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1.
Gut ; 68(9): 1676-1687, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31315892

RESUMO

BACKGROUND & OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC. DESIGN: Expression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice. RESULTS: ITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis. CONCLUSIONS: These results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.


Assuntos
Carcinoma Hepatocelular/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias Hepáticas/metabolismo , Adulto , Animais , Apoptose/fisiologia , Sinalização do Cálcio/fisiologia , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/fisiologia , Células Cultivadas , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Hepatócitos/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/deficiência , Receptores de Inositol 1,4,5-Trifosfato/genética , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Regeneração Hepática/fisiologia , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Análise de Sobrevida
2.
Adv Exp Med Biol ; 1124: 103-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183824

RESUMO

The ureter acts as a functional syncytium and is controlled by a propagating plateau-type action potential (AP) which gives rise to a wave of contraction (ureteral peristalsis) via a process called excitation-contraction (E-C)coupling. The second messenger Ca2+ activates Ca2+/calmodulin-dependent myosin light chain kinase-dependent phosphorylation of 20-kDa regulatory light chains of myosin which leads to ureteric contraction. Ca2+ entry from the extracellular space via voltage-gated L-type Ca2+ channels (VGCCs) provides the major source of activator Ca2+, responsible for generation of both the AP and a Ca2+ transient that appears as an intercellular Ca2+ wave. The AP, inward Ca2+ current, Ca2+ transient and twitch contraction are all fully blocked by the selective L-type Ca2+ channel blocker nifedipine. Ca2+ entry via VGCCs, coupled to activation of Ca2+-sensitive K+ (KCa) or Cl- (ClCa) channels, acts as a negative or positive feedback mechanism, respectively, to control excitability and the amplitude and duration of the plateau component of the AP, Ca2+ transient and twitch contraction. The ureter, isolated from the pelvis, is not spontaneously active. However, spontaneous activity can be initiated in the proximal and distal ureter by a variety of biological effectors such as neurotransmitters, paracrine, endocrine and inflammatory factors. Applied agonists depolarise ureteric smooth muscles cells to threshold of AP activation, initiating propagating intercellular AP-mediated Ca2+ waves to produce antegrade and/or retrograde ureteric peristalsis. Several mechanisms have been proposed to describe agonist-induced depolarization of ureteric smooth muscle, which include suppression of K+ channels, stimulation of ClCa current and activation of non-selective cation receptor/store operated channels.


Assuntos
Sinalização do Cálcio , Canais Iônicos/fisiologia , Contração Muscular , Músculo Liso/fisiologia , Peristaltismo , Ureter/fisiologia , Cálcio/fisiologia , Humanos
3.
Adv Exp Med Biol ; 1124: 171-194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183827

RESUMO

The corpus cavernosum smooth muscle is important for both erection of the penis and for maintaining penile flaccidity. Most of the time, the smooth muscle cells are in a contracted state, which limits filling of the corpus sinuses with blood. Occasionally, however, they relax in a co-ordinated manner, allowing filling to occur. This results in an erection. When contractions of the corpus cavernosum are measured, it can be deduced that the muscle cells work together in a syncytium, for not only do they spontaneously contract in a co-ordinated manner, but they also synchronously relax. It is challenging to understand how they achieve this.In this review we will attempt to explain the activity of the corpus cavernosum, firstly by summarising current knowledge regarding the role of ion channels and how they influence tone, and secondly by presenting data on the intracellular Ca2+ signals that interact with the ion channels. We propose that spontaneous Ca2+ waves act as a primary event, driving transient depolarisation by activating Ca2+-activated Cl- channels. Depolarisation then facilitates Ca2+ influx via L-type voltage-dependent Ca2+ channels. We propose that the spontaneous Ca2+ oscillations depend on Ca2+ release from both ryanodine- and inositol trisphosphate (IP3)-sensitive stores and that modulation by signalling molecules is achieved mainly by interactions with the IP3-sensitive mechanism. This pacemaker mechanism is inhibited by nitric oxide (acting through cyclic GMP) and enhanced by noradrenaline. By understanding these mechanisms better, it might be possible to design new treatments for erectile dysfunction.


Assuntos
Sinalização do Cálcio , Canais Iônicos/fisiologia , Músculo Liso/fisiologia , Pênis/fisiologia , Cálcio/fisiologia , Humanos , Masculino , Ereção Peniana
4.
Adv Exp Med Biol ; 1124: 195-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183828

RESUMO

Spontaneous myogenic contractions have been shown to be significantly upregulated in prostate tissue collected from men with Benign Prostatic Hyperplasia (BPH), an extremely common disorder of the ageing male. Although originally thought likely to be involved in 'housekeeping' functions, mixing prostatic secretions to prevent stagnation, these spontaneous myogenic contractions provide a novel opportunity to understand and treat BPH. This treatment potential differs from previous models, which focused exclusively on attenuating nerve-mediated neurogenic contractions. Previous studies in the rodent prostate have provided an insight into the mechanisms underlying the regulation of myogenic contractions. 'Prostatic Interstitial Cells' (PICs) within the prostate appear to generate pacemaker potentials, which arise from the summation of number of spontaneous transient depolarisations triggered by the spontaneous release of Ca2+ from internal stores and the opening of Ca2+-activated Cl- channels. Pacemaker potentials then conduct into neighbouring smooth muscle cells to generate spontaneous slow waves. These slow waves trigger the firing of 'spike-like' action potentials, Ca2+ entry and contraction, which are not attenuated by blockers of neurotransmission. However, these spontaneous prostatic contractions can be modulated by the autonomic nervous system. Here, we discuss the mechanisms underlying rodent and human prostate myogenic contractions and the actions of existing and novel pharmacotherapies for the treatment of BPH. Understanding the generation of human prostatic smooth muscle tone will confirm the mechanism of action of existing drugs, inform the identification and effectiveness of new pharmacotherapies, as well as predict patient outcomes.


Assuntos
Sinalização do Cálcio , Células Intersticiais de Cajal/fisiologia , Canais Iônicos/fisiologia , Contração Muscular , Músculo Liso/fisiologia , Animais , Cálcio/fisiologia , Humanos , Masculino , Hiperplasia Prostática
5.
Adv Exp Med Biol ; 1124: 217-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183829

RESUMO

Seminal vesicles (SVs), a pair of male accessory glands, contract upon sympathetic nerve excitation during ejaculation while developing spontaneous phasic constrictions in the inter-ejaculatory storage phase. Recently, the fundamental role of the mucosa in generating spontaneous activity in SV of the guinea pig has been revealed. Stretching the mucosa-intact but not mucosa-denuded SV smooth muscle evokes spontaneous phasic contractions arising from action potential firing triggered by electrical slow waves and associated Ca2+ flashes. These spontaneous events primarily depend on sarco-endoplasmic reticulum (SR/ER) Ca2+ handling linked with the opening of Ca2+-activated chloride channels (CaCCs) resulting in the generation of slow waves. Slow waves in mucosa-intact SV smooth muscle are abolished upon blockade of gap junctions, suggesting that seminal smooth muscle cells are driven by cells distributed in the mucosa. In the SV mucosal preparations dissected free from the smooth muscle layer, a population of cells located just beneath the epithelium develop spontaneous Ca2+ transients relying on SR/ER Ca2+ handling. In the lamina propria of the SV mucosa, vimentin-immunoreactive interstitial cells including platelet-derived growth factor receptor α (PDGFRα)-immunoreactive cells are distributed, while known pacemaker cells in other smooth muscle tissues, e.g. c-Kit-positive interstitial cells or α-smooth muscle actin-positive atypical smooth muscle cells, are absent. The spontaneously-active subepithelial cells appear to drive spontaneous activity in SV smooth muscle either by sending depolarizing signals or by releasing humoral substances. Interstitial cells in the lamina propria may act as intermediaries of signal transmission from the subepithelial cells to the smooth muscle cells.


Assuntos
Células Intersticiais de Cajal/fisiologia , Membrana Mucosa/fisiologia , Contração Muscular , Músculo Liso/fisiologia , Glândulas Seminais/fisiologia , Animais , Sinalização do Cálcio , Cobaias , Canais Iônicos/fisiologia , Masculino , Miócitos de Músculo Liso/fisiologia
6.
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
7.
Adv Exp Med Biol ; 1124: 297-312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183832

RESUMO

Rhythmical contractility of blood vessels was first observed in bat wing veins by Jones (Philos Trans R Soc Lond 1852:142, 131-136), and subsequently described in arteries and arterioles of multiple vascular beds in several species. Despite an abundance of descriptive literature regarding the presence of vasomotion, to date we do not have an accurate picture of the cellular and ionic basis of these oscillations in tone, or the physiological relevance of the changes in pulsatile blood flow arising from vasomotion. This chapter reviews our current understanding of the cellular and ionic mechanisms underlying vasomotion in resistance arteries and arterioles. Focus is directed to the ion channels, changes in cytosolic Ca2+ concentration, and involvement of intercellular gap junctions in the development and synchronization of rhythmic changes in membrane potential and cytosolic Ca2+ concentration within the vessel wall that contribute to vasomotion. The physiological consequences of vasomotion are discussed with a focus on the cerebral vasculature, as recent advances show that rhythmic oscillations in cerebral arteriolar diameter appear to be entrained by cortical neural activity to increase the local supply of blood flow to active regions of the brain.


Assuntos
Artérias/fisiologia , Sinalização do Cálcio , Canais Iônicos/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Arteríolas , Junções Comunicantes , Potenciais da Membrana , Fluxo Pulsátil
8.
Adv Exp Med Biol ; 1124: 313-328, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183833

RESUMO

Veins exhibit spontaneous contractile activity, a phenomenon generally termed vasomotion. This is mediated by spontaneous rhythmical contractions of mural cells (i.e. smooth muscle cells (SMCs) or pericytes) in the wall of the vessel. Vasomotion occurs through interconnected oscillators within and between mural cells, entraining their cycles. Pharmacological studies indicate that a key oscillator underlying vasomotion is the rhythmical calcium ion (Ca2+) release-refill cycle of Ca2+ stores. This occurs through opening of inositol 1,4,5-trisphosphate receptor (IP3R)- and/or ryanodine receptor (RyR)-operated Ca2+ release channels in the sarcoplasmic/endoplasmic (SR/ER) reticulum and refilling by the SR/ER reticulum Ca2+ATPase (SERCA). Released Ca2+ from stores near the plasma membrane diffuse through the cytosol to open Ca2+-activated chloride (Cl-) channels, this generating inward current through an efflux of Cl-. The resultant depolarisation leads to the opening of voltage-dependent Ca2+ channels and possibly increased production of IP3, which through Ca2+-induced Ca2+ release (CICR) of IP3Rs and/or RyRs and IP3R-mediated Ca2+ release provide a means by which store oscillators entrain their activity. Intercellular entrainment normally involves current flow through gap junctions that interconnect mural cells and in many cases this is aided by additional connectivity through the endothelium. Once entrainment has occurred the substantial Ca2+ entry that results from the near-synchronous depolarisations leads to rhythmical contractions of the mural cells, this often leading to vessel constriction. The basis for venous/venular vasomotion has yet to be fully delineated but could improve both venous drainage and capillary/venular absorption of blood plasma-associated fluids.


Assuntos
Sinalização do Cálcio , Contração Muscular , Miócitos de Músculo Liso/fisiologia , Veias/fisiologia , Cálcio/fisiologia , Membrana Celular , Retículo Endoplasmático/fisiologia , Humanos , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Retículo Sarcoplasmático/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia
9.
Adv Exp Med Biol ; 1124: 329-356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183834

RESUMO

The microvasculature is composed of arterioles, capillaries and venules. Spontaneous arteriolar constrictions reduce effective vascular resistance to enhance tissue perfusion, while spontaneous venular constrictions facilitate the drainage of tissue metabolites by pumping blood. In the venules of visceral organs, mural cells, i.e. smooth muscle cells (SMCs) or pericytes, periodically generate spontaneous phasic constrictions, Ca2+ transients and transient depolarisations. These events arise from spontaneous Ca2+ release from the sarco-endoplasmic reticulum (SR/ER) and the subsequent opening of Ca2+-activated chloride channels (CaCCs). CaCC-dependent depolarisation further activates L-type voltage-dependent Ca2+ channels (LVDCCs) that play a critical role in maintaining the synchrony amongst mural cells. Mural cells in arterioles or capillaries are also capable of developing spontaneous activity. Non-contractile capillary pericytes generate spontaneous Ca2+ transients primarily relying on SR/ER Ca2+ release. Synchrony amongst capillary pericytes depends on gap junction-mediated spread of depolarisations resulting from the opening of either CaCCs or T-type VDCCs (TVDCCs) in a microvascular bed-dependent manner. The propagation of capillary Ca2+ transients into arterioles requires the opening of either L- or TVDCCs again depending on the microvascular bed. Since the blockade of gap junctions or CaCCs prevents spontaneous Ca2+ transients in arterioles and venules but not capillaries, capillary pericytes appear to play a primary role in generating spontaneous activity of the microvasculature unit. Pericytes in capillaries where the interchange of substances between tissues and the circulation takes place may provide the fundamental drive for upstream arterioles and downstream venules so that the microvasculature network functions as an integrated unit.


Assuntos
Sinalização do Cálcio , Canais Iônicos/fisiologia , Microvasos/fisiologia , Pericitos/fisiologia , Arteríolas/fisiologia , Cálcio/fisiologia , Humanos , Microvasos/citologia , Vênulas/fisiologia
10.
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
11.
Adv Exp Med Biol ; 1124: 381-422, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183836

RESUMO

Airway smooth muscle (ASM) extends from the trachea throughout the bronchial tree to the terminal bronchioles. In utero, spontaneous phasic contraction of fetal ASM is critical for normal lung development by regulating intraluminal fluid movement, ASM differentiation, and release of key growth factors. In contrast, phasic contraction appears to be absent in the adult lung, and regulation of tonic contraction and airflow is under neuronal and humoral control. Accumulating evidence suggests that changes in ASM responsiveness contribute to the pathophysiology of lung diseases with lifelong health impacts.Functional assessments of fetal and adult ASM and airways have defined pharmacological responses and signaling pathways that drive airway contraction and relaxation. Studies using precision-cut lung slices, in which contraction of intrapulmonary airways and ASM calcium signaling can be assessed simultaneously in situ, have been particularly informative. These combined approaches have defined the relative importance of calcium entry into ASM and calcium release from intracellular stores as drivers of spontaneous phasic contraction in utero and excitation-contraction coupling.Increased contractility of ASM in asthma contributes to airway hyperresponsiveness. Studies using animal models and human ASM and airways have characterized inflammatory and other mechanisms underlying increased reactivity to contractile agonists and reduced bronchodilator efficacy of ß2-adrenoceptor agonists in severe diseases. Novel bronchodilators and the application of bronchial thermoplasty to ablate increased ASM within asthmatic airways have the potential to overcome limitations of current therapies. These approaches may directly limit excessive airway contraction to improve outcomes for difficult-to-control asthma and other chronic lung diseases.


Assuntos
Sinalização do Cálcio , Contração Muscular , Músculo Liso/fisiologia , Músculo Liso/fisiopatologia , Fenômenos Fisiológicos Respiratórios , Animais , Asma/fisiopatologia , Broncodilatadores , Humanos , Pulmão , Sistema Respiratório/fisiopatologia
12.
Nat Commun ; 10(1): 2516, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175287

RESUMO

Store-operated Ca2+ entry, involving endoplasmic reticulum Ca2+ sensing STIM proteins and plasma membrane Orai1 channels, is a widespread and evolutionary conserved Ca2+ influx pathway. This form of Ca2+ influx occurs at discrete loci where peripheral endoplasmic reticulum juxtaposes the plasma membrane. Stimulation evokes numerous STIM1-Orai1 clusters but whether distinct signal transduction pathways require different cluster numbers is unknown. Here, we show that two Ca2+-dependent transcription factors, NFAT1 and c-fos, have different requirements for the number of STIM1-Orai1 clusters and on the Ca2+ flux through them. NFAT activation requires fewer clusters and is more robustly activated than c-fos by low concentrations of agonist. For similar cluster numbers, transcription factor recruitment occurs sequentially, arising from intrinsic differences in Ca2+ sensitivities. Variations in the number of STIM1-Orai1 clusters and Ca2+ flux through them regulate the robustness of signalling to the nucleus whilst imparting a mechanism for selective recruitment of different Ca2+-dependent transcription factors.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Ratos , Transdução de Sinais
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.
Chin J Physiol ; 62(3): 123-130, 2019 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31249266

RESUMO

Chlorzoxazone is a skeletal muscle relaxant. However, the effect of chlorzoxazone on intracellular Ca2+ concentrations ([Ca2+]i) in oral cancer cells is unclear. This study examined whether chlorzoxazone altered Ca2+ signaling and cell viability in OC2 human oral cancer cells. [Ca2+]iin suspended cells was measured using the fluorescent Ca2+-sensitive dye fura-2. Cell viability was examined by water-soluble tetrazolium-1 assay. Chlorzoxazone (250-1000 µM) induced [Ca2+]irises in a concentration-dependent manner. Ca2+ removal reduced the signal by approximately 50%. Mn2+ has been shown to enter cells through similar mechanisms as Ca2+ but quenches fura-2 fluorescence at all excitation wavelengths. Chlorzoxazone (1000 µM) induced Mn2+ influx, suggesting that Ca2+ entry occurred. Chlorzoxazone-induced Ca2+ entry was inhibited by 20% by inhibitors of store-operated Ca2+ channels and protein kinase C (PKC) modulators. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) inhibited chlorzoxazone-evoked [Ca2+]irises by 88%. Conversely, treatment with chlorzoxazone-suppressed TG-evoked [Ca2+]irises 75%. Chlorzoxazone induced [Ca2+]irises by exclusively releasing Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C (PLC) with U73122 did not alter chlorzoxazone-induced [Ca2+]irises. PLC activity was not involved in chlorzoxazone-evoked [Ca2+]irises. Chlorzoxazone at 200-700 µM decreased cell viability, which was not reversed by pretreatment with Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl. In sum, in OC2 cells, chlorzoxazone induced [Ca2+]irises by evoking PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ entry. Chlorzoxazone also caused Ca2+-independent cell death. Since [Ca2+]irises play a triggering or modulatory role in numerous cellular phenomena, the effect of chlorzoxazone on [Ca2+]iand cell viability should be taken into account in other in vitro studies.


Assuntos
Sinalização do Cálcio , Neoplasias Bucais , Apoptose , Cálcio , Linhagem Celular Tumoral , Sobrevivência Celular , Clorzoxazona , Humanos , Fosfolipases Tipo C
15.
Toxicol Lett ; 313: 42-49, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31154016

RESUMO

Astrocytes are the major glial cell type in the central nervous system (CNS), and the distal part of the astrocyte forms the blood-brain barrier with nearby blood vessels. They maintain the overall metabolism, growth, homeostasis of neurons, and signaling in the CNS. Ochratoxin A is considered a carcinogen and immunotoxic, nephrotoxic, and neurotoxic mycotoxin. Specifically, it exhibits neurotoxicity with high affinity for the brain. Despite some previous studies about the effects of ochratoxin A in glial cells, the intracellular working mechanism in astrocytes is not fully understood. In this study, we studied the specific working mechanism of ochratoxin A in the human astrocyte cell line, NHA-SV40LT. Ochratoxin A reduced cell proliferation with sub G0/G1 cell cycle arrest by inhibiting CCND1, CCNE1, CDK4, and MYC expression. It induced apoptosis of NHA-SV40LT cells through mitochondrial membrane potential (MMP) loss and up-regulation of BAX and TP53. In addition, ochratoxin A increased cytosolic and mitochondrial calcium levels, resulting in an increase in MMP2 and PLAUR mRNA expression in NHA-SV40LT cells. Furthermore, ochratoxin A regulated the phosphorylation of AKT, ERK1/2, and JNK signal molecules of human astrocytes. Collectively, ochratoxin A exerts neurotoxicity through anti-proliferation and mitochondria-dependent apoptosis in human astrocytes.


Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Cálcio/metabolismo , Mitocôndrias/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Ocratoxinas/toxicidade , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Sinalização do Cálcio/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo
16.
Nat Neurosci ; 22(7): 1110-1121, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31160741

RESUMO

Learning to predict rewards based on environmental cues is essential for survival. The orbitofrontal cortex (OFC) contributes to such learning by conveying reward-related information to brain areas such as the ventral tegmental area (VTA). Despite this, how cue-reward memory representations form in individual OFC neurons and are modified based on new information is unknown. To address this, using in vivo two-photon calcium imaging in mice, we tracked the response evolution of thousands of OFC output neurons, including those projecting to VTA, through multiple days and stages of cue-reward learning. Collectively, we show that OFC contains several functional clusters of neurons distinctly encoding cue-reward memory representations, with only select responses routed downstream to VTA. Unexpectedly, these representations were stably maintained by the same neurons even after extinction of the cue-reward pairing, and supported behavioral learning and memory. Thus, OFC neuronal activity represents a long-term cue-reward associative memory to support behavioral adaptation.


Assuntos
Adaptação Psicológica/fisiologia , Aprendizagem por Associação/fisiologia , Sinalização do Cálcio , Condicionamento Clássico/fisiologia , Memória de Longo Prazo/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Recompensa , Estimulação Acústica , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Sinais (Psicologia) , Comportamento de Ingestão de Líquido/fisiologia , Extinção Psicológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/fisiologia , Neurônios/enzimologia , Optogenética , Técnicas de Patch-Clamp , Córtex Pré-Frontal/citologia , Análise de Célula Única , Área Tegmentar Ventral/fisiologia
17.
Pestic Biochem Physiol ; 157: 152-160, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153463

RESUMO

Malathion, one of commonly used organophosphate insecticides, has a wide range of toxic actions in different models. However, the effect of this compound on Ca2+ homeostasis and its related cytotoxicity in glial cells is elusive. This study examined whether malathion evoked intracellular Ca2+ concentration ([Ca2+]i) rises and established the relationship between Ca2+ signaling and cytotoxicity in normal human astrocytes, rat astrocytes and human glioblastoma cells. The data show that malathion induced concentration-dependent [Ca2+]i rises in Gibco® Human Astrocytes (GHA cells), but not in DI TNC1 normal rat astrocytes and DBTRG-05MG human glioblastoma cells. In GHA cells, this Ca2+ signal response was reduced by removing extracellular Ca2+. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished malathion-induced [Ca2+]i rises. Conversely, incubation with malathion abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 also blocked malathion-induced [Ca2+]i rises. In Ca2+-containing medium, malathion-induced [Ca2+]i rises was inhibited by store-operated Ca2+ channel blockers (2-APB, econazole or SKF96365) and the protein kinase C (PKC) inhibitor GF109203X. Malathion (5-25 µM) concentration-dependently caused cytotoxicity in GHA, DI TNC1 and DBTRG-05MG cells. This cytotoxic effect was partially prevented by prechelating cytosolic Ca2+ with BAPTA-AM (a selective Ca2+ chelator) only in GHA cells. Together, in GHA but not in DI TNC1 and DBTRG-05MG cells, malathion induced [Ca2+]i rises by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ channels. Furthermore, malathion induced Ca2+-associated cytotoxicity, suggesting that Ca2+ chelating may have a protective effect on malathion-induced cytotoxicity in normal human astrocytes.


Assuntos
Cálcio/metabolismo , Malation/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quelantes , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Ratos
18.
Life Sci ; 231: 116555, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31194991

RESUMO

AIMS: Caffeine is a methylxanthine with multiple actions in vascular smooth muscle cells (VSMCs), including the increase in the intracellular Ca2+ (iCa2+) concentration by the activation of ryanodine receptors (RyRs). The present study aimed at investigating the participation of Ca2+-influx through different Ca2+-channels on the transient contraction (TC) induced by caffeine in mice mesenteric arteries. MAIN METHODS: Second-order of mesenteric arteries was isolated from male Swiss mice. Vessels without functional endothelium were stimulated with caffeine (10 mM). The caffeine-induced TC was evaluated after the incubation of artery rings for 30 min with the following drugs: nifedipine (10 µM), a Cav1.2 blocker; 2-aminoethoxydiphenyl borate (2-APB; 10 µM) and ruthenium red (RuR; 10 µM), transient receptor potential (TRPs) channels blockers; capsazepine (10 µM) and HC067047 (10 µM), TRPV1 and TRPV4 antagonists, respectively; paxilline (1 µM), a selective BKCa blocker; and SKF-96365 (30 µM), an Orai blocker. Ca2+-fluorescence measurements were also performed on the investigated arteries. KEY FINDINGS: The TC induced by caffeine was partially dependent on Ca2+-influx. However, the blockage of Cav1.2 increased the TC while reduced the iCa2+ signal. Similar results were observed after the blockage of TRPs or BKCa. Therefore, caffeine promoted Ca2+-influx via TRPs and Cav1.2, and hyperpolarization through the activation of BKCa, inducing negative feedback of TC. SIGNIFICANCE: Our results indicate an alternative mechanism for the control of VSMCs contraction in resistance arteries. The evidence of the negative feedback of contraction via TRP-Cav1.2-BKCa provides a new perspective for understanding the mechanism involved in the vascular responses triggered by caffeine.


Assuntos
Cafeína/farmacologia , Canais de Cálcio Tipo L/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Masculino , Artérias Mesentéricas/metabolismo , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Canais de Cátion TRPV/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
19.
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
20.
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
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