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1.
Nat Rev Neurosci ; 21(4): 213-229, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32161339

RESUMO

Chemical synapses are heterogeneous junctions formed between neurons that are specialized for the conversion of electrical impulses into the exocytotic release of neurotransmitters. Voltage-gated Ca2+ channels play a pivotal role in this process as they are the major conduits for the Ca2+ ions that trigger the fusion of neurotransmitter-containing vesicles with the presynaptic membrane. Alterations in the intrinsic function of these channels and their positioning within the active zone can profoundly alter the timing and strength of synaptic output. Advances in optical and electron microscopic imaging, structural biology and molecular techniques have facilitated recent breakthroughs in our understanding of the properties of voltage-gated Ca2+ channels that support their presynaptic functions. Here we examine the nature of these channels, how they are trafficked to and anchored within presynaptic boutons, and the mechanisms that allow them to function optimally in shaping the flow of information through neural circuits.


Assuntos
Canais de Cálcio/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Humanos , Transporte Proteico
2.
Curr Opin Cell Biol ; 63: 154-161, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32097833

RESUMO

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.


Assuntos
Motilidade Espermática/fisiologia , Cauda do Espermatozoide/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Humanos , Masculino , Transdução de Sinais/fisiologia , Motilidade Espermática/genética , Cauda do Espermatozoide/metabolismo , Espermatozoides/metabolismo , Espermatozoides/fisiologia
3.
J Neurosci ; 40(12): 2403-2415, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32086258

RESUMO

α2δ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) predominantly, if not exclusively, express one isoform of this family, α2δ-2 (Cacna2d2), we used PCs as a model system to examine roles of α2δ in excitatory synaptic function in male and female Cacna2d2 knock-out (KO) mice. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked EPSCs. CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2+ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wild-type as a result of increased glutamate reuptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of α2δ proteins.SIGNIFICANCE STATEMENT α2δ proteins (Cacna2d1-4) regulate synaptic transmission and synaptogenesis, but coexpression of multiple α2δ isoforms has obscured a clear understanding of how various α2δ proteins control synaptic function. We focused on roles of the α2δ-2 protein (Cacna2d2), the deletion of which causes cerebellar ataxia and epilepsy in mice and humans. Because cerebellar Purkinje cells (PCs) only express this single isoform, we studied excitatory climbing fiber synaptic function onto PCs in Cacna2d2 KO mice. Using optical and electrophysiological analysis, we provide a detailed description of the changes in PCs lacking α2δ-2, and provide a comprehensive mechanistic explanation for how functional synaptic phenotypes contribute to the altered cerebellar output.


Assuntos
Canais de Cálcio/fisiologia , Cerebelo/fisiologia , Fibras Nervosas/fisiologia , Células de Purkinje/fisiologia , Sinapses/fisiologia , Animais , Canais de Cálcio Tipo L , Cerebelo/citologia , Simulação por Computador , Potenciais Pós-Sinápticos Excitadores/fisiologia , Ácido Glutâmico/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Neurológicos , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura
4.
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
5.
Cells ; 9(1)2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878335

RESUMO

The skeletal muscle and myocardial cells present highly specialized structures; for example, the close interaction between the sarcoplasmic reticulum (SR) and mitochondria-responsible for excitation-metabolism coupling-and the junction that connects the SR with T-tubules, critical for excitation-contraction (EC) coupling. The mechanisms that underlie EC coupling in these two cell types, however, are fundamentally distinct. They involve the differential expression of Ca2+ channel subtypes: CaV1.1 and RyR1 (skeletal), vs. CaV1.2 and RyR2 (cardiac). The CaV channels transform action potentials into elevations of cytosolic Ca2+, by activating RyRs and thus promoting SR Ca2+ release. The high levels of Ca2+, in turn, stimulate not only the contractile machinery but also the generation of mitochondrial reactive oxygen species (ROS). This forward signaling is reciprocally regulated by the following feedback mechanisms: Ca2+-dependent inactivation (of Ca2+ channels), the recruitment of Na+/Ca2+ exchanger activity, and oxidative changes in ion channels and transporters. Here, we summarize both well-established concepts and recent advances that have contributed to a better understanding of the molecular mechanisms involved in this bidirectional signaling.


Assuntos
Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/fisiologia , Citosol/metabolismo , Acoplamento Excitação-Contração/fisiologia , Humanos , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcolema/fisiologia , Retículo Sarcoplasmático/fisiologia , Transdução de Sinais
6.
Life Sci ; 239: 116909, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31689439

RESUMO

Cytosolic calcium [Ca2+]cyt signaling plays a critical role in the regulation of multiple cellular functions, and Ca2+ channels/transporters are important to regulate calcium homeostasis whose abnormality may contribute human tumorogenesis including colorectal cancer (CRC). In this review, we summarized and discussed the current knowledge on pathogenic roles of the altered [Ca2+]cyt and Ca2+ channels/transporters like SOCE, TRP channels, SERCA and Na+/Ca2+ exchangers in CRC tumorigenesis and progression. Understanding the detailed molecular mechanisms underlying the effects of [Ca2+]cyt on CRC is essential to develop Ca2+ channels/transporters as diagnostic and therapeutic targets. Targeting Ca2+ signaling for cancer therapy has become an emerging research area nowadays, although our knowledge about the roles of Ca2+ channels/transporters in tumorigenesis is still in the early stage, we still believe that they will act as novel preventive/therapeutic targets for CRC with potentially extensive clinical significance.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Neoplasias do Colo/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Carcinogênese/metabolismo , Colo/metabolismo , Neoplasias do Colo/patologia , Citosol/metabolismo , Homeostase , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Transdução de Sinais
7.
Biol Pharm Bull ; 42(10): 1741-1745, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582662

RESUMO

Our previous studies have shown that phenylephrine-induced contraction of cutaneous arteries is primarily mediated via α1A-adrenoceptors, but not α1D-adrenoceptors that generally mediate vascular contraction, and that the larger part of the contraction is induced in a voltage-dependent Ca2+ channel (VDCC)-independent manner. Here, we investigated the mechanism underlying the smaller part of the α1A-adrenoceptor-mediated contraction, i.e., VDCC-dependent one, in cutaneous arteries. Isometric contraction was measured with wire myograph in endothelium-denuded tail and iliac arterial rings isolated from male Wistar rats. LOE908 (10 µM), a cation channel blocker, partially inhibited the contraction induced by phenylephrine in tail and iliac arteries. Nifedipine (10 µM) further suppressed the phenylephrine-induced contraction that remained in the presence of LOE908 (10 µM) in iliac arteries but barely in tail arteries, suggesting that phenylephrine-induced depolarization in tail arteries is due to the activation of LOE908-sensitive cation channels. In iliac arteries, the contraction induced by A-61603, a specific α1A-adrenoceptor agonist, was also partially inhibited by LOE908 (10 µM); however, nifedipine had little effect on the A-61603-induced contraction that remained in the presence of LOE908 (10 µM), suggesting that depolarization mediated via α1A-adrenoceptors is due to the activation of LOE908-sensitive cation channels even in iliac arteries. These results suggest that membrane depolarization mediated via α1Α-adrenoceptors is caused by cation influx through LOE908-sensitive cation channels. Less contribution of VDCC to phenylephrine-induced contraction in tail arteries compared to in iliac arteries is likely due to that α1Α-adrenoceptor-mediated activation of VDCC is caused only by depolarization via cation influx through LOE908-sensitive cation channels.


Assuntos
Canais de Cálcio/fisiologia , Artéria Ilíaca/fisiologia , Receptores Adrenérgicos alfa 1/fisiologia , Cauda/irrigação sanguínea , Acetamidas/farmacologia , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Imidazóis/farmacologia , Isoquinolinas/farmacologia , Masculino , Nifedipino/farmacologia , Fenilefrina/farmacologia , Ratos Wistar , Cauda/fisiologia , Tetra-Hidronaftalenos/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia
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.
PLoS One ; 14(8): e0220473, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31369604

RESUMO

Simvastatin reduces pulmonary arterial pressure and right ventricular hypertrophy in animal models of pulmonary arterial hypertension (PAH) and is thought to restore endothelial dysfunction. In vivo effects of drugs are complicated by several factors and little is known of the direct effects of statins on pulmonary arteries. This study investigated the direct effects of simvastatin on pulmonary arteries isolated from rats with or without monocrotaline-induced PAH. Simvastatin suppressed contractions evoked by the thromboxane A2 receptor agonist U46619 (30 nM), the α1-adrenergic agonist phenylephrine (5 µM) and KCl (50 mM) by ~50% in healthy and diseased arteries, but did not reduce contraction evoked by sarco/endoplasmic reticulum ATPase blockers. It relaxed hypertensive arteries in the absence of stimulation. Removing the endothelium or inhibiting eNOS did not prevent the inhibition by simvastatin. Inhibiting RhoA/rho kinase (ROCK) with Y27632 (10 µM) suppressed contractions to U46619 and phenylephrine by ~80% and prevented their inhibition by simvastatin. Y27632 reduced KCl-induced contraction by ~30%, but did not prevent simvastatin inhibition. Simvastatin suppressed Ca2+ entry into smooth muscle cells, as detected by Mn2+ quench of fura-2 fluorescence. The calcium antagonist, nifedipine (1 µM), almost abolished K+-induced contraction with less effect against U46619 and phenylephrine. We conclude that simvastatin relaxes pulmonary arteries by acting on smooth muscle to interfere with signalling through G-protein coupled receptors and voltage-dependent Ca2+ entry. Its actions likely include inhibition of ROCK-dependent Ca2+ sensitisation and voltage-gated Ca2+ channels. These are likely to contribute to the beneficial effects of simvastatin in animal models of PAH.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Sinvastatina/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Canais de Cálcio/fisiologia , Modelos Animais de Doenças , Endotélio Vascular/fisiologia , Masculino , Relaxamento Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Fenilefrina/farmacologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/fisiologia , Ratos , Ratos Wistar
10.
Semin Nephrol ; 39(4): 353-367, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31300091

RESUMO

The epithelium of the kidney collecting duct (CD) is composed mainly of two different types of cells with distinct and complementary functions. CD principal cells traditionally have been considered to have a major role in Na+ and water regulation, while intercalated cells (ICs) were thought to largely modulate acid-base homeostasis. In recent years, our understanding of IC function has improved significantly owing to new research findings. Thus, we now have a new model for CD transport that integrates mechanisms of salt and water reabsorption, K+ homeostasis, and acid-base status between principal cells and ICs. There are three main types of ICs (type A, type B, and non-A, non-B), which first appear in the late distal convoluted tubule or in the connecting segment in a species-dependent manner. ICs can be detected in CD from cortex to the initial part of the inner medulla, although some transport proteins that are key components of ICs also are present in medullary CD, cells considered inner medullary. Of the three types of ICs, each has a distinct morphology and expresses different complements of membrane transport proteins that translate into very different functions in homeostasis and contributions to CD luminal pro-urine composition. This review includes recent discoveries in IC intracellular and paracrine signaling that contributes to acid-base regulation as well as Na+, Cl-, K+, and Ca2+ homeostasis. Thus, these new findings highlight the potential role of ICs as targets for potential hypertension treatments.


Assuntos
Equilíbrio Ácido-Base/fisiologia , Células Epiteliais/fisiologia , Túbulos Renais Coletores/fisiologia , Animais , Canais de Cálcio/fisiologia , Canais de Cloreto/fisiologia , Células Epiteliais/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/fisiologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia
11.
Methods Mol Biol ; 2029: 257-271, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31273748

RESUMO

Calcium imaging in an ex-vivo setup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380 nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the endoplasmic reticulum (ER)-driven store-operated calcium entry (SOCE) in human neural precursors (NPCs) and spontaneously differentiated neurons derived from a pluripotent human embryonic stem cell (hESC) line. The derivation of neural precursors from stem cells and their subsequent spontaneous neural differentiation is also explained. The method can be used for various non-excitable and excitable cell types including neurons, be it freshly isolated, from frozen vials, or derived from different stem cell lines.


Assuntos
Cálcio/metabolismo , Diferenciação Celular/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Humanos
12.
J Ethnopharmacol ; 242: 112039, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31252093

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Parsley (Petroselinum crispum; P. crispum) is among the popular aromatic vegetables and a part of the daily diet in the Mediterranean area. This plant is widely used in alternative medicine as a remedy against hypertension. THE AIM OF THE STUDY: The aim of the study was to evaluate the antihypertensive activity of the aqueous extract of this plant. MATERIAL AND METHODS: In the current study, the aqueous extract of the aerial parts of parsley (AEPC) was prepared and its antihypertensive activity was evaluated using in vivo and in vitro studies. In the in vivo investigation, anesthetized L-NAME-hypertensive and normotensive rats received orally AEPC (160 mg/kg) during 6 h for the acute experiment and during seven days for the sub-chronic treatment. Thereafter, systolic, diastolic, mean arterial blood pressure and heart rate were recorded using a tail cuff and a computer-assisted monitoring device. Concerning the in vitro investigation, isolated thoracic aortic rings were suspended in a tissue bath and the tension changes were recorded to a data acquisition system. RESULTS: The results indicated that AEPC extract decreased the systolic, diastolic, mean arterial blood pressure in normotensive and hypertensive rats. The data revealed that parsley extract exerts its hypotensive effects through vasodilatory properties via an endothelium-independent pathway. More interestingly, the study demonstrated here that the vasorelaxing ability of AEPC is exerted through both Voltage Operated and Receptor Operated Calcium Channels (VOCC and ROCC). CONCLUSION: The study illustrates the beneficial action of P. crispum as an antihypertensive agent.


Assuntos
Anti-Hipertensivos , Bloqueadores dos Canais de Cálcio , Canais de Cálcio/fisiologia , Hipertensão/tratamento farmacológico , Petroselinum , Extratos Vegetais , Vasodilatadores , Animais , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Aorta/efeitos dos fármacos , Aorta/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Masculino , NG-Nitroarginina Metil Éster , Componentes Aéreos da Planta , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ratos Wistar , Vasodilatadores/farmacologia , Vasodilatadores/uso terapêutico
13.
Cell ; 177(6): 1495-1506.e12, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31150622

RESUMO

The L-type voltage-gated Ca2+ (Cav) channels are modulated by various compounds exemplified by 1,4-dihydropyridines (DHP), benzothiazepines (BTZ), and phenylalkylamines (PAA), many of which have been used for characterizing channel properties and for treatment of hypertension and other disorders. Here, we report the cryoelectron microscopy (cryo-EM) structures of Cav1.1 in complex with archetypal antagonistic drugs, nifedipine, diltiazem, and verapamil, at resolutions of 2.9 Å, 3.0 Å, and 2.7 Å, respectively, and with a DHP agonist Bay K 8644 at 2.8 Å. Diltiazem and verapamil traverse the central cavity of the pore domain, directly blocking ion permeation. Although nifedipine and Bay K 8644 occupy the same fenestration site at the interface of repeats III and IV, the coordination details support previous functional observations that Bay K 8644 is less favored in the inactivated state. These structures elucidate the modes of action of different Cav ligands and establish a framework for structure-guided drug discovery.


Assuntos
Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/ultraestrutura , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil) , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Canais de Cálcio/ultraestrutura , Canais de Cálcio Tipo L/fisiologia , Microscopia Crioeletrônica , Diltiazem , Ligantes , Masculino , Modelos Moleculares , Nifedipino , Coelhos , Verapamil
14.
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
15.
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
16.
Am J Respir Cell Mol Biol ; 61(4): 501-511, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30943377

RESUMO

The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke (CS), the major risk factor for developing chronic obstructive pulmonary disease (COPD). CS leads to bronchial epithelial damage on ciliated, goblet, and club cells, which could involve calcium (Ca2+) signaling. Ca2+ is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating, and epithelial repair. In this study, we analyzed Ca2+ signaling in air-liquid interface-reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired Ca2+ signaling. First, we showed that the endoplasmic reticulum (ER) depletion of Ca2+ stores was decreased in patients with COPD and that the Ca2+ influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute CS exposure led to a decrease in ER Ca2+ release, significant in smoker subjects, and to a decrease in Ca2+ influx only in control subjects. Furthermore, the differential expression of 55 genes involved in Ca2+ signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered Ca2+ influx and ciliary beating, but not mucus and cytokine secretion or epithelial repair, in control subjects. Our data suggest that Ca2+ signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.


Assuntos
Brônquios/citologia , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/metabolismo , Fumar/metabolismo , Adulto , Idoso , Benzamidas/farmacologia , Brônquios/metabolismo , Canais de Cálcio/biossíntese , Canais de Cálcio/genética , Sinalização do Cálcio , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/fisiologia , Citocinas/metabolismo , Retículo Endoplasmático/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-8/biossíntese , Masculino , Pessoa de Meia-Idade , Mucina-5AC/biossíntese , Muco/metabolismo , Pirazóis/farmacologia , Mucosa Respiratória/patologia , Transdução de Sinais/fisiologia , Fumaça , Fumantes
17.
Bioelectrochemistry ; 128: 155-164, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31003054

RESUMO

In nerve cells, changes in local membrane potentials are generated and propagated along a nerve axon mainly by the function of K+ and Na+ channels. Generally, concurrent monitoring of multi-points on an axon is performed based on the voltage-clamp method. As the respective membrane potentials have been evaluated by considering the relations between the applied potential, the local current, and conductance, experimental values are not directly evaluated. We directly measured the actual membrane potentials and local currents of the respective cells using a nerve-model system comprising liquid-membrane cells. It was then proven that the action potential spreads along the axon toward the axon terminal due to the function of both the channel-type receptors in the synapse and voltage-gated Na+ channels on the axon, and that hyperpolarization cannot be caused by only the operation of the delayed-K+ and the voltage-gated Na+ channels.


Assuntos
Potenciais de Ação , Modelos Neurológicos , Neurônios/fisiologia , Animais , Canais de Cálcio/fisiologia , Transporte de Íons , Potenciais da Membrana , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Sinapses/fisiologia
18.
Anesthesiology ; 130(5): 804-819, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30839350

RESUMO

BACKGROUND: Chronic use of µ-opioid receptor agonists paradoxically causes both hyperalgesia and the loss of analgesic efficacy. Opioid treatment increases presynaptic N-methyl-D-aspartate receptor activity to potentiate nociceptive input to spinal dorsal horn neurons. However, the mechanism responsible for this opioid-induced activation of presynaptic N-methyl-D-aspartate receptors remains unclear. α2δ-1, formerly known as a calcium channel subunit, interacts with N-methyl-D-aspartate receptors and is primarily expressed at presynaptic terminals. This study tested the hypothesis that α2δ-1-bound N-methyl-D-aspartate receptors contribute to presynaptic N-methyl-D-aspartate receptor hyperactivity associated with opioid-induced hyperalgesia and analgesic tolerance. METHODS: Rats (5 mg/kg) and wild-type and α2δ-1-knockout mice (10 mg/kg) were treated intraperitoneally with morphine twice/day for 8 consecutive days, and nociceptive thresholds were examined. Presynaptic N-methyl-D-aspartate receptor activity was recorded in spinal cord slices. Coimmunoprecipitation was performed to examine protein-protein interactions. RESULTS: Chronic morphine treatment in rats increased α2δ-1 protein amounts in the dorsal root ganglion and spinal cord. Chronic morphine exposure also increased the physical interaction between α2δ-1 and N-methyl-D-aspartate receptors by 1.5 ± 0.3 fold (means ± SD, P = 0.009, n = 6) and the prevalence of α2δ-1-bound N-methyl-D-aspartate receptors at spinal cord synapses. Inhibiting α2δ-1 with gabapentin or genetic knockout of α2δ-1 abolished the increase in presynaptic N-methyl-D-aspartate receptor activity in the spinal dorsal horn induced by morphine treatment. Furthermore, uncoupling the α2δ-1-N-methyl-D-aspartate receptor interaction with an α2δ-1 C terminus-interfering peptide fully reversed morphine-induced tonic activation of N-methyl-D-aspartate receptors at the central terminal of primary afferents. Finally, intraperitoneal injection of gabapentin or intrathecal injection of an α2δ-1 C terminus-interfering peptide or α2δ-1 genetic knockout abolished the mechanical and thermal hyperalgesia induced by chronic morphine exposure and largely preserved morphine's analgesic effect during 8 days of morphine treatment. CONCLUSIONS: α2δ-1-Bound N-methyl-D-aspartate receptors contribute to opioid-induced hyperalgesia and tolerance by augmenting presynaptic N-methyl-D-aspartate receptor expression and activity at the spinal cord level.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio/fisiologia , Hiperalgesia/induzido quimicamente , Morfina/farmacologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Tolerância a Medicamentos , Feminino , Gabapentina/farmacologia , Gânglios Espinais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Medula Espinal/fisiologia
19.
Nat Cell Biol ; 21(4): 476-486, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858581

RESUMO

The capacity of cells to alter bioenergetics in response to the demands of various biological processes is essential for normal physiology. The coordination of energy sensing and production with highly energy-demanding cellular processes, such as cell division, is poorly understood. Here, we show that a cell cycle-dependent mitochondrial Ca2+ transient connects energy sensing to mitochondrial activity for mitotic progression. The mitochondrial Ca2+ uniporter (MCU) mediates a rapid mitochondrial Ca2+ transient during mitosis. Inhibition of mitochondrial Ca2+ transients via MCU depletion causes spindle checkpoint-dependent mitotic delay. Cellular ATP levels drop during early mitosis, and the mitochondrial Ca2+ transients boost mitochondrial respiration to restore energy homeostasis. This is achieved through mitosis-specific MCU phosphorylation and activation by the mitochondrial translocation of energy sensor AMP-activated protein kinase (AMPK). Our results establish a critical role for AMPK- and MCU-dependent mitochondrial Ca2+ signalling in mitosis and reveal a mechanism of mitochondrial metabolic adaptation to acute cellular energy stress.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Mitocôndrias/metabolismo , Mitose , Trifosfato de Adenosina/biossíntese , Animais , Canais de Cálcio/genética , Linhagem Celular , Células Cultivadas , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Mitocôndrias/enzimologia
20.
Science ; 363(6430): 948-955, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30819957

RESUMO

We investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII. Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by l-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.


Assuntos
Sinalização do Cálcio , Proteínas de Drosophila/fisiologia , Glutamatos/fisiologia , Discos Imaginais/fisiologia , Receptores Ionotrópicos de Glutamato/fisiologia , Sinapses/fisiologia , Animais , Animais Geneticamente Modificados , Canais de Cálcio/fisiologia , Drosophila melanogaster/fisiologia , Imagem Óptica , Pseudópodes/fisiologia , Proteínas R-SNARE/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia , Sinaptotagmina I/fisiologia , Técnicas de Cultura de Tecidos
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