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BACKGROUND: The conventional "whole-cell patch-clamp" recording technique is widely used to measure the resting membrane potential (VM) and to dissect the underlying membrane ionic conductances in isolated vascular endothelial cells. NEW METHOD: Herein, we assessed whether the automated patch-clamp (APC) technology, which replaces the traditional patch-pipette with a planar substrate to permit researchers lacking formal training in electrophysiology to generate large amounts of data in a relatively short time, can be used to characterize the bioelectrical activity of vascular endothelial cells. We assessed whether the Port-a-Patch planar patch-clamp system, which is regarded as the smallest electrophysiological rig available on the market, can be used to measure the VM and resting membrane currents in the human cerebrovascular endothelial cell line, hCMEC/D3. COMPARISON WITH EXISTING METHODS: We demonstrated that the Port-a-Patch planar patch-clamp system provides the same values of the resting VM as those provided by the conventional patch-clamp technique. Furthermore, the APC technology provides preliminary data demonstrating that the resting VM of hCMEC/D3 cells is primarily contributed by Cl- and Na+, as demonstrated with the patch-clamp technique for many other endothelial cell types. CONCLUSIONS: The Port-a-Patch planar patch-clamp system can be successfully used to measure the resting VM and the underlying membrane ionic conductances in hCMEC/D3 cells. We envisage that this easy-to-use APC system could also be extremely useful for the investigation of the membrane currents that can be activated by chemical, thermal, optical, and mechanical stimuli in this cell line as well as in other types of isolated vascular endothelial cells.
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Células Endoteliales , Potenciales de la Membrana , Técnicas de Placa-Clamp , Humanos , Técnicas de Placa-Clamp/métodos , Técnicas de Placa-Clamp/instrumentación , Células Endoteliales/fisiología , Células Endoteliales/citología , Potenciales de la Membrana/fisiología , Línea CelularRESUMEN
Lysosomal Ca2+ signaling is emerging as a crucial regulator of endothelial Ca2+ dynamics. Ca2+ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP3) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca2+ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca2+ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) induced a significant increase in [Ca2+]i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca2+ release was sustained both by lysosomal Ca2+ release and ER Ca2+- release through inositol-1,4,5-trisphophate receptors and store-operated Ca2+ entry. Notably, interfering with TRPML1-mediated lysosomal Ca2+ mobilization led to a decrease in the free ER Ca2+ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca2+-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca2+ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca2+ store can shape endothelial Ca2+ signaling and Ca2+-dependent functions in vascular endothelial cells.
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Purinergic signaling plays a crucial role in vascular endothelium functions. In particular, ionotropic P2X receptors (P2XRs) are engaged in various intracellular pathways through which endothelial cells (ECs) adapt to external stimuli. However, very little is known about the impact of P2XRs on vascular remodeling during carcinogenesis. We previously demonstrated that high purinergic stimulation impairs the migratory phenotype of tumor-derived endothelial cells (TECs) but not of normal ECs. Since P2XRs are sensitive to different physical and chemical factors, we investigated the impact of tumor microenvironment (TME) on healthy ECs to verify the ability of cancer cells to affect endothelial migratory phenotype through purinergic signaling tuning. More specifically, we focused on P2XR modulation by two different types of TME, mimicking breast and pancreas cancer milieux, which show very different features in terms of vascularization and composition. ECs conditioning with both cancer cell types induced a significant upregulation of some of the most represented P2XR. However, only conditioning with MCF-7 cells and not that with PANC-1 cells was able to alter the migratory phenotype of normal ECs supporting a P2XR-mediated inhibition of cell migration. The differences observed between the two cancer cells could be due to their different proliferative potential and the subsequent different extracellular pH. In addition, in agreement with some of our previous data, the P2XR-induced inhibition of EC migration seems to be independent of calcium signals, as conditioned ECs didn't reveal any changes in the long-lasting responses evoked by purinergic agonists. Collectively, highlighting a significant P2RX modulation by TME, our data strengthen the hypothesis that purinergic signaling may play a central role in vascular remodeling during carcinogenesis. However, the molecular routes upstream and downstream of this modulation remain to be elucidated.
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Neoplasias de la Mama , Movimiento Celular , Células Endoteliales , Receptores Purinérgicos P2X , Transducción de Señal , Microambiente Tumoral , Humanos , Movimiento Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Receptores Purinérgicos P2X/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Células MCF-7 , Femenino , FenotipoAsunto(s)
Señalización del Calcio , Células Endoteliales , Microvasos , Receptores Acoplados a Proteínas G , Sodio , Humanos , Señalización del Calcio/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Sodio/metabolismo , Microvasos/metabolismo , Microvasos/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Calcio/metabolismoRESUMEN
Glutamate is the major excitatory neurotransmitter in the central nervous system. Glutamatergic transmission can be mediated by ionotropic glutamate receptors (iGluRs), which mediate rapid synaptic depolarization that can be associated with Ca2+ entry and activity-dependent change in the strength of synaptic transmission, as well as by metabotropic glutamate receptors (mGluRs), which mediate slower postsynaptic responses through the recruitment of second messenger systems. A wealth of evidence reported over the last three decades has shown that this dogmatic subdivision between iGluRs and mGluRs may not reflect the actual physiological signaling mode of the iGluRs, i.e., α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA) receptors (AMPAR), kainate receptors (KARs), and N-methyl-D-aspartate (NMDA) receptors (NMDARs). Herein, we review the evidence available supporting the notion that the canonical iGluRs can recruit flux-independent signaling pathways not only in neurons, but also in brain astrocytes and cerebrovascular endothelial cells. Understanding the signaling versatility of iGluRs can exert a profound impact on our understanding of glutamatergic synapses. Furthermore, it may shed light on novel neuroprotective strategies against brain disorders.
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A monolayer of endothelial cells lines the innermost surface of all blood vessels, thereby coming into close contact with every region of the body and perceiving signals deriving from both the bloodstream and parenchymal tissues. An increase in intracellular Ca2+ concentration ([Ca2+]i) is the main mechanism whereby vascular endothelial cells integrate the information conveyed by local and circulating cues. Herein, we describe the dynamics and spatial distribution of endothelial Ca2+ signals to understand how an array of spatially restricted (at both the subcellular and cellular levels) Ca2+ signals is exploited by the vascular intima to fulfill this complex task. We then illustrate how local endothelial Ca2+ signals affect the most appropriate vascular function and are integrated to transmit this information to more distant sites to maintain cardiovascular homeostasis. Vasorelaxation and sprouting angiogenesis were selected as an example of functions that are finely tuned by the variable spatio-temporal profile endothelial Ca2+ signals. We further highlighted how distinct Ca2+ signatures regulate the different phases of vasculogenesis, i.e., proliferation and migration, in circulating endothelial precursors.
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Calcio , Células Endoteliales , Células Endoteliales/metabolismo , Calcio/metabolismo , Señalización del Calcio/fisiología , Endotelio/metabolismo , Calcio de la DietaRESUMEN
The array of ion channels and transporters expressed in cell membranes, collectively referred to as the transportome, is a complex and multifunctional molecular machinery; in particular, at the plasma membrane level it finely tunes the exchange of biomolecules and ions, acting as a functionally adaptive interface that accounts for dynamic plasticity in the response to environmental fluctuations and stressors. The transportome is responsible for the definition of membrane potential and its variations, participates in the transduction of extracellular signals, and acts as a filter for most of the substances entering and leaving the cell, thus enabling the homeostasis of many cellular parameters. For all these reasons, physiologists have long been interested in the expression and functionality of ion channels and transporters, in both physiological and pathological settings and across the different domains of life. Today, thanks to the high-throughput technologies of the postgenomic era, the omics approach to the study of the transportome is becoming increasingly popular in different areas of biomedical research, allowing for a more comprehensive, integrated, and functional perspective of this complex cellular apparatus. This article represents a first effort for a systematic review of the scientific literature on this topic. Here we provide a brief overview of all those studies, both primary and meta-analyses, that looked at the transportome as a whole, regardless of the biological problem or the models they used. A subsequent section is devoted to the methodological aspect by reviewing the most important public databases annotating ion channels and transporters, along with the tools they provide to retrieve such information. Before conclusions, limitations and future perspectives are also discussed.
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Investigación Biomédica , Humanos , Homeostasis , Potenciales de la MembranaRESUMEN
Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder that may lead patients to sudden cell death through the occurrence of ventricular arrhythmias. ACM is characterised by the progressive substitution of cardiomyocytes with fibrofatty scar tissue that predisposes the heart to life-threatening arrhythmic events. Cardiac mesenchymal stromal cells (C-MSCs) contribute to the ACM by differentiating into fibroblasts and adipocytes, thereby supporting aberrant remodelling of the cardiac structure. Flecainide is an Ic antiarrhythmic drug that can be administered in combination with ß-adrenergic blockers to treat ACM due to its ability to target both Nav1.5 and type 2 ryanodine receptors (RyR2). However, a recent study showed that flecainide may also prevent fibro-adipogenic differentiation by inhibiting store-operated Ca2+ entry (SOCE) and thereby suppressing spontaneous Ca2+ oscillations in C-MSCs isolated from human ACM patients (ACM C-hMSCs). Herein, we briefly survey ACM pathogenesis and therapies and then recapitulate the main molecular mechanisms targeted by flecainide to mitigate arrhythmic events, including Nav1.5 and RyR2. Subsequently, we describe the role of spontaneous Ca2+ oscillations in determining MSC fate. Next, we discuss recent work showing that spontaneous Ca2+ oscillations in ACM C-hMSCs are accelerated to stimulate their fibro-adipogenic differentiation. Finally, we describe the evidence that flecainide suppresses spontaneous Ca2+ oscillations and fibro-adipogenic differentiation in ACM C-hMSCs by inhibiting constitutive SOCE.
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Living organisms are multiscale complex systems that have evolved high degrees of multifunctionality and redundancy in the structure-function relationship. A number of factors, only in part determined genetically, affect the jobs of proteins. The overall structural organization confers unique molecular properties that provide the potential to perform a pattern of activities, some of which are co-opted by specific environments. The variety of multifunctional proteins is expanding, but most cases are handled individually and according to the still dominant 'one structure-one function' approach, which relies on the attribution of canonical names typically referring to the first task identified for a given protein. The present topical review focuses on the multifunctionality of ion channels as a paradigmatic example. Mounting evidence reports the ability of many ion channels (including members of voltage-dependent, ligand-gated and transient receptor potential families) to exert biological effects independently of their ion conductivity. 'Functionally based' nomenclature (the practice of naming a protein or family of proteins based on a single purpose) is a conceptual bias for three main reasons: (i) it increases the amount of ambiguity, deceiving our understanding of the multiple contributions of biomolecules that is the heart of the complexity; (ii) it is in stark contrast to protein evolution dynamics, largely based on multidomain arrangement; and (iii) it overlooks the crucial role played by the microenvironment in adjusting the actions of cell structures and in tuning protein isoform diversity to accomplish adaptational requirements. Biological information in protein physiology is distributed among different entwined layers working as the primary 'locus' of natural selection and of evolutionary constraints.
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The human cytomegalovirus (HCMV) US12 gene family contributes to virus-host interactions by regulating the virus' cell tropism and its evasion of host innate immune responses. US21, one of the 10 US12 genes (US12-US21), is a descendant of a captured cellular transmembrane BAX inhibitor motif-containing gene. It encodes a 7TMD endoplasmic reticulum (ER)-resident viroporin (pUS21) capable of reducing the Ca2+ content of ER stores, which, in turn, protects cells against apoptosis. Since regulation of Ca2+ homeostasis affects a broad range of cellular responses, including cell motility, we investigated whether pUS21 might also interfere with this cytobiological consequence of Ca2+ signaling. Indeed, deletion of the US21 gene impaired the ability of HCMV-infected cells to migrate, whereas expression of US21 protein stimulated cell migration and adhesion, as well as focal adhesion (FA) dynamics, in a way that depended on its ability to manipulate ER Ca2+ content. Mechanistic studies revealed pUS21-mediated cell migration to involve calpain 2 activation since its inhibition prevented the viroporin's effects on cell motility. Pertinently, pUS21 expression stimulated a store-operated Ca2+ entry (SOCE) mechanism that may determine the activation of calpain 2 by promoting Ca2+ entry. Furthermore, pUS21 was observed to interact with talin-1, a calpain 2 substrate, and crucial protein component of FA complexes. A functional consequence of this interaction was confirmed by talin-1 knockdown, which abrogated the pUS21-mediated increase in cell migration. Together, these results indicate the US21-encoded viroporin to be a viral regulator of cell adhesion and migration in the context of HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic pathogen that owes part of its success to the capture, duplication, and tuning of cellular genes to generate modern viral proteins which promote infection and persistence in the host by interfering with many cell biochemical and physiological pathways. The US21 viral protein provides an example of this evolutionary strategy: it is a cellular-derived calcium channel that manipulates intracellular calcium homeostasis to confer edges to HCMV replication. Here, we report on the characterization of a novel function of the US21 protein as a viral regulator of cell migration and adhesion through mechanisms involving its calcium channel activity. Characterization of HCMV multifunctional regulatory proteins, like US21, supports the better understanding of viral pathogenesis and may open avenues for the design of new antiviral strategies that exploit their functions.
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Citomegalovirus , Proteínas Viroporinas , Humanos , Citomegalovirus/fisiología , Proteínas Viroporinas/metabolismo , Calpaína/genética , Calpaína/metabolismo , Talina/metabolismo , Proteínas Virales/metabolismo , Canales de Calcio/metabolismo , Movimiento CelularRESUMEN
Nitric oxide (NO) represents a crucial mediator to regulate cerebral blood flow (CBF) in the human brain both under basal conditions and in response to somatosensory stimulation. An increase in intracellular Ca2+ concentrations ([Ca2+]i) stimulates the endothelial NO synthase to produce NO in human cerebrovascular endothelial cells. Therefore, targeting the endothelial ion channel machinery could represent a promising strategy to rescue endothelial NO signalling in traumatic brain injury and neurodegenerative disorders. Allyl isothiocyanate (AITC), a major active constituent of cruciferous vegetables, was found to increase CBF in non-human preclinical models, but it is still unknown whether it stimulates NO release in human brain capillary endothelial cells. In the present investigation, we showed that AITC evoked a Ca2+-dependent NO release in the human cerebrovascular endothelial cell line, hCMEC/D3. The Ca2+ response to AITC was shaped by both intra- and extracellular Ca2+ sources, although it was insensitive to the pharmacological blockade of transient receptor potential ankyrin 1, which is regarded to be among the main molecular targets of AITC. In accord, AITC failed to induce transmembrane currents or to elicit membrane hyperpolarization, although NS309, a selective opener of the small- and intermediate-conductance Ca2+-activated K+ channels, induced a significant membrane hyperpolarization. The AITC-evoked Ca2+ signal was triggered by the production of cytosolic, but not mitochondrial, reactive oxygen species (ROS), and was supported by store-operated Ca2+ entry (SOCE). Conversely, the Ca2+ response to AITC did not require Ca2+ mobilization from the endoplasmic reticulum, lysosomes or mitochondria. However, pharmacological manipulation revealed that AITC-dependent ROS generation inhibited plasma membrane Ca2+-ATPase (PMCA) activity, thereby attenuating Ca2+ removal across the plasma membrane and resulting in a sustained increase in [Ca2+]i. In accord, the AITC-evoked NO release was driven by ROS generation and required ROS-dependent inhibition of PMCA activity. These data suggest that AITC could be exploited to restore NO signalling and restore CBF in brain disorders that feature neurovascular dysfunction.
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Células Endoteliales , Óxido Nítrico , Humanos , Especies Reactivas de Oxígeno/metabolismo , Células Endoteliales/metabolismo , Óxido Nítrico/metabolismo , Línea CelularRESUMEN
Tens of thousands of gene expression data sets describing a variety of model organisms in many different pathophysiological conditions are currently stored in publicly available databases such as the Gene Expression Omnibus (GEO) and ArrayExpress (AE). As microarray technology is giving way to RNA-seq, it becomes strategic to develop high-level tools of analysis to preserve access to this huge amount of information through the most sophisticated methods of data preparation and processing developed over the years, while ensuring, at the same time, the reproducibility of the results. To meet this need, here we present bioTEA (biological Transcript Expression Analyzer), a novel software tool that combines ease of use with the versatility and power of an R/Bioconductor-based differential expression analysis, starting from raw data retrieval and preparation to gene annotation. BioTEA is an R-coded pipeline, wrapped in a Python-based command line interface and containerized with Docker technology. The user can choose among multiple options-including gene filtering, batch effect handling, sample pairing, statistical test type-to adapt the algorithm flow to the structure of the particular data set. All these options are saved in a single text file, which can be easily shared between different laboratories to deterministically reproduce the results. In addition, a detailed log file provides accurate information about each step of the analysis. Overall, these features make bioTEA an invaluable tool for both bioinformaticians and wet-lab biologists interested in transcriptomics. BioTEA is free and open-source.
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The tumoral microenvironment often displays peculiar features, including accumulation of extracellular ATP, hypoxia, low pH-acidosis, as well as an imbalance in zinc (Zn2+) and calcium (Ca2+). We previously reported the ability of some purinergic agonists to exert an anti-migratory activity on tumor-derived human endothelial cells (TEC) only when applied at a high concentration. They also trigger calcium signals associated with release from intracellular stores and calcium entry from the external medium. Here, we provide evidence that high concentrations of BzATP (100 µM), a potent agonist of P2X receptors, decrease migration in TEC from different tumors, but not in normal microvascular ECs (HMEC). The same agonist evokes a calcium increase in TEC from the breast and kidney, as well as in HMEC, but not in TEC from the prostate, suggesting that the intracellular pathways responsible for the P2X-induced impairment of TEC migration could vary among different tumors. The calcium signal is mainly due to a long-lasting calcium entry from outside and is strictly dependent on the presence of the receptor occupancy. Low pH, as well as high extracellular Zn2+ and Ca2+, interfere with the response, a distinctive feature typically found in some P2X purinergic receptors. This study reveals that a BzATP-sensitive pathway impairs the migration of endothelial cells from different tumors through mechanisms finely tuned by environmental factors.
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Calcium (Ca2+)-permeable channels are key players in different processes leading to blood vessel formation via sprouting angiogenesis, including endothelial cell (EC) proliferation and migration, as well as in controlling vascular features which are typical of the tumor vasculature.In this review we present an up-to-date and critical view on the role of Ca2+-permeable channels in tumor vascularization, emphasizing on the dual communication between growth factors (mainly VEGF) and Ca2+ signals. Due to the complexity of the tumor microenvironment (TME) as a source of multiple stimuli acting on the endothelium, we aim to discuss the close interaction between chemical and physical challenges (hypoxia, oxidative stress, mechanical stress) and endothelial Ca2+-permeable channels, focusing on transient receptor potential (TRP), store-operated Ca2+ channels (SOCs), and mechanosensitive Piezo channels. This approach will depict their crucial contribution in regulating key properties of tumor blood vessels, such as recruitment of endothelial progenitors cells (EPCs) in the early steps of tumor vascularization, abnormal EC migration and proliferation, and increased vascular permeability. Graphical abstract depicting the functional role of Ca2+-permeable TRP, SOCs and Piezo channels in the biological processes regulating tumor angiogenesis in presence of both chemical (oxidative stress and oxygen levels) and mechanical stimuli (ECM stiffness). SOCs store-operated Ca2+ channels, TRPA transient receptor potential ankyrin, TRPV transient receptor potential vanilloid, TRPC transient receptor potential canonical, TRPM transient receptor potential melastatin, TRPM transient receptor potential vanilloid, O2 oxygen, ECM extracellular matrix.
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Neoplasias , Canales Catiónicos TRPM , Canales de Potencial de Receptor Transitorio , Calcio/metabolismo , Canales de Calcio/metabolismo , Señales (Psicología) , Humanos , Oxígeno/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Microambiente TumoralRESUMEN
Bone formation involves a complex crosstalk between endothelial cells (EC) and osteodifferentiating stem cells. This functional interplay is greatly mediated by the paracrine and autocrine action of soluble factors released at the vasculature-bone interface. This study elucidates the molecular and functional responses triggered by this intimate interaction. In this study, we showed that human dermal microvascular endothelial cells (HMEC) induced the expression of pro-angiogenic factors in stem cells from human exfoliated deciduous teeth (SHED) and sustain their osteo-differentiation at the same time. In contrast, osteodifferentiating SHED increased EC recruitment and promoted the formation of complex vascular networks. Moreover, HMEC enhanced anaerobic glycolysis in proliferating SHED without compromising their ability to undergo the oxidative metabolic shift required for adequate osteo-differentiation. Taken together, these findings provide novel insights into the molecular mechanism underlying the synergistic cooperation between EC and stem cells during bone tissue renewal.
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Tumor microenvironment is particularly enriched with extracellular ATP (eATP), but conflicting evidence has been provided on its functional effects on tumor growth and vascular remodeling. We have previously shown that high eATP concentrations exert a strong anti-migratory, antiangiogenic and normalizing activity on human tumor-derived endothelial cells (TECs). Since both metabotropic and ionotropic purinergic receptors trigger cytosolic calcium increase ([Ca2+]c), the present work investigated the properties of [Ca2+]c events elicited by high eATP in TECs and their role in anti-migratory activity. In particular, the quantitative and kinetic properties of purinergic-induced Ca2+ release from intracellular stores and Ca2+ entry from extracellular medium were investigated. The main conclusions are: (1) stimulation of TECs with high eATP triggers [Ca2+]c signals which include Ca2+ mobilization from intracellular stores (mainly ER) and Ca2+ entry through the plasma membrane; (2) the long-lasting Ca2+ influx phase requires both store-operated Ca2+ entry (SOCE) and non-SOCE components; (3) SOCE is not significantly involved in the antimigratory effect of high ATP stimulation; (4) ER is the main source for intracellular Ca2+ release by eATP: it is required for the constitutive migratory potential of TECs but is not the only determinant for the inhibitory effect of high eATP; (5) a complex interplay occurs among ER, mitochondria and lysosomes upon purinergic stimulation; (6) high eUTP is unable to inhibit TEC migration and evokes [Ca2+]c signals very similar to those described for eATP. The potential role played by store-independent Ca2+ entry and Ca2+-independent events in the regulation of TEC migration by high purinergic stimula deserves future investigation.
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BACKGROUND: Purinergic signalling is involved in several physiological and pathophysiological processes. P2X7 Receptor (P2X7R) is a calcium-permeable ion channel that is gaining interest as a potential therapeutic target for the treatment of different diseases including inflammation, pain, psychiatric disorders and cancer. P2X7R is ubiquitously expressed and sensitive to high ATP levels, usually found in tumor microenvironment. P2X7R regulates several cell functions, from migration to cell death, but its selective contribution to tumor progression remains controversial. OBJECTIVE: Current review was conducted to check involvement of P2X7R use in cancer treatment. METHODS: We review the most recent patents focused on the use of P2X7R in the treatment of cancer. RESULTS: P2X7R is an intriguing purinergic receptor that plays different roles in tumor progression. CONCLUSION: Powerful strategies able to selectively interfere with its expression and function should reveal helpful in the development of new anti-cancer therapies.
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Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Receptores Purinérgicos P2X7/biosíntesis , Microambiente Tumoral/efectos de los fármacos , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Humanos , Neoplasias/metabolismo , Patentes como Asunto , Antagonistas del Receptor Purinérgico P2X/farmacología , Microambiente Tumoral/fisiologíaRESUMEN
The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]i , which was maximally activated at 200 µM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.
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Encéfalo/irrigación sanguínea , Señalización del Calcio/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Ácido Glutámico/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , NADP/análogos & derivados , Acoplamiento Neurovascular/efectos de los fármacos , Óxido Nítrico/metabolismo , Animales , Canales de Calcio/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Humanos , Ratones , NADP/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Factores de TiempoRESUMEN
Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cß (PLCß) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.