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The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.
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Células Madre Pluripotentes Inducidas , Proteoma , Animales , Humanos , Pez Cebra , Genética Humana , Mamíferos , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Plasmacytoid dendritic cells (pDCs) display an increased abundance in visceral adipose tissue (VAT) of humans with obesity. In the current study, we set out to decipher the molecular mechanisms of their recruitment to VAT and the functional relevance of this process. We observed increased pDC numbers in murine blood, liver, spleen, and VAT after feeding a high-fat diet (HFD) for 3 wk when compared with a standard diet. pDCs were enriched in fat-associated lymphoid clusters representing highly specific lymphoid regions within VAT. HFD led to an enlargement of fat-associated lymphoid clusters with an increased density and migratory speed of pDCs as shown by intravital multiphoton microscopy. For their recruitment into VAT, pDCs employed P-selectin with E-selectin and L-selectin being only critical in response to HFD, indicating that the molecular cues underlying pDC trafficking were dependent on the nutritional state. Subsequent recruitment steps required α4ß1 and α4ß7 integrins and engagement of CCR7. Application of fingolimod (FTY720) abrogated egress of pDCs from VAT, indicating the involvement of sphingosine-1-phosphate in this process. Furthermore, HFD altered pDC functions by promoting their activation and type 1 IFN expression. Blocking pDC infiltration into VAT prevented weight gain and improved glucose tolerance during HFD. In summary, a HFD fundamentally alters pDC biology by promoting their trafficking, retention, and activation in VAT, which in turn seems to regulate metabolism.
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Dieta Alta en Grasa , Grasa Intraabdominal , Tejido Adiposo , Animales , Células Dendríticas , Grasa Intraabdominal/metabolismo , Ratones , FenotipoRESUMEN
Signaling via ß2 integrins (CD11/CD18) as well as TCRs and BCRs involves similar pathways. However, the activation of the same signaling molecule can result in opposing effects. One such example is the hematopoietic progenitor kinase 1 (HPK1), which negatively regulates T and B cell activation but enforces neutrophil adhesion via ß2 integrins. This difference may be defined by specific HPK1 interacting networks in different leukocyte subsets which have already been described in the adaptive immune system. Here, we set out to identify interacting proteins of HPK1 in neutrophil-like differentiated HL-60 cells exposed to immobilized fibrinogen and left nonactivated or Mn2+ -activated to allow ß2 integrin-dependent adhesion. Co-IP experiments followed by mass spectrometry led to the identification of 115 HPK1-interacting proteins. A total of 58 proteins were found only in nonactivated cells and 39 proteins only in Mn2+ -activated adherent cells. From these results, we decoded a pre-existing signaling cluster of HPK1 in nonactivated cells encompassing proteins essential for ß2 integrin-mediated signaling during neutrophil trafficking, namely DNAX-activation protein 12 (DAP12), spleen tyrosine kinase (Syk), and Rac1. Thus, our study provides novel insights into the complex architecture of the signaling processes during neutrophil activation and the complex signaling profile of HPK1 in leukocytes.
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Proteómica , Receptores de Antígenos de Linfocitos T , Humanos , Inmunidad Innata , Integrinas/metabolismo , Proteínas Serina-Treonina QuinasasRESUMEN
BACKGROUND: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. METHODS: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines' representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. RESULTS: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. CONCLUSION: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression.
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Seminoma , Neoplasias Testiculares , Masculino , Humanos , Conexina 43/metabolismo , Seminoma/patología , Cadherinas/metabolismo , Células de Sertoli/metabolismo , Células de Sertoli/patología , Neoplasias Testiculares/patología , Línea Celular , Biopsia , ARN Mensajero/genéticaRESUMEN
Neutrophils, the most abundant white blood cells in the human circulation, play crucial roles in various diseases, including kidney disease. Traditionally viewed as short-lived pro-inflammatory phagocytes that release reactive oxygen species, cytokines and neutrophil extracellular traps, recent studies have revealed their complexity and heterogeneity, thereby challenging this perception. Neutrophils are now recognized as transcriptionally active cells capable of proliferation and reverse migration, displaying phenotypic and functional heterogeneity. They respond to a wide range of signals and deploy various cargo to influence the activity of other cells in the circulation and in tissues. They can regulate the behavior of multiple immune cell types, exhibit innate immune memory, and contribute to both acute and chronic inflammatory responses while also promoting inflammation resolution in a context-dependent manner. Here, we explore the origin and heterogeneity of neutrophils, their functional diversity, and the cues that regulate their effector functions. We also examine their emerging role in infectious and non-infectious diseases with a particular emphasis on kidney disease. Understanding the complex behavior of neutrophils during tissue injury and inflammation may provide novel insights, thereby paving the way for potential therapeutic strategies to manage acute and chronic conditions. By deciphering their multifaceted role, targeted interventions can be developed to address the intricacies of neutrophil-mediated immune responses and improve disease outcomes.
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Neutrophils are the first leukocytes to arrive at sites of injury during the acute inflammatory response. To maintain the polarized morphology during migration, nonmuscle myosins class II are essential, but studies using genetic models to investigate the role of Myh9 for neutrophil migration were missing. In this study, we analyzed the functional role of Myh9 on neutrophil trafficking using genetic downregulation of Myh9 in Vav-iCre+/Myh9wt/fl mice because the complete knockout of Myh9 in the hematopoietic system was lethal. Migration velocity and Euclidean distance were significantly diminished during mechanotactic migration of Vav-iCre+/Myh9wt/fl neutrophils compared with Vav-iCre-/Myh9wt/fl control neutrophils. Similar results were obtained for transmigration and migration in confined three-dimensional environments. Stimulated emission depletion nanoscopy revealed that a certain threshold of Myh9 was required to maintain proper F-actin dynamics in the front of the migrating cell. In laser-induced skin injury and in acute peritonitis, reduced Myh9 expression in the hematopoietic system resulted in significantly diminished neutrophil extravasation. Investigation of bone marrow chimeric mice in the peritonitis model revealed that the migration defect was cell intrinsic. Expression of Myh9-EGFP rescued the Myh9-related defects in two-dimensional and three-dimensional migration of Hoxb8-SCF cell-derived neutrophils generated from fetal liver cells with a Myh9 knockdown. Live cell imaging provided evidence that Myh9 was localized in branching lamellipodia and in the uropod where it may enable fast neutrophil migration. In summary, the severe migration defects indicate an essential and fundamental role of Myh9 for neutrophil trafficking in innate immunity.
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Movimiento Celular/inmunología , Inmunidad Innata , Infiltración Neutrófila , Neutrófilos/inmunología , Miosina Tipo IIA no Muscular/inmunología , Seudópodos/inmunología , Actinas/genética , Actinas/inmunología , Animales , Movimiento Celular/genética , Ratones , Ratones Transgénicos , Cadenas Pesadas de Miosina , Neutrófilos/patología , Miosina Tipo IIA no Muscular/genética , Peritonitis/genética , Peritonitis/inmunología , Peritonitis/patología , Seudópodos/genética , Piel/inmunología , Piel/lesiones , Piel/patologíaRESUMEN
Trafficking of polymorphonuclear neutrophils (PMNs) during inflammation critically depends on the ß2 integrins lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) and macrophage-1 antigen (CD11b/CD18). Here, we identify coronin 1A (Coro1A) as a novel regulator of ß2 integrins that interacts with the cytoplasmic tail of CD18 and is crucial for induction of PMN adhesion and postadhesion events, including adhesion strengthening, spreading, and migration under flow conditions. Transition of PMN rolling to firm adhesion critically depends on Coro1A by regulating the accumulation of high-affinity LFA-1 in focal zones of adherent cells. Defective integrin affinity regulation in the genetic absence of Coro1A impairs leukocyte adhesion and extravasation in inflamed cremaster muscle venules in comparison with control animals. In a Helicobacter pylori mouse infection model, PMN infiltration into the gastric mucosa is dramatically reduced in Coro1A-/- mice, resulting in an attenuated gastric inflammation. Thus, Coro1A represents an important novel player in integrin biology, with key functions in PMN trafficking during innate immunity.
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4-Butirolactona/análogos & derivados , Antígenos CD18/metabolismo , Movimiento Celular , Inmunidad Innata , Neutrófilos/citología , Neutrófilos/metabolismo , 4-Butirolactona/metabolismo , Actinas/metabolismo , Animales , Señalización del Calcio , Adhesión Celular , Gastritis/inmunología , Gastritis/microbiología , Gastritis/patología , Helicobacter pylori/fisiología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/metabolismo , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/metabolismo , ReologíaRESUMEN
BACKGROUND: Neutrophil recruitment during acute inflammation critically depends on the spatial and temporal regulation of ß2 integrins (CD11/CD18). This regulation occurs by inside-out and outside-in signalling via interaction of cytoplasmic proteins with the intracellular domains of the integrin α- and ß-subunits. The underlying molecular mechanisms regulating ß2 integrins in neutrophils are still incompletely understood. AIM: This review provides a comprehensive overview of our current knowledge on proteins interacting with the cytoplasmic tail of CD18, the conserved ß-subunit of ß2 integrins, their regulation and their functional importance for neutrophil trafficking during acute inflammation. RESULTS: A total of 22 proteins including Talin, Kindlin 3 and Coronin 1A have been reported to interact with the CD18 cytoplasmic tail. Here, proteins binding to the cytoplasmic domain of CD18 in experiments using purified, recombinant proteins or peptides in, for example, pull-down assays, are defined as direct interactors. Proteins that have been shown to interact with the cytoplasmic domain of CD18 using whole cell lysates in, for example, pull-down experiments are claimed as interacting proteins without evidence for direct interaction. In summary, ß2 integrin activation and signalling depend on a specific subset of proteins interacting with CD18 and their precise regulation. If disturbed, profound defects of neutrophil recruitment and activation become evident compromising the innate immune response. CONCLUSIONS: The knowledge of proteins interacting with ß2 integrins and their regulation during neutrophil trafficking does not only improve our basic understanding of innate immunity but may pave the way to novel therapeutic strategies in the treatment of inflammatory diseases.
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Antígenos CD18/fisiología , Neutrófilos/fisiología , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Humanos , Infiltración Neutrófila/fisiología , Unión Proteica/fisiología , Proteínas/fisiología , Transducción de Señal/fisiologíaRESUMEN
KEY POINTS: Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. ABSTRACT: The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.
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Calcio/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Células Endoteliales/metabolismo , Uniones Comunicantes/metabolismo , Microvasos/metabolismo , Receptor de Adenosina A2B/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Línea Celular , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Células Endoteliales/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Microvasos/efectos de los fármacosRESUMEN
Control of blood flow distribution and tissue homeostasis depend on the tight regulation of and coordination between the microvascular network and circulating blood cells. Channels formed by connexins or pannexins that connect the intra- and extracellular compartments allow the release of paracrine signals, such as ATP and prostaglandins, and thus play a central role in achieving fine regulation and coordination of vascular function. This review focuses on vascular connexin hemichannels and pannexin channels. We review their expression pattern within the arterial and venous system with a special emphasis on how post-translational modifications by phosphorylation and S-nitrosylation of these channels modulate their function and contribute to vascular homeostasis. Furthermore, we highlight the contribution of these channels in smooth muscle cells and endothelial cells in the regulation of vasomotor tone as well as how these channels in endothelial cells regulate inflammatory responses such as during ischemic and hypoxic conditions. In addition, this review will touch on recent evidence implicating a role for these proteins in regulating red blood cell and platelet function.
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Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiología , Conexinas/metabolismo , Inflamación/metabolismo , Inflamación/patología , Proteínas del Tejido Nervioso/metabolismo , Animales , Conexinas/química , Conexinas/genética , Células Endoteliales/metabolismo , Humanos , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genéticaRESUMEN
Using the double whole-cell patch-clamp technique, we found that the absence of intracellular ATP led to gap junction uncoupling in cochlear-supporting Hensen cells. The uncoupling was observed as a progressive reduction of the gap junctional electrical conductance from a starting value of approximately 40 nS to less than 0.04 nS within 10-20 min. The conductance rundown was partly avoided by at least 3 mM ATP and completely suppressed by 5 mM ATP or 5'-adenylyl-imidodiphosphate (AMP-PNP), the non-hydrolysable ATP analog, in the pipette filling solution, suggesting that ATP was needed as ligand and not as a hydrolysable energy supplier or substrate for enzymatic reactions. The effect of intracellular ATP was mimicked by the external application of barium, a nonselective blocker of inwardly rectifying K(+) (Kir) channels, and glibenclamide, an inhibitor of the ATP-sensitive Kir channels (KATP). Moreover a Ba(2+)-sensitive whole-cell inward current was observed in absence of internal ATP. We propose that the internal ATP kept the KATP channels in a closed state, thereby maintaining the gap junction coupling of Hensen cells. The immunostaining of guinea pig cochlear tissue revealed for the first time the expression of the KATP channel subunits Kir6.1 and SUR1 in Hensen cells and supported the proposed hypothesis. The results suggest that KATP channels, as regulator of the gap junction coupling in Hensen cells, could be the physiological link between the metabolic state of the supporting cells and K(+) recycling in the organ of Corti.
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Adenosina Trifosfato/metabolismo , Cóclea/metabolismo , Uniones Comunicantes/metabolismo , Canales KATP/metabolismo , Receptores de Sulfonilureas/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Cóclea/efectos de los fármacos , Electrofisiología/métodos , Uniones Comunicantes/efectos de los fármacos , Gliburida/farmacología , Cobayas , Potenciales de la Membrana/efectos de los fármacos , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismoRESUMEN
Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.
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Microcirculación/fisiología , Óxido Nítrico Sintasa de Tipo III/fisiología , Secuencia de Aminoácidos , Endotelio Vascular/metabolismo , Eritrocitos/enzimología , Humanos , Datos de Secuencia Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/química , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Vasculitis/metabolismo , Vasodilatación/fisiologíaRESUMEN
Pannexin channels are newly discovered ATP release channels expressed throughout the body. Pannexin 1 (Panx1) channels have become of great interest as they appear to participate in a multitude of signalling cascades, including regulation of vascular function. Although numerous Panx1 pharmacological inhibitors have been discovered, these inhibitors are not specific for Panx1 and have additional effects on other proteins. Therefore, molecular tools, such as RNA interference and knockout animals, are needed to demonstrate the role of pannexins in various cellular functions. This review focuses on the known roles of Panx1 related to purinergic signalling in the vasculature focusing on post-translational modifications and channel gating mechanisms that may participate in the regulated release of ATP.
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Conexinas/genética , Músculo Liso Vascular/metabolismo , Proteínas del Tejido Nervioso/genética , Procesamiento Proteico-Postraduccional/genética , Receptores Purinérgicos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Conexinas/metabolismo , Humanos , Músculo Liso Vascular/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Interferencia de ARN , Receptores Purinérgicos/genética , Transducción de SeñalRESUMEN
The present report evaluates the advantages of using the gold nanoparticle-mediated laser perforation (GNOME LP) technique as a computer-controlled cell optoperforation to introduce Lucifer yellow (LY) into cells in order to analyze the gap junction coupling in cell monolayers. To permeabilize GM-7373 endothelial cells grown in a 24 multiwell plate with GNOME LP, a laser beam of 88 µm in diameter was applied in the presence of gold nanoparticles and LY. After 10 min to allow dye uptake and diffusion through gap junctions, we observed a LY-positive cell band of 179 ± 8 µm width. The presence of the gap junction channel blocker carbenoxolone during the optoperforation reduced the LY-positive band to 95 ± 6 µm. Additionally, a forskolin-related enhancement of gap junction coupling, recently found using the scrape loading technique, was also observed using GNOME LP. Further, an automatic cell imaging and a subsequent semi-automatic quantification of the images using a java-based ImageJ-plugin were performed in a high-throughput sequence. Moreover, the GNOME LP was used on cells such as RBE4 rat brain endothelial cells, which cannot be mechanically scraped as well as on three-dimensionally cultivated cells, opening the possibility to implement the GNOME LP technique for analysis of gap junction coupling in tissues. We conclude that the GNOME LP technique allows a high-throughput automated analysis of gap junction coupling in cells. Moreover this non-invasive technique could be used on monolayers that do not support mechanical scraping as well as on cells in tissue allowing an in vivo/ex vivo analysis of gap junction coupling.
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Células Endoteliales/metabolismo , Uniones Comunicantes/metabolismo , Oro/química , Rayos Láser , Nanopartículas del Metal/química , Animales , Carbenoxolona/farmacología , Bovinos , Línea Celular , Células Endoteliales/citología , RatasRESUMEN
Previous data showed that dipyridamole enhanced gap junction coupling in vascular endothelial and smooth muscle cell lines by a cAMP-dependent mechanism. The present study investigates the level at which dipyridamole affects gap junction coupling. In the GM-7373 endothelial cell line, scrape loading/dye transfer experiments revealed a rapid increase in gap junction coupling induced during the first 6 h of dipyridamole treatment, followed by a slow increase induced by further incubation. Immunostaining analyses showed that the rapid enhancement of gap junction coupling correlated with an increased amount of Cx43 gap junction plaques and a reduced amount of Cx43 containing vesicles, while the amount of Cx43 mRNA or protein was not changed during this period, as found by semiquantitative RT-PCR and Western blot. Additionally, brefeldin A did not block this short-term-induced enhancement of gap junction coupling. Along with the dipyridamole-induced long-term enhancement of gap junction coupling, the amount of Cx43 mRNA and protein additionally to the amount of Cx43 gap junction plaques were increased. Furthermore, the anti-Cx43 antibody detected only two bands at 42 kDa and 44 kDa in control cells and cells treated with dipyridamole for 6 h, while long-term dipyridamole-treated cells showed a third band at 46 kDa. We propose that a dipyridamole-induced cAMP synthesis increased gap junction coupling in the GM-7373 endothelial cell line at different levels: the short-term effect is related to already oligomerised connexins beyond the Golgi apparatus and the long-term effect involves new expression and synthesis as well as posttranslational modification of Cx43.
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Conexina 43/metabolismo , Dipiridamol/farmacología , Células Endoteliales/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , ARN Mensajero/metabolismo , Células Cultivadas , Conexina 43/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Uniones Comunicantes/metabolismo , Humanos , Fosforilación , ARN Mensajero/genéticaRESUMEN
The expression and physiology of purine receptors of the human blood-brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca(2+)-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y(2)-, P2Y(6)-, P2Y(11)- as well as the ionotropic P2X(4)-, P2X(5)- and P2X(7)-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca(2+) concentration ([Ca(2+)](i)) from 150 to 300 nM in single cells. The change in [Ca(2+)](i) corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, αß-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP(3) cascade-mediated Ca(2+) release, indicating that the nucleotide-induced Ca(2+) signal was mainly related to P2Y(2, 6 and 11) receptors. The gene silencing of the P2Y(2) receptor reduced the ATP- or UTP-induced Ca(2+) signal and suppressed the Ca(2+) signal mediated by P2Y(6) and P2Y(11) more specific agonists like UDP (P2Y(6)), BzATP (P2Y(11)) and ATPγS (P2Y(11)). This report identifies the P2Y(2) receptor subtype as the main purine receptor involved in Ca(2+) signalling of the hCMEC/D3 cells.
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The lumen of blood vessels is covered by endothelial cells, which regulate their permeability to ions and solutes. Endothelial permeability depends on the vascular bed and cell phenotype, and is influenced by different disease states. Most characterization of endothelial permeability has been carried out using isolated cells in culture. While analysis of cultured cells is a valuable approach, it does not account for factors of the native cell environment. Building on Ussing chamber studies of intact tissue specimens, here we describe a method to measure the electrophysiological properties of intact arteriole and venule endothelia, including transendothelial electrical resistance (TEER) and ion permselectivity. As an example, vessels isolated from the mesentery were treated ex vivo, then mounted in a custom-made tissue cassette that enable their analysis by classical approaches with an Ussing chamber. This method enables a detailed analysis of electrophysiological vessel responses to stresses such as proinflammatory cytokines, in the context of an intact vessel. Graphic abstract.
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Whole-cell patch-clamp analysis revealed a resting membrane potential of -60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of -20 mV and a repolarization between the spikes to -45 mV. Expressed channels were characterized by application of voltage pulses between -150 mV and 90 mV in 10 mV steps, from a holding potential of -40 mV. Voltages below -60 mV induced an inward current. Depolarizing voltages above -30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between -30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above -30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K(+) (K(ir)) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na(+) (Na(v)) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K(+) (K(v)) channels. In addition, RT-PCR showed expression of Na(v)1.3, Na(v)1.4, Na(v)1.5, Na(v)1.6, Na(v)1.7, and K(ir)2.1, K(ir)2.3, and K(ir)2.4 as well as K(v)2.1. We conclude that osteoblasts express channels that allow firing of action potentials.
Asunto(s)
Osteoblastos/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Neoplasias Óseas , Línea Celular , Línea Celular Tumoral , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteosarcoma , Técnicas de Placa-Clamp , Canales de Potasio/biosíntesis , Canales de Potasio/fisiología , Canales de Sodio/biosíntesis , Canales de Sodio/fisiologíaRESUMEN
Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the ß2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of ß2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of ß2 integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the ß subunit of ß2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.
Asunto(s)
Antígenos CD18/metabolismo , Adhesión Celular/genética , Movimiento Celular/genética , Infiltración Neutrófila/genética , Neutrófilos/inmunología , Pez Cebra/genética , Pez Cebra/inmunología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Antígenos CD11/química , Antígenos CD11/genética , Antígenos CD11/metabolismo , Antígenos CD18/química , Antígenos CD18/genética , Adhesión Celular/inmunología , Movimiento Celular/inmunología , Modelos Animales de Enfermedad , Eliminación de Gen , Técnicas de Inactivación de Genes , Inflamación/genética , Inflamación/inmunología , Integrinas/metabolismo , Larva/genética , Larva/inmunología , Síndrome de Deficiencia de Adhesión del Leucocito/inmunología , Infiltración Neutrófila/inmunologíaRESUMEN
The endothelial cell barrier regulates the passage of fluid between the bloodstream and underlying tissues, and barrier function impairment exacerbates the severity of inflammatory insults. To understand how inflammation alters vessel permeability, we studied the effects of the proinflammatory cytokine TNFα on transendothelial permeability and electrophysiology in ex vivo murine veins and arteries. We found that TNFα specifically decreased the barrier function of venous endothelium without affecting that of arterial endothelium. On the basis of RNA expression profiling and protein analysis, we found that claudin-11 (CLDN11) was the predominant claudin in venous endothelial cells and that there was little, if any, CLDN11 in arterial endothelial cells. Consistent with a difference in claudin composition, TNFα increased the permselectivity of Cl- over Na+ in venous but not arterial endothelium. The vein-specific effects of TNFα also required the activation of Pannexin 1 (Panx1) channels and the CD39-mediated hydrolysis of ATP to adenosine, which subsequently stimulated A2A adenosine receptors. Moreover, the increase in vein permeability required the activation of the Ca2+ channel TRPV4 downstream of Panx1 activation. Panx1-deficient mice resisted the pathologic effects of sepsis induced by cecal ligation and puncture on life span and lung vascular permeability. These data provide a targetable pathway with the potential to promote vein barrier function and prevent the deleterious effects of vascular leak in response to inflammation.