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1.
Adv Exp Med Biol ; 1131: 73-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646507

RESUMO

Imaging techniques may overcome the limitations of electrode techniques to measure locally not only membrane potential changes, but also ionic currents. Here, we review a recently developed approach to image native neuronal Ca2+ currents from brain slices. The technique is based on combined fluorescence recordings using low-affinity Ca2+ indicators possibly in combination with voltage sensitive dyes. We illustrate how the kinetics of a Ca2+ current can be estimated from the Ca2+ fluorescence change and locally correlated with the change of membrane potential, calibrated on an absolute scale, from the voltage fluorescence change. We show some representative measurements from the dendrites of CA1 hippocampal pyramidal neurons, from olfactory bulb mitral cells and from cerebellar Purkinje neurons. We discuss the striking difference in data analysis and interpretation between Ca2+ current measurements obtained using classical electrode techniques and the physiological currents obtained using this novel approach. Finally, we show how important is the kinetic information on the native Ca2+ current to explore the potential molecular targets of the Ca2+ flux from each individual Ca2+ channel.


Assuntos
Canais de Cálcio , Neuroimagem , Animais , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Dendritos/fisiologia , Humanos , Potenciais da Membrana/fisiologia , Imagem Óptica , Células Piramidais/fisiologia
2.
Adv Exp Med Biol ; 1131: 445-469, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646520

RESUMO

Store-operated Ca2+ entry (SOCE) is a ubiquitous mechanism for Ca2+ influx in mammalian cells with important physiological implications. Since the discovery of SOCE more than three decades ago, the mechanism that communicates the information about the amount of Ca2+ accumulated in the intracellular Ca2+ stores to the plasma membrane channels and the nature of these channels have been matters of intense investigation and debate. The stromal interaction molecule-1 (STIM1) has been identified as the Ca2+ sensor of the intracellular Ca2+ compartments that activates the store-operated channels. STIM1 regulates two types of store-dependent channels: the Ca2+ release-activated Ca2+ (CRAC) channels, formed by Orai1 subunits, that conduct the highly Ca2+ selective current I CRAC and the cation permeable store-operated Ca2+ (SOC) channels, which consist of Orai1 and TRPC1 proteins and conduct the non-selective current I SOC. While the crystal structure of Drosophila CRAC channel has already been solved, the architecture of the SOC channels still remains unclear. The dynamic interaction of STIM1 with the store-operated channels is modulated by a number of proteins that either support the formation of the functional STIM1-channel complex or protect the cell against Ca2+ overload.


Assuntos
Canais de Cálcio , Cálcio , Transporte de Íons , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Transporte de Íons/genética , Molécula 1 de Interação Estromal/metabolismo
3.
Adv Exp Med Biol ; 1131: 489-504, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646522

RESUMO

Store-Operated Ca2+ Entry (SOCE) is an important Ca2+ influx pathway expressed by several excitable and non-excitable cell types. SOCE is recognized as relevant signaling pathway not only for physiological process, but also for its involvement in different pathologies. In fact, independent studies demonstrated the implication of essential protein regulating SOCE, such as STIM, Orai and TRPCs, in different pathogenesis and cell disorders, including cardiovascular disease, muscular dystrophies and angiogenesis. Compelling evidence showed that dysregulation in the function and/or expression of isoforms of STIM, Orai or TRPC play pivotal roles in cardiac hypertrophy and heart failure, vascular remodeling and hypertension, skeletal myopathies, and angiogenesis. In this chapter, we summarized the current knowledge concerning the mechanisms underlying abnormal SOCE and its involvement in some diseases, as well as, we discussed the significance of STIM, Orai and TRPC isoforms as possible therapeutic targets for the treatment of angiogenesis, cardiovascular and skeletal muscle diseases.


Assuntos
Cálcio , Doenças Cardiovasculares , Doenças Musculares , Neovascularização Patológica , Cálcio/metabolismo , Canais de Cálcio , Sinalização do Cálcio , Doenças Cardiovasculares/fisiopatologia , Humanos , Transporte de Íons , Doenças Musculares/fisiopatologia , Neovascularização Patológica/fisiopatologia
4.
Adv Exp Med Biol ; 1131: 625-648, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646528

RESUMO

Free Calcium (Ca2+) is an important and universal signalling entity in all cells, red blood cells included. Although mature mammalian red blood cells are believed to not contain organelles as Ca2+ stores such as the endoplasmic reticulum or mitochondria, a 20,000-fold gradient based on a intracellular Ca2+ concentration of approximately 60 nM vs. an extracellular concentration of 1.2 mM makes Ca2+-permeable channels a major signalling tool of red blood cells. However, the internal Ca2+ concentration is tightly controlled, regulated and maintained primarily by the Ca2+ pumps PMCA1 and PMCA4. Within the last two decades it became evident that an increased intracellular Ca2+ is associated with red blood cell clearance in the spleen and promotes red blood cell aggregability and clot formation. In contrast to this rather uncontrolled deadly Ca2+ signals only recently it became evident, that a temporal increase in intracellular Ca2+ can also have positive effects such as the modulation of the red blood cells O2 binding properties or even be vital for brief transient cellular volume adaptation when passing constrictions like small capillaries or slits in the spleen. Here we give an overview of Ca2+ channels and Ca2+-regulated channels in red blood cells, namely the Gárdos channel, the non-selective voltage dependent cation channel, Piezo1, the NMDA receptor, VDAC, TRPC channels, CaV2.1, a Ca2+-inhibited channel novel to red blood cells and i.a. relate these channels to the molecular unknown sickle cell disease conductance Psickle. Particular attention is given to correlation of functional measurements with molecular entities as well as the physiological and pathophysiological function of these channels. This view is in constant progress and in particular the understanding of the interaction of several ion channels in a physiological context just started. This includes on the one hand channelopathies, where a mutation of the ion channel is the direct cause of the disease, like Hereditary Xerocytosis and the Gárdos Channelopathy. On the other hand it applies to red blood cell related diseases where an altered channel activity is a secondary effect like in sickle cell disease or thalassemia. Also these secondary effects should receive medical and pharmacologic attention because they can be crucial when it comes to the life-threatening symptoms of the disease.


Assuntos
Canais de Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Eritrócitos/fisiologia , Doenças Hematológicas/fisiopatologia , Humanos , Mutação
5.
Adv Exp Med Biol ; 1131: 771-797, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646534

RESUMO

In this article, we present an overview of simulation strategies in the context of subcellular domains where calcium-dependent signaling plays an important role. The presentation follows the spatial and temporal scales involved and represented by each algorithm. As an exemplary cell type, we will mainly cite work done on striated muscle cells, i.e. skeletal and cardiac muscle. For these cells, a wealth of ultrastructural, biophysical and electrophysiological data is at hand. Moreover, these cells also express ubiquitous signaling pathways as they are found in many other cell types and thus, the generalization of the methods and results presented here is straightforward.The models considered comprise the basic calcium signaling machinery as found in most excitable cell types including Ca2+ ions, diffusible and stationary buffer systems, and calcium regulated calcium release channels. Simulation strategies can be differentiated in stochastic and deterministic algorithms. Historically, deterministic approaches based on the macroscopic reaction rate equations were the first models considered. As experimental methods elucidated highly localized Ca2+ signaling events occurring in femtoliter volumes, stochastic methods were increasingly considered. However, detailed simulations of single molecule trajectories are rarely performed as the computational cost implied is too large. On the mesoscopic level, Gillespie's algorithm is extensively used in the systems biology community and with increasing frequency also in models of microdomain calcium signaling. To increase computational speed, fast approximations were derived from Gillespie's exact algorithm, most notably the chemical Langevin equation and the τ-leap algorithm. Finally, in order to integrate deterministic and stochastic effects in multiscale simulations, hybrid algorithms are increasingly used. These include stochastic models of ion channels combined with deterministic descriptions of the calcium buffering and diffusion system on the one hand, and algorithms that switch between deterministic and stochastic simulation steps in a context-dependent manner on the other. The basic assumptions of the listed methods as well as implementation schemes are given in the text. We conclude with a perspective on possible future developments of the field.


Assuntos
Sinalização do Cálcio , Cálcio , Simulação por Computador , Algoritmos , Animais , Cálcio/metabolismo , Canais de Cálcio , Fenômenos Eletrofisiológicos , Humanos , Modelos Biológicos , Processos Estocásticos
6.
Adv Exp Med Biol ; 1131: 799-826, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646535

RESUMO

Transient rises and falls of the intracellular calcium concentration have been observed in numerous cell types and under a plethora of conditions. There is now a growing body of evidence that these whole-cell calcium oscillations are stochastic, which poses a significant challenge for modelling. In this review, we take a closer look at recently developed statistical approaches to calcium oscillations. These models describe the timing of whole-cell calcium spikes, yet their parametrisations reflect subcellular processes. We show how non-stationary calcium spike sequences, which e.g. occur during slow depletion of intracellular calcium stores or in the presence of time-dependent stimulation, can be analysed with the help of so-called intensity functions. By utilising Bayesian concepts, we demonstrate how values of key parameters of the statistical model can be inferred from single cell calcium spike sequences and illustrate what information whole-cell statistical models can provide about the subcellular mechanistic processes that drive calcium oscillations. In particular, we find that the interspike interval distribution of HEK293 cells under constant stimulation is captured by a Gamma distribution.


Assuntos
Sinalização do Cálcio , Cálcio , Modelos Biológicos , Teorema de Bayes , Cálcio/metabolismo , Canais de Cálcio , Células HEK293 , Humanos
7.
BMC Plant Biol ; 19(1): 368, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429706

RESUMO

BACKGROUND: We previously reported the involvement of nitric oxide (NO) and cyclic nucleotide-gated ion channel 6 (CNGC6) in the responses of plants to heat shock (HS) exposure. To elucidate their relationship with heat tolerance in Arabidopsis thaliana, we examined the effects of HS on several groups of seedlings: wild type, cngc6, and cngc6 complementation and overexpression lines. RESULTS: After HS exposure, the level of NO was lower in cngc6 seedlings than in wild-type seedlings but significantly elevated in the transgenic lines depending on CNGC6 expression level. The treatment of seeds with calcium ions (Ca2+) enhanced the NO level in Arabidopsis seedlings under HS conditions, whereas treatment with EGTA (a Ca2+ chelator) reduced it, implicating that CNGC6 stimulates the accumulation of NO depending on an increase in cytosolic Ca2+ ([Ca2+]cyt). This idea was proved by phenotypic observations and thermotolerance testing of transgenic plants overexpressing NIA2 and NOA1, respectively, in a cngc6 background. Western blotting indicated that CNGC6 stimulated the accumulation of HS proteins via NO. CONCLUSION: These data indicate that CNGC6 acts upstream of NO in the HS pathway, which improves our insufficient knowledge of the initiation of plant responses to high temerature.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Óxido Nítrico/metabolismo , Termotolerância , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Canais de Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Citosol/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Mutação , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Plântula/genética , Plântula/metabolismo
8.
Biol Res ; 52(1): 45, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426853

RESUMO

BACKGROUND: Resveratrol was reported to trigger the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats but the subcellular mechanism remains unclear. Since ER stress, mitochondrial dysfunction and oxidative stress were involved in the effects of resveratrol with imbalance of calcium bio-transmission, store operated calcium entry (SOCE), a novel intracellular calcium regulatory pathway, may also participate in this process. RESULTS: In the present study, Resveratrol was found to suppress ORAI1 expression of a dose dependent manner while have no evident effects on STIM1 expressive level. Besides, resveratrol had no effects on ATP or TG induced calcium depletion but present partly dose-dependent suppression of SOCE. On the one hand, microinjection of ORAI1 overexpressed vector in sick toe partly counteracted the therapeutic effects of resveratrol on adjuvant arthritis and serum inflammatory cytokine including IL-1, IL-6, IL-8, IL-10 and TNF-α. On the other hand, ORAI1 SiRNA injection provided slight relief to adjuvant arthritis in rats. In addition, ORAI1 overexpression partly diminished the alleviation of hemogram abnormality induced by adjuvant arthritis after resveratrol treatment while ORAI1 knockdown presented mild resveratrol-like effect on hemogram in rats model. CONCLUSION: These results indicated that resveratrol reduced store-operated Ca2+ entry and enhanced the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex, providing a theoretical basis for ORAI1 targeted therapy in future treatment with resveratrol on rheumatoid arthritis.


Assuntos
Apoptose/efeitos dos fármacos , Artrite Experimental/fisiopatologia , Canais de Cálcio/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Proteína ORAI1/efeitos dos fármacos , Resveratrol/farmacologia , Molécula 1 de Interação Estromal/efeitos dos fármacos , Sinoviócitos/efeitos dos fármacos , Animais , Canais de Cálcio/fisiologia , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Resveratrol/administração & dosagem
9.
Biochemistry (Mosc) ; 84(6): 593-607, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238859

RESUMO

Mitochondria are among the most important cell organelles involved in the regulation of intracellular calcium homeostasis. During the last decade, a number of molecular structures responsible for the mitochondrial calcium transport have been identified including the mitochondrial Ca2+ uniporter (MCU), Na+/Ca2+ exchanger (NCLX), and Ca2+/H+ antiporter (Letm1). The review summarizes the data on the structure, regulation, and physiological role of such structures. The pathophysiological mechanism of Ca2+ transport through the cyclosporine A-sensitive mitochondrial permeability transition pore is discussed. An alternative mechanism for the mitochondrial pore opening, namely, formation of the lipid pore induced by saturated fatty acids, and its role in Ca2+ transport are described in detail.


Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Animais , Canais de Cálcio/metabolismo , Citoplasma/metabolismo , Humanos , Transporte de Íons , Metabolismo dos Lipídeos , Membranas Mitocondriais/metabolismo , Estrutura Molecular , Trocador de Sódio e Cálcio/metabolismo
10.
Nat Plants ; 5(6): 581-588, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182842

RESUMO

Chloroplasts are integral to sensing biotic and abiotic stress in plants, but their role in transducing Ca2+-mediated stress signals remains poorly understood1,2. Here we identify cMCU, a member of the mitochondrial calcium uniporter (MCU) family, as an ion channel mediating Ca2+ flux into chloroplasts in vivo. Using a toolkit of aequorin reporters targeted to chloroplast stroma and the cytosol in cMCU wild-type and knockout lines, we provide evidence that stress-stimulus-specific Ca2+ dynamics in the chloroplast stroma correlate with expression of the channel. Fast downstream signalling events triggered by osmotic stress, involving activation of the mitogen-activated protein kinases (MAPK) MAPK3 and MAPK6, and the transcription factors MYB60 and ethylene-response factor 6 (ERF6), are influenced by cMCU activity. Relative to wild-type plants, cMCU knockouts display increased resistance to long-term water deficit and improved recovery on rewatering. Modulation of stromal Ca2+ in specific processing of stress signals identifies cMCU as a component of plant environmental sensing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas Mitocondriais/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Escherichia coli , Técnicas de Inativação de Genes , Sistema de Sinalização das MAP Quinases , Proteínas Mitocondriais/genética , Pressão Osmótica
11.
Nat Plants ; 5(6): 559-560, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182841
12.
J Physiol Pharmacol ; 70(1)2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31172971

RESUMO

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Mucosa Respiratória/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo
13.
Nat Commun ; 10(1): 2659, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201323

RESUMO

In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K+-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca2+ levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca2+, can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K+ transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K+-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca2+- and voltage-induced electrical excitability to the central organelle of plant cells.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Canais de Cálcio/metabolismo , Potenciais da Membrana/fisiologia , Vacúolos/fisiologia , Proteínas de Arabidopsis/genética , Canais de Cálcio/genética , Sinalização do Cálcio/fisiologia , Membranas Intracelulares/fisiologia , Mutação com Perda de Função , Células do Mesofilo/citologia , Células do Mesofilo/fisiologia , Plantas Geneticamente Modificadas , Potássio/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo
14.
Life Sci ; 231: 116567, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202839

RESUMO

AIMS: Metabotropic glutamate receptor 5 (mGluR5), a member of group I mGluR, exerts its effect via elevation of intracellular Ca2+ level. We here characterized Ca2+ signals in the tsA201 cells transfected with mGluR5 and investigated the role of passages for mGluR5-induced Ca2+ signals in synaptic plasticity. MAIN METHODS: Using a genetically encoded Ca2+ indicator, GCamp2, Ca2+ signals were reliably induced by bath application of (S)-3,5-dihydroxyphenylglycine, the group I mGluR agonist, in the tsA201 cells transfected with mGluR5. Using whole-cell recordings in the substantia gelatinosa (SG) neurons of the spinal trigeminal subnucleus caudalis (Vc), excitatory postsynaptic currents were recorded by stimulating the trigeminal tract. KEY FINDINGS: Ca2+ signals were mediated by "classical" or "canonical" transient receptor potential (TRPC) channels, particularly TRPC1/3/4/6, but not TRPC5, naturally existing in the tsA201 cells. Interestingly, the induction of Ca2+ signals was independent of the phospholipase C signaling pathway; instead, it critically involves the cyclic adenosine diphosphate ribose/ryanodine receptor-dependent signaling pathway and only partially protein kinase C. On the other hand, both TRPC3 and TRPC4 mediated mGluR1/5-induced long-lasting potentiation of excitatory synaptic transmission from the trigeminal primary afferents to the SG neurons of the Vc. SIGNIFICANCE: This study demonstrates that endogenous TRPC channels contribute to mGluR5-induced Ca2+ signals in tsA201 cells and synaptic plasticity at excitatory synapses.


Assuntos
Sinalização do Cálcio/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Receptor de Glutamato Metabotrópico 5/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Nervo Trigêmeo/metabolismo , Núcleo Espinal do Trigêmeo/metabolismo
15.
Life Sci ; 231: 116587, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31220526

RESUMO

The endoplasmic reticulum (ER) and mitochondria are two important organelles in cells. Mitochondria-associated membranes (MAMs) are lipid raft-like domains formed in the ER membranes that are in close apposition to mitochondria. They play an important role in signal transmission between these two essential organelles. When cells are exposed to internal or external stressful stimuli, the ER will activate an adaptive response called the ER stress response, which has a significant effect on mitochondrial function. Mitochondrial quality control is an important mechanism to ensure the functional integrity of mitochondria and the effect of ER stress on mitochondrial quality control through MAMs is of great significance. Therefore, in this review, we introduce ER stress and mitochondrial quality control, and discuss how ER stress signals are transmitted to mitochondria through MAMs. We then review the important roles of MAMs in mitochondrial quality control under ER stress.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Microdomínios da Membrana , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia
16.
Br J Anaesth ; 123(2): e226-e238, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31253357

RESUMO

BACKGROUND: The long-term use of opioid analgesics is limited by the development of unwanted side-effects, such as tolerance. The molecular mechanisms of morphine anti-nociceptive tolerance are still unclear. The mitochondrial calcium uniporter (MCU) is involved in painful hyperalgesia, but the role of MCU in morphine tolerance has not been uncharacterised. METHODS: Rats received intrathecal injection of morphine for 7 days to induce morphine tolerance. The mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. The effects of an MCU inhibitor, antisense oligodeoxynucleotide against cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) or cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in morphine tolerance were examined. RESULTS: Spinal morphine tolerance was associated with an increased expression of neuronal MCU, phospho-CREB (pCREB), and CPEB1 in the spinal cord dorsal horn. MCU inhibition increased the mechanical threshold and thermal latency, and reduced the accumulation of mitochondrial calcium in morphine tolerance. Intrathecal antisense oligodeoxynucleotide against CREB or CPEB1 restored the anti-nociceptive effects of morphine compared with mismatch oligodeoxynucleotide in von Frey test and hotplate test. Chromatin immunoprecipitation with quantitative PCR assay showed that CREB knockdown reduced the interaction of pCREB with the ccdc109a gene (encoding MCU expression) promoter and decreased the MCU mRNA transcription. RNA immunoprecipitation assay suggested that CPEB1 binds to the MCU mRNA 3' untranslated region. CPEB1 knockdown decreased the expression of MCU protein. CONCLUSIONS: These findings suggest that spinal MCU is regulated by pCREB and CPEB1 in morphine tolerance, and that inhibition of MCU, pCREB, or CPEB1 may be useful in preventing the development of opioid tolerance.


Assuntos
Proteína de Ligação a CREB/genética , Canais de Cálcio/metabolismo , Tolerância a Medicamentos/genética , Morfina/farmacologia , Proteínas de Ligação a RNA/genética , Corno Dorsal da Medula Espinal/metabolismo , Analgésicos Opioides/farmacologia , Animais , Masculino , Modelos Animais , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley
17.
Adv Exp Med Biol ; 1124: 233-263, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183830

RESUMO

We start by describing the functions of the uterus, its structure, both gross and fine, innervation and blood supply. It is interesting to note the diversity of the female's reproductive tract between species and to remember it when working with different animal models. Myocytes are the overwhelming cell type of the uterus (>95%) and our focus. Their function is to contract, and they have an intrinsic pacemaker and rhythmicity, which is modified by hormones, stretch, paracrine factors and the extracellular environment. We discuss evidence or not for pacemaker cells in the uterus. We also describe the sarcoplasmic reticulum (SR) in some detail, as it is relevant to calcium signalling and excitability. Ion channels, including store-operated ones, their contributions to excitability and action potentials, are covered. The main pathway to excitation is from depolarisation opening voltage-gated Ca2+ channels. Much of what happens downstream of excitability is common to other smooth muscles, with force depending upon the balance of myosin light kinase and phosphatase. Mechanisms of maintaining Ca2+ balance within the myocytes are discussed. Metabolism, and how it is intertwined with activity, blood flow and pH, is covered. Growth of the myometrium and changes in contractile proteins with pregnancy and parturition are also detailed. We finish with a description of uterine activity and why it is important, covering progression to labour as well as preterm and dysfunctional labours. We conclude by highlighting progress made and where further efforts are required.


Assuntos
Canais de Cálcio/fisiologia , Sinalização do Cálcio , Miométrio/fisiologia , Contração Uterina , Útero/fisiologia , Animais , Cálcio/fisiologia , Feminino , Gravidez , Retículo Sarcoplasmático/fisiologia
18.
Adv Exp Med Biol ; 1124: 357-377, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183835

RESUMO

The lymphatic system extends its network of vessels throughout most of the body. Lymphatic vessels carry a fluid rich in proteins, immune cells, and long-chain fatty acids known as lymph. It results from an excess of interstitial tissue fluid collected from the periphery and transported centrally against hydrostatic pressure and protein concentration gradients. Thus, this one-way transport system is a key component in the maintenance of normal interstitial tissue fluid volume, protein concentration and fat metabolism, as well as the mounting of adequate immune responses as lymph passes through lymph nodes. In most cases, lymph is actively propelled via rhythmical phasic contractions through a succession of valve-bordered chambers constituting the lymphatic vessels. This contraction/relaxation cycle, or lymphatic pumping, is initiated in the smooth muscle cells present in the vessel wall by a pacemaker mechanism generating voltage-gated Ca2+ channel-induced action potentials. The action potentials provide the depolarization and Ca2+ influx essential for the engagement of the contractile machinery leading to the phasic constrictions of the lymphatic chambers and forward movement of lymph. The spontaneous lymphatic constrictions can be observed in isolated vessels in the absence of any external stimulation, while they are critically regulated by physical means, such as lymph-induced transmural pressure and flow rate, as well as diffusible molecules released from the lymphatic endothelium, perivascular nerve varicosities, blood and surrounding tissues/cells. In this chapter, we describe the latest findings which are improving our understanding of the mechanisms underlying spontaneous lymphatic pumping and discuss current theories about their physiological initiation.


Assuntos
Sinalização do Cálcio , Sistema Linfático/fisiologia , Vasos Linfáticos/fisiologia , Contração Muscular , Potenciais de Ação , Canais de Cálcio/fisiologia , Líquido Extracelular , Humanos , Linfonodos
19.
Neuron ; 103(1): 66-79.e12, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31104951

RESUMO

The precision and reliability of synaptic information transfer depend on the molecular organization of voltage-gated calcium channels (VGCCs) within the presynaptic membrane. Alternative splicing of exon 47 affects the C-terminal structure of VGCCs and their affinity to intracellular partners and synaptic vesicles (SVs). We show that hippocampal synapses expressing VGCCs either with exon 47 (CaV2.1+47) or without (CaV2.1Δ47) differ in release probability and short-term plasticity. Tracking single channels revealed transient visits (∼100 ms) of presynaptic VGCCs in nanodomains (∼80 nm) that were controlled by neuronal network activity. Surprisingly, despite harboring prominent binding sites to scaffold proteins, CaV2.1+47 persistently displayed higher mobility within nanodomains. Synaptic accumulation of CaV2.1 was accomplished by optogenetic clustering, but only CaV2.1+47 increased transmitter release and enhanced synaptic short-term depression. We propose that exon 47-related alternative splicing of CaV2.1 channels controls synapse-specific release properties at the level of channel mobility-dependent coupling between VGCCs and SVs.


Assuntos
Canais de Cálcio/genética , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio/efeitos da radiação , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Células HEK293 , Humanos , Luz , Neurotransmissores/metabolismo , Optogenética , Gravidez , Isoformas de Proteínas/genética , Ratos , Vesículas Sinápticas/fisiologia
20.
Br J Anaesth ; 123(2): 219-227, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31056238

RESUMO

BACKGROUND: P/Q- and N-type voltage-gated calcium channels (VGCC) are the principal subtypes mediating synaptic vesicle (SV) exocytosis. Both the degree of isoflurane inhibition of SV exocytosis and VGCC subtype expression vary between brain regions and neurotransmitter phenotype. We hypothesised that differences in VGCC subtype expression contribute to synapse-selective presynaptic effects of isoflurane. METHODS: We used quantitative live-cell imaging to measure exocytosis in cultured rat hippocampal neurones after transfection of the fluorescent biosensor vGlut1-pHluorin. Selective inhibitors of P/Q- and N-type VGCCs were used to isolate subtype-specific effects of isoflurane. RESULTS: Inhibition of N-type channels by 1 µM ω-conotoxin GVIA reduced SV exocytosis to 81±5% of control (n=10). Residual exocytosis mediated by P/Q-type channels was further inhibited by isoflurane to 42±4% of control (n=10). The P/Q-type channel inhibitor ω-agatoxin IVA at 0.4 µM inhibited SV exocytosis to 29±3% of control (n=10). Residual exocytosis mediated by N-type channels was further inhibited by isoflurane to 17±3% of control (n=10). Analysis of isoflurane effects at the level of individual boutons revealed no difference in sensitivity to isoflurane between P/Q- or N-type channel-mediated SV exocytosis (P=0.35). There was no correlation between the effect of agatoxin (P=0.91) or conotoxin (P=0.15) and the effect of isoflurane on exocytosis. CONCLUSIONS: Sensitivity of SV exocytosis to isoflurane in rat hippocampal neurones is independent of the specific VGCC subtype coupled to exocytosis. The differential sensitivity of VGCC subtypes to isoflurane does not explain the observed neurotransmitter-selective effects of isoflurane in hippocampus.


Assuntos
Anestésicos Inalatórios/farmacologia , Canais de Cálcio/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Isoflurano/farmacologia , Vesículas Sinápticas/efeitos dos fármacos , Animais , Células Cultivadas , Técnicas In Vitro , Modelos Animais , Neurônios/efeitos dos fármacos , Ratos
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