The results showed an unexpected effect of BAPTA-AM in decreasing cellular pH and inhibiting acidocalcisome acidification in starved cells. The implication of these results in the role of Ca2+ signaling and cellular/organellar pH in T. brucei autophagy is discussed.


Assuntos
Aminoácidos/metabolismo , Autofagia , Quelantes/metabolismo , Ácido Egtázico/análogos & derivados , Organelas/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ácido Egtázico/metabolismo , Concentração de Íons de Hidrogênio
6.
Sci Rep ; 6: 38281, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27910894

RESUMO

Chromosome condensation is essential for the faithful transmission of genetic information to daughter cells during cell division. The depletion of chromosome scaffold proteins does not prevent chromosome condensation despite structural defects. This suggests that other factors contribute to condensation. Here we investigated the contribution of divalent cations, particularly Ca2+, to chromosome condensation in vitro and in vivo. Ca2+ depletion caused defects in proper mitotic progression, particularly in chromosome condensation after the breakdown of the nuclear envelope. Fluorescence lifetime imaging microscopy-Förster resonance energy transfer and electron microscopy demonstrated that chromosome condensation is influenced by Ca2+. Chromosomes had compact globular structures when exposed to Ca2+ and expanded fibrous structures without Ca2+. Therefore, we have clearly demonstrated a role for Ca2+ in the compaction of chromatin fibres.


Assuntos
Cálcio/farmacologia , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Empacotamento do DNA/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Cálcio/metabolismo , Quelantes de Cálcio/metabolismo , Quelantes de Cálcio/farmacologia , Cromatina/metabolismo , Cromatina/ultraestrutura , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Ácido Egtázico/farmacologia , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Fura-2/análogos & derivados , Fura-2/química , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura
7.
Cell Physiol Biochem ; 40(3-4): 597-607, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27889774

RESUMO

BACKGROUND/AIMS: The CDC25B inhibitor NSC-95397 triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The substance is effective in part by modification of gene expression. Similar to apoptosis of nucleated cells erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of protein kinases. The present study explored, whether NSC-95397 induces eryptosis and, if so, to shed some light on the mechanisms involved. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to NSC-95397 significantly increased the percentage of annexin-V-binding cells (≥ 1 µM), significantly decreased forward scatter (≥ 2.5 µM), and significantly increased Fluo3-fluorescence (≥ 1 µM), DCFDA fluorescence (5 µM) and ceramide abundance (≥ 5 µM). The effect of NSC-95397 (5 µM) on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+ and by addition of the protein kinase C inhibitor staurosporine (1 µM). CONCLUSIONS: NSC-95397 triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring entry of Ca2+ and activation of staurosporine sensitive kinase(s).


Assuntos
Inibidores Enzimáticos/farmacologia , Eriptose/efeitos dos fármacos , Naftoquinonas/farmacologia , Fosfatases cdc25/antagonistas & inibidores , Cálcio/metabolismo , Ceramidas/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Espalhamento de Radiação , Fosfatases cdc25/metabolismo
8.
J Neurophysiol ; 115(6): 3101-12, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27009157

RESUMO

Individual neurons in several sensory systems receive synaptic inputs organized according to subcellular topographic maps, yet the fine structure of this topographic organization and its relation to dendritic morphology have not been studied in detail. Subcellular topography is expected to play a role in dendritic integration, particularly when dendrites are extended and active. The lobula giant movement detector (LGMD) neuron in the locust visual system is known to receive topographic excitatory inputs on part of its dendritic tree. The LGMD responds preferentially to objects approaching on a collision course and is thought to implement several interesting dendritic computations. To study the fine retinotopic mapping of visual inputs onto the excitatory dendrites of the LGMD, we designed a custom microscope allowing visual stimulation at the native sampling resolution of the locust compound eye while simultaneously performing two-photon calcium imaging on excitatory dendrites. We show that the LGMD receives a distributed, fine retinotopic projection from the eye facets and that adjacent facets activate overlapping portions of the same dendritic branches. We also demonstrate that adjacent retinal inputs most likely make independent synapses on the excitatory dendrites of the LGMD. Finally, we show that the fine topographic mapping can be studied using dynamic visual stimuli. Our results reveal the detailed structure of the dendritic input originating from individual facets on the eye and their relation to that of adjacent facets. The mapping of visual space onto the LGMD's dendrites is expected to have implications for dendritic computation.


Assuntos
Dendritos/fisiologia , Percepção de Movimento/fisiologia , Células Receptoras Sensoriais/fisiologia , Células Receptoras Sensoriais/ultraestrutura , Campos Visuais/fisiologia , Vias Visuais/citologia , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Gafanhotos , Masculino , Modelos Neurológicos , Estimulação Luminosa , Vias Visuais/fisiologia
9.
J Gen Physiol ; 147(1): 95-102, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26712852

RESUMO

The divalent cation chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), often used to buffer physiological changes in cytosolic Ca(2+), also binds Zn(2+) with high affinity. In a recently published method (Lamboley et al. 2015. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201411250), the absorbance shift of BAPTA at 292 nm was successfully used to determine the total calcium concentrations of various skeletal muscle tissues. In the present study, we show that endogenous Zn(2+) in rat skeletal muscle tissue can be unknowingly measured as "Ca(2+)," unless appropriate measures are taken to eliminate Zn(2+) interference. We analyzed two rat skeletal muscle tissues, soleus and plantaris, for total calcium and zinc using either inductively coupled plasma mass spectrometry (ICP-MS) or the BAPTA method described above. ICP-MS analysis showed that total zinc contents in soleus and plantaris were large enough to affect the determination of total calcium by the BAPTA method (calcium = 1.72 ± 0.31 and 1.96 ± 0.14, and zinc = 0.528 ± 0.04 and 0.192 ± 0.01; mean ± standard error of the mean [SEM]; n = 5; mmole/kg, respectively). We next analyzed total calcium using BAPTA but included the Zn(2+)-specific chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) that buffers Zn(2+) without affecting Ca(2+)/BAPTA binding. We found that estimated concentrations of total calcium ([CaT]WM) in soleus and plantaris were reduced after TPEN addition ([CaT]WM = 3.71 ± 0.62 and 3.57 ± 0.64 without TPEN and 3.39 ± 0.64 and 3.42 ± 0.62 with TPEN; mean ± SEM; n = 3; mmole/kg, respectively). Thus, we show that a straightforward correction can be applied to the BAPTA method to improve the accuracy of the determination of total calcium that should be applicable to most any tissue studied. In addition, we show that using TPEN in combination with the BAPTA method allows one to make reasonable estimates of total zinc concentration that are in agreement with the direct determination of zinc concentration by ICP-MS.


Assuntos
Cálcio/metabolismo , Músculo Esquelético/metabolismo , Zinco/metabolismo , Animais , Quelantes/metabolismo , Citosol/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Etilenodiaminas/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344
10.
Toxicol Lett ; 240(1): 1-9, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26476400

RESUMO

Hyperbilirubinemia is a common clinical phenomenon observed in human newborns. A high level of bilirubin can result in severe jaundice and bilirubin encephalopathy. However, the cellular mechanisms underlying bilirubin excitotoxicity are unclear. Our previous studies showed the action of gamma-aminobutyric acid (GABA)/glycine switches from excitatory to inhibitory during development in the ventral cochlear nucleus (VCN), one of the most sensitive auditory nuclei to bilirubin toxicity. In the present study, we investigated the roles of GABAA/glycine receptors in the induction of bilirubin hyperexcitation in early developing neurons. Using the patch clamp technique, GABAA/glycine receptor-mediated spontaneous inhibitory synaptic currents (sIPSCs) were recorded from bushy and stellate cells in acute brainstem slices from young mice (postnatal day 2-6). Bilirubin significantly increased the frequency of sIPSCs, and this effect was prevented by pretreatments of slices with either fast or slow Ca(2+) chelators BAPTA-AM and EGTA-AM suggesting that bilirubin can increase the release of GABA/glycine via Ca(2+)-dependent mechanisms. Using cell-attached recording configuration, we found that antagonists of GABAA and glycine receptors strongly attenuated spontaneous spiking firings in P2-6 neurons but produced opposite effect in P15-19 neurons. Furthermore, these antagonists reversed bilirubin-evoked hyperexcitability in P2-6 neurons, indicating that excitatory action of GABA/glycinergic transmission specifically contribute to bilirubin-induced hyperexcitability in the early stage of development. Our results suggest that bilirubin-induced enhancement of presynaptic release GABA/Glycine via Ca(2+)-dependent mechanisms may play a critical role in mediating neuronal hyperexcitation associated with jaundice, implicating potential new strategies for predicting, preventing, and treating bilirubin neurotoxicity.


Assuntos
Bilirrubina/efeitos adversos , Glicina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Animais , Núcleo Coclear/efeitos dos fármacos , Núcleo Coclear/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Hiperbilirrubinemia/induzido quimicamente , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Receptores da Glicina/metabolismo
11.
Mol Neurobiol ; 53(10): 7078-7088, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-26676572

RESUMO

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants and are ubiquitous in the environment and human tissues. Recent evidence has demonstrated that PBDE-induced neurotoxicity is associated with neuronal apoptosis via interfering with the calcium ion (Ca2+) homeostasis; however, the underlying mechanisms remain elusive. Thus, we sought to investigate the role of Ca2+ homeostasis in PBDE-47-induced neuronal apoptosis. Here, we showed that PBDE-47 significantly decreased neuronal number while increased neuronal apoptosis in vitro and in vivo, as manifested by an increased percentage of Annexin V-positive staining cells and caspase-3 activation in human neuroblastoma SH-SY5Y cells and hippocampal neurons of rats. Further study identified that PBDE-47 elicited ΔΨm collapse following an early and sustained [Ca2+] i, overload, as well as stimulated cytochrome c release from mitochondria into the cytosol in SH-SY5Y cells and rat hippocampal tissue. Interestingly, the extracellular Ca2+ chelator ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) blocked PBDE-47-induced [Ca2+] i elevation, ΔΨm collapse, cytochrome c release, and caspase-3 activation in SH-SY5Y cells, whereas the intracellular Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) had no influences on them, indicating that the [Ca2+] i overload originates primarily from extracellular Ca2+ component rather than from intracellular calcium storage and that the increase in [Ca2+] i is a major contributor to ΔΨm collapse and subsequent neuronal apoptosis. Overall, these findings suggest that PBDE-47 affects Ca2+ homeostasis as a crucial event in activation of neuronal death associated with mitochondria and provide novel insight into the mechanism of action underlying PBDE neurotoxicity.


Assuntos
Cálcio/metabolismo , Éteres Difenil Halogenados/toxicidade , Homeostase , Neurônios/patologia , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Hipocampo/patologia , Homeostase/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Ratos Sprague-Dawley
12.
Cell Mol Life Sci ; 73(8): 1699-713, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26588916

RESUMO

Apoptosis, necrosis, or autophagy-it is the mode of cell demise that defines the response of surrounding cells and organs. In case of one of the most toxic substances known to date, cadmium (Cd), and despite a large number of studies, the mode of cell death induced is still unclear. As there exists conflicting data as to which cell death mode is induced by Cd both across various cell types and within a single one, we chose to analyse Cd-induced cell death in primary human endothelial cells by investigating all possibilities that a cell faces in undergoing cell death. Our results indicate that Cd-induced death signalling starts with the causation of DNA damage and a cytosolic calcium flux. These two events lead to an apoptosis signalling-related mitochondrial membrane depolarisation and a classical DNA damage response. Simultaneously, autophagy signalling such as ER stress and phagosome formation is initiated. Importantly, we also observed lysosomal membrane permeabilization. It is the integration of all signals that results in DNA degradation and a disruption of the plasma membrane. Our data thus suggest that Cd causes the activation of multiple death signals in parallel. The genotype (for example, p53 positive or negative) as well as other factors may determine the initiation and rate of individual death signals. Differences in the signal mix and speed may explain the differing results recorded as to the Cd-induced mode of cell death thus far. In human endothelial cells it is the sum of most if not all of these signals that determine the mode of Cd-induced cell death: programmed necrosis.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cádmio/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Necrose/patologia , Cádmio/metabolismo , Cálcio/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quelantes/metabolismo , Dano ao DNA/efeitos dos fármacos , Ácido Egtázico/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/patologia , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo
13.
Proc Natl Acad Sci U S A ; 112(36): 11377-82, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26305966

RESUMO

In vivo Ca2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ (Kd=561 nM), single-action potential-evoked Ca2+ transients were discernable in most neurons with a good signal-to-noise ratio. Action potential-dependent Ca2+ transients were recorded in neurons of all six layers of the cortex at depths of up to -900 µm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca2+ indicators. Ca2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca2+ indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.


Assuntos
Cálcio/metabolismo , Fluorometria/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neuroimagem/métodos , Potenciais de Ação/fisiologia , Animais , Cálcio/análise , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Dendritos/metabolismo , Dendritos/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/química , Ácido Egtázico/metabolismo , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp , Reprodutibilidade dos Testes
14.
J Neurochem ; 135(4): 777-86, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26263185

RESUMO

The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic-day-16 fetuses. To calibrate acid-induced intracelluar [Zn(2+) ] ([Zn(2+) ]i ) elevations, a low affinity (Kd = 39 µM at pH 6.1) ratiometric Zn(2+) probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn(2+) ]i elevations reaching 2 µM; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn(2+) ]i increased further to 5.6 µM; analogous acid- and NEM-induced [Zn(2+) ]i elevations could also be detected but not calibrated, using the high affinity Zn(2+) probe FluoZin-3. The data indicate that NEM causes Zn(2+) release from ligands that chelate Zn(2+) at pH 6.1. ATP could also chelate Zn(2+) at pH 6.1 because its pKa is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn(2+) ]i elevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid-induced [Zn(2+) ]i increases. These data suggest that the total amount of Zn(2+) accumulated in intracellular ATP-dependent stores (Zn(2+) -ATP complexes and organelles that accumulate Zn(2+) in an ATP-dependent manner) is negligible compared to the amount of Zn(2+) accumulated in the acid-sensitive intracellular ligands. In vitro, upon acidification, Zn(2+) -cysteine complexes release Zn(2+) and ATP chelates the released Zn(2+) . However, in vivo (cultured neurons), an ATP depletion failed to enhance acid-induced [Zn(2+) ]i elevations. These [Zn(2+) ]i elevations were calibrated using a low affinity ratiometric probe FuraZin-1; they reached 2 µM levels and increased to 5 µM when a thiol-reactive agent, N-ethylmaleimide, compromised Zn(2+) binding by cysteines.


Assuntos
Citosol/metabolismo , Neurônios/metabolismo , Zinco/metabolismo , Ácidos/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Quelantes/farmacologia , Cisteína , Citosol/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Etilenodiaminas , Etilmaleimida/farmacologia , Magnésio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Compostos Policíclicos , Ionóforos de Próton/farmacologia
15.
Curr Protoc Cytom ; 72: 9.8.1-9.8.21, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25827486

RESUMO

Using flow cytometry, single-cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation.


Assuntos
Citometria de Fluxo/métodos , Espaço Intracelular/metabolismo , Animais , Tampões (Química) , Cálcio/metabolismo , Calibragem , Ácido Egtázico/metabolismo , Fluorescência , Humanos , Íons , Camundongos , Poloxâmero/farmacologia , Espectrometria de Fluorescência
16.
PLoS One ; 10(3): e0118750, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25775468

RESUMO

Shank and GKAP are scaffold proteins and binding partners at the postsynaptic density (PSD). The distribution and dynamics of Shank and GKAP were studied in dissociated hippocampal cultures by pre-embedding immunogold electron microscopy. Antibodies against epitopes containing their respective mutual binding sites were used to verify the expected juxtapositioning of Shank and GKAP. If all Shank and GKAP molecules at the PSD were bound to each other, the distribution of label for the two proteins should coincide. However, labels for the mutual binding sites showed significant differences in distribution, with a narrow distribution for GKAP located close to the postsynaptic membrane, and a wider distribution for Shank extending deeper into the cytoplasm. Upon depolarization with high K+, neither the intensity nor distribution of label for GKAP changed, but labeling intensity for Shank at the PSD increased to ~150% of controls while the median distance of label from postsynaptic membrane increased by 7.5 nm. These results indicate a preferential recruitment of Shank to more distal parts of the PSD complex. Conversely, upon incubation in Ca2+-free medium containing EGTA, the labeling intensity of Shank at the PSD decreased to ~70% of controls and the median distance of label from postsynaptic membrane decreased by 9 nm, indicating a preferential loss of Shank molecules in more distal parts of the PSD complex. These observations identify two pools of Shank at the PSD complex, one relatively stable pool, closer to the postsynaptic membrane that can bind to GKAP, and another more dynamic pool at a location too far away to bind to GKAP.


Assuntos
Hipocampo/citologia , Proteínas do Tecido Nervoso/metabolismo , Densidade Pós-Sináptica/metabolismo , Animais , Células Cultivadas , Ácido Egtázico/metabolismo , Proteínas do Tecido Nervoso/análise , Neurônios/citologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Densidade Pós-Sináptica/ultraestrutura , Potássio/metabolismo , Ratos Sprague-Dawley , Proteínas Associadas SAP90-PSD95
17.
Biochem Biophys Res Commun ; 458(2): 251-5, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25640842

RESUMO

We have shown that Mg/EGTA (5 mM Mg(2+) and 1.5 mM EGTA) could effectively promote the adhesion of integrin αLß2 to its ligand ICAM-1 but could not promote that of the αMß2 to denatured BSA. In order to determine the structural differences between αL and αM that specifically contribute to Mg/EGTA sensitivity, a series of αL/αM chimeras were constructed. Our results showed that αLß2 with αM calf-1 domain completely lost the response to Mg/EGTA activation. In the reverse experiment, αMß2 would require the presence of both the αL calf-1 and calf-2 domain to initiate the Mg/EGTA sensitivity.


Assuntos
Adesão Celular/fisiologia , Ácido Egtázico/química , Ácido Egtázico/metabolismo , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Magnésio/química , Magnésio/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Ligação Proteica , Relação Estrutura-Atividade
18.
Cereb Cortex ; 25(9): 2594-609, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24692513

RESUMO

Neural activity regulates local increases in cerebral blood flow (ΔCBF) and the cortical metabolic rate of oxygen (ΔCMRO2) that constitutes the basis of BOLD functional neuroimaging signals. Glutamate signaling plays a key role in brain vascular and metabolic control; however, the modulatory effect of GABA is incompletely understood. Here we performed in vivo studies in mice to investigate how THIP (which tonically activates extrasynaptic GABAARs) and Zolpidem (a positive allosteric modulator of synaptic GABAARs) impact stimulation-induced ΔCBF, ΔCMRO2, local field potentials (LFPs), and fluorescent cytosolic Ca(2+) transients in neurons and astrocytes. Low concentrations of THIP increased ΔCBF and ΔCMRO2 at low stimulation frequencies. These responses were coupled to increased synaptic activity as indicated by LFP responses, and to Ca(2+) activities in neurons and astrocytes. Intermediate and high concentrations of THIP suppressed ΔCBF and ΔCMRO2 at high stimulation frequencies. Zolpidem had similar but less-pronounced effects, with similar dependence on drug concentration and stimulation frequency. Our present findings suggest that slight increases in both synaptic and extrasynaptic GABAAR activity might selectively gate and amplify transient low-frequency somatosensory inputs, filter out high-frequency inputs, and enhance vascular and metabolic responses that are likely to be reflected in BOLD functional neuroimaging signals.


Assuntos
Cálcio/metabolismo , Circulação Cerebrovascular/fisiologia , Consumo de Oxigênio/fisiologia , Receptores de GABA-A/metabolismo , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Potenciais de Ação/fisiologia , Animais , Biofísica , Circulação Cerebrovascular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Estimulação Elétrica , Lateralidade Funcional , Agonistas GABAérgicos/farmacologia , Isoxazóis/farmacologia , Camundongos , Consumo de Oxigênio/efeitos dos fármacos , Pressão Parcial , Piridinas/farmacologia , Sulfonamidas/metabolismo , Tiazóis/metabolismo , Vibrissas/inervação , Zolpidem , Ácido gama-Aminobutírico/farmacologia
19.
Proc Natl Acad Sci U S A ; 111(41): 14918-23, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25228765

RESUMO

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca(2+) entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca(2+)-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca(2+) reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca(2+) concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca(2+) buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca(2+), intracellular Ca(2+) buffering, and membrane potential, by their common effect on intracellular free Ca(2+).


Assuntos
Adaptação Fisiológica , Cálcio/metabolismo , Células Ciliadas Auditivas/fisiologia , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Mecanotransdução Celular , Estereocílios/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Animais , Cálcio/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Espaço Extracelular/metabolismo , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/fisiologia , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Estereocílios/efeitos dos fármacos
20.
Methods Mol Biol ; 1187: 131-41, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25053486

RESUMO

Signaling assays in Drosophila cell lines are a valuable method for investigating whether other proteins influence the function of the Notch pathway and for assessing whether specific enhancers or genes are regulated by Notch. In this chapter, we will describe two different types of assays that can be used to monitor Notch activation in Kc167 and S2 cells. One involves activating Notch in cultured cells and measuring the change in endogenous gene expression levels. The other uses luciferase reporters and measures their response to Notch, by co-transfecting with NICD.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Ácido Edético/metabolismo , Ácido Egtázico/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Luciferases/genética , Reação em Cadeia da Polimerase/métodos , Receptores Notch/genética , Renilla/enzimologia , Transfecção
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