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GITDs are among the most common causes of death in adult and young horses in the United States (US). Previous studies have indicated a connection between GITDs and the equine gut microbiome. However, the low taxonomic resolution of the current microbiome sequencing methods has hampered the identification of specific bacterial changes associated with GITDs in horses. Here, we have compared TEHC, a new approach for 16S rRNA gene selection and sequencing, with conventional 16S rRNA gene amplicon sequencing for the characterization of the equine fecal microbiome. Both sequencing approaches were used to determine the fecal microbiome of four adult horses and one commercial mock microbiome. Our results show that TEHC yielded significantly more operational taxonomic units (OTUs) than conventional 16S amplicon sequencing when the same number of reads were used in the analysis. This translated into a deeper and more accurate characterization of the fecal microbiome when the samples were sequenced with TEHC according to the relative abundance analysis. Alpha and beta diversity metrics corroborated these findings and demonstrated that the microbiome of the fecal samples was significantly richer when sequenced with TEHC compared to 16S amplicon sequencing. Altogether, our study suggests that the TEHC strategy provides a more extensive characterization of the fecal microbiome of horses than the current alternative based on the PCR amplification of a portion of the 16S rRNA gene.
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Excess consumption of carbohydrates, fat and calories leads to non-alcoholic fatty liver disease (NAFLD) and hepatic insulin resistance; these are major factors in the pathogenesis of type II diabetes. Hormones and catecholamines acting through G-protein coupled receptors (GPCRs) linked to phospholipase C (PLC) and increases in cytosolic Ca2+ ([Ca2+ ]c ) regulate many metabolic functions of the liver. In the intact liver, catabolic hormones such as glucagon, catecholamines and vasopressin integrate and synergize to regulate the frequency and extent to which [Ca2+ ]c waves propagate across hepatic lobules to control metabolism. Dysregulation of hepatic Ca2+ homeostasis has been implicated in the development of metabolic disease, but changes in hepatic GPCR-dependent Ca2+ signalling have been largely unexplored in this context. We show that short-term, 1-week, high-fat diet (HFD) feeding of mice attenuates noradrenaline-stimulated Ca2+ signalling, reducing the number of cells responding and suppressing the frequency of [Ca2+ ]c oscillations in both isolated hepatocytes and intact liver. The 1-week HFD feeding paradigm did not change basal Ca2+ homeostasis; endoplasmic reticulum Ca2+ load, store-operated Ca2+ entry and plasma membrane Ca2+ pump activity were unchanged compared to low-fat diet (LFD)-fed controls. However, noradrenaline-induced inositol 1,4,5-trisphosphate production was significantly reduced after HFD feeding, demonstrating an effect of HFD on receptor-stimulated PLC activity. Thus, we have identified a lesion in the PLC signalling pathway induced by short-term HFD feeding, which interferes with hormonal Ca2+ signalling in isolated hepatocytes and the intact liver. These early events may drive adaptive changes in signalling, which lead to pathological consequences in fatty liver disease. KEY POINTS: Non-alcoholic fatty liver disease (NAFLD) is a growing epidemic. In healthy liver, the counteracting effects of catabolic and anabolic hormones regulate metabolism and energy storage as fat. Hormones and catecholamines promote catabolic metabolism via increases in cytosolic Ca2+ ([Ca2+ ]c ). We show that 1 week high-fat diet (HFD) feeding of mice attenuated the Ca2+ signals induced by physiological concentrations of noradrenaline. Specifically, HFD suppressed the normal pattern of periodic [Ca2+ ]c oscillations in isolated hepatocytes and disrupted the propagation of intralobular [Ca2+ ]c waves in the intact perfused liver. Short-term HFD inhibited noradrenaline-induced inositol 1,4,5-trisphosphate generation, but did not change basal endoplasmic reticulum Ca2+ load or plasma membrane Ca2+ fluxes. We propose that impaired Ca2+ signalling plays a key role in the earliest phases of the etiology of NAFLD, and is responsible for many of the ensuing metabolic and related dysfunctional outcomes at the cellular and whole tissue level.
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Diabetes Mellitus Tipo 2 , Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Dieta Alta en Grasa/efectos adversos , Inositol 1,4,5-Trifosfato/metabolismo , Catecolaminas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Glucagón , Norepinefrina/farmacología , Ratones Endogámicos C57BLRESUMEN
There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.
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Escherichia coli/genética , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Listeria/genética , Salmonella/genética , Secuenciación Completa del Genoma/métodos , Animales , Bacterias/genética , ADN Bacteriano/genética , Infecciones por Escherichia coli/microbiología , Enfermedades Transmitidas por los Alimentos/microbiología , Biblioteca de Genes , Genómica , Laboratorios , Infecciones por Salmonella/microbiología , Virulencia/genéticaRESUMEN
The current treatment of leishmaniasis is based on a few drugs that present several drawbacks, such as high toxicity, difficult administration route, and low efficacy. These disadvantages raise the necessity to develop novel antileishmanial compounds allied with a comprehensive understanding of their mechanisms of action. Here, we elucidate the probable mechanism of action of the antileishmanial binuclear cyclopalladated complex [Pd(dmba)(µ-N3)]2 (CP2) in Leishmania amazonensis. CP2 causes oxidative stress in the parasite, resulting in disruption of mitochondrial Ca2+ homeostasis, cell cycle arrest at the S-phase, increasing the reactive oxygen species (ROS) production and overexpression of stress-related and cell detoxification proteins, and collapsing the Leishmania mitochondrial membrane potential, and promotes apoptotic-like features in promastigotes, leading to necrosis, or directs programmed cell death (PCD)-committed cells toward necrotic-like destruction. Moreover, CP2 reduces the parasite load in both liver and spleen in Leishmania infantum-infected hamsters when treated for 15 days with 1.5 mg/kg body weight/day CP2, expanding its potential application in addition to the already known effectiveness on cutaneous leishmaniasis for the treatment of visceral leishmaniasis, showing the broad spectrum of action of this cyclopalladated complex. The data presented here bring new insights into the CP2 molecular mechanisms of action, assisting the promotion of its rational modification to improve both safety and efficacy.
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Antiprotozoarios , Leishmania infantum , Leishmaniasis Cutánea , Animales , Antiprotozoarios/uso terapéutico , Calcio/metabolismo , Muerte Celular , Leishmaniasis Cutánea/tratamiento farmacológico , Macrófagos , Ratones , Ratones Endogámicos BALB C , MitocondriasRESUMEN
Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists.
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Receptor-coupled phospholipase C (PLC) is an important target for the actions of ethanol. In the ex vivo perfused rat liver, concentrations of ethanol >100 mM were required to induce a rise in cytosolic calcium (Ca2+) suggesting that these responses may only occur after binge ethanol consumption. Conversely, pharmacologically achievable concentrations of ethanol (≤30 mM) decreased the frequency and magnitude of hormone-stimulated cytosolic and nuclear Ca2+ oscillations and the parallel translocation of protein kinase C-ß to the membrane. Ethanol also inhibited gap junction communication resulting in the loss of coordinated and spatially organized intercellular Ca2+ waves in hepatic lobules. Increasing the hormone concentration overcame the effects of ethanol on the frequency of Ca2+ oscillations and amplitude of the individual Ca2+ transients; however, the Ca2+ responses in the intact liver remained disorganized at the intercellular level, suggesting that gap junctions were still inhibited. Pretreating hepatocytes with an alcohol dehydrogenase inhibitor suppressed the effects of ethanol on hormone-induced Ca2+ increases, whereas inhibiting aldehyde dehydrogenase potentiated the inhibitory actions of ethanol, suggesting that acetaldehyde is the underlying mediator. Acute ethanol intoxication inhibited the rate of rise and the magnitude of hormone-stimulated production of inositol 1,4,5-trisphosphate (IP3), but had no effect on the size of Ca2+ spikes induced by photolysis of caged IP3. These findings suggest that ethanol inhibits PLC activity, but does not affect IP3 receptor function. We propose that by suppressing hormone-stimulated PLC activity, ethanol interferes with the dynamic modulation of [IP3] that is required to generate large, amplitude Ca2+ oscillations.
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Señalización del Calcio , Inositol 1,4,5-Trifosfato , Ratas , Animales , Inositol 1,4,5-Trifosfato/metabolismo , Etanol/farmacología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado , Hormonas/metabolismoRESUMEN
Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Monitoreo Epidemiológico/veterinaria , Genómica/métodos , Infecciones Estafilocócicas/veterinaria , Staphylococcus/efectos de los fármacos , Staphylococcus/genética , Animales , Proteínas Bacterianas/genética , Canadá , Enfermedades de los Perros/microbiología , Perros/microbiología , Genómica/normas , Genotipo , Pruebas de Sensibilidad Microbiana , Fenotipo , Filogenia , Reproducibilidad de los Resultados , Infecciones Estafilocócicas/microbiología , Staphylococcus/aislamiento & purificación , Estados Unidos , Secuenciación Completa del GenomaRESUMEN
Calcium (Ca2+) oscillations in hepatocytes have a wide dynamic range. In particular, recent experimental evidence shows that agonist stimulation of the P2Y family of receptors leads to qualitatively diverse Ca2+ oscillations. We present a new model of Ca2+ oscillations in hepatocytes based on these experiments to investigate the mechanisms controlling P2Y-activated Ca2+ oscillations. The model accounts for Ca2+ regulation of the IP3 receptor (IP3R), the positive feedback from Ca2+ on phospholipase C (PLC) and the P2Y receptor phosphorylation by protein kinase C (PKC). Furthermore, PKC is shown to control multiple cellular substrates. Utilising the model, we suggest the activity and intensity of PLC and PKC necessary to explain the qualitatively diverse Ca2+ oscillations in response to P2Y receptor activation.
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Señalización del Calcio , Proteína Quinasa C , Receptores Purinérgicos P2Y/metabolismo , Fosfolipasas de Tipo C , Calcio/metabolismo , Hepatocitos , Humanos , Fosforilación , Transducción de Señal , Fosfolipasas de Tipo C/metabolismoRESUMEN
Cytosolic Ca2+ regulates multiple steps in the host-cell invasion, growth, proliferation, and egress of blood-stage Plasmodium falciparum, yet our understanding of Ca2+ signaling in this endemic malaria parasite is incomplete. By using a newly generated transgenic line of P. falciparum (PfGCaMP3) that expresses constitutively the genetically encoded Ca2+ indicator GCaMP3, we have investigated the dynamics of Ca2+ release and influx elicited by inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pumps, cyclopiazonic acid (CPA), and thapsigargin (Thg). Here we show that in isolated trophozoite phase parasites: (i) both CPA and Thg release Ca2+ from intracellular stores in P. falciparum parasites; (ii) Thg is able to induce Ca2+ release from an intracellular compartment insensitive to CPA; (iii) only Thg is able to activate Ca2+ influx from extracellular media, through a mechanism resembling store-operated Ca2+ entry, typical of mammalian cells; and (iv) the Thg-sensitive Ca2+ pool is unaffected by collapsing the mitochondria membrane potential with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone or the release of acidic Ca2+ stores with nigericin. These data suggest the presence of two Ca2+ pools in P. falciparum with differential sensitivity to the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pump inhibitors, and only the release of the Thg-sensitive Ca2+ store induces Ca2+ influx. Activation of the store-operated Ca2+ entry-like Ca2+ influx may be relevant for controlling processes such as parasite invasion, egress, and development mediated by kinases, phosphatases, and proteases that rely on Ca2+ levels for their activation.
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Calcio/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Animales Modificados Genéticamente , Señalización del Calcio , Humanos , Transporte Iónico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Calcium (Ca2+) oscillations in hepatocytes control many critical cellular functions, including glucose metabolism and bile secretion. The mechanisms underlying repetitive Ca2+ oscillations and how these mechanisms regulate these oscillations is not fully understood. Recent experimental evidence has shown that both Ca2+ regulation of the inositol 1,4,5-trisphosphate (IP3) receptor and IP3 metabolism generate Ca2+ oscillations and co-exist in hepatocytes. To investigate the effects of these feedback mechanisms on the Ca2+ response, we construct a mathematical model of the Ca2+ signalling network in hepatocytes. The model accounts for the biphasic regulation of Ca2+ on the IP3 receptor (IP3R) and the positive feedback from Ca2+ on IP3 metabolism, via activation of phospholipase C (PLC) by agonist and Ca2+. Model simulations show that Ca2+ oscillations exist for both constant [IP3] and for [IP3] changing dynamically. We show, both experimentally and in the model, that as agonist concentration increases, Ca2+ oscillations transition between simple narrow-spike oscillations and complex broad-spike oscillations. The model predicts that narrow-spike oscillations persist when Ca2+ transport across the plasma membrane is blocked. This prediction has been experimentally validated. In contrast, broad-spike oscillations are terminated when plasma membrane transport is blocked. We conclude that multiple feedback mechanisms participate in regulating Ca2+ oscillations in hepatocytes.
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Calcio , Inositol 1,4,5-Trifosfato , Calcio/metabolismo , Señalización del Calcio , Hepatocitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transducción de SeñalRESUMEN
Ca2+ oscillations that depend on inositol-1,4,5-trisphosphate (IP3) have been ascribed to biphasic Ca2+ regulation of the IP3 receptor (IP3R) or feedback mechanisms controlling IP3 levels in different cell types. IP3 uncaging in hepatocytes elicits Ca2+ transients that are often localized at the subcellular level and increase in magnitude with stimulus strength. However, this does not reproduce the broad baseline-separated global Ca2+ oscillations elicited by vasopressin. Addition of hormone to cells activated by IP3 uncaging initiates a qualitative transition from high-frequency spatially disorganized Ca2+ transients, to low-frequency, oscillatory Ca2+ waves that propagate throughout the cell. A mathematical model with dual coupled oscillators that integrates Ca2+-induced Ca2+ release at the IP3R and mutual feedback mechanisms of cross-coupling between Ca2+ and IP3 reproduces this behavior. Thus, multiple Ca2+ oscillation modes can coexist in the same cell, and hormonal stimulation can switch from the simpler to the more complex to yield robust signaling.
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Two closely related isolates, 27335T and 24999, of rapidly growing, non-pigmented mycobacteria, were cultured from two clinically ill fish of the family Syngnathidae. Whole genome sequencing of the two isolates revealed low sequence homology to documented mycobacteria within public databases such as the NCBI. Evaluation of targeted housekeeping genes, including 16S rRNA, ITS, rpoB and hsp65, related the two bacteria distantly to Mycobacterium senegalense CK2 M4421 and Mycobacterium farcinogenes DSM 43637. Phenotypic, biochemical and dDNA-DNA hybridization tests demonstrated that Mycobacterium syngnathidarum is a new species distinct from other recognized rapidly growing mycobacterial species. Phenotypic, chemotaxonomic and phylogenetic data evaluation provided evidence that the two strains represent one novel species. We propose the formal recognition of Mycobacterium syngnathidarum sp. nov., with isolate 27335T as the type strain (=ATCC TSD-89T,=DSM 105112T).
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Infecciones por Mycobacterium/veterinaria , Mycobacterium/clasificación , Filogenia , Smegmamorpha/microbiología , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Genes Bacterianos , Georgia , Mycobacterium/genética , Mycobacterium/aislamiento & purificación , Infecciones por Mycobacterium/microbiología , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , South CarolinaRESUMEN
BACKGROUND: Phosphoinositides (PIs) and their derivatives are essential cellular components that form the building blocks for cell membranes and regulate numerous cell functions. Specifically, the ability to generate myo-inositol 1,4,5-trisphosphate (InsP3) via phospholipase C (PLC) dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to InsP3 and diacylglycerol (DAG) initiates intracellular calcium signaling events representing a fundamental signaling mechanism dependent on PIs. InsP3 produced by PI turnover as a second messenger causes intracellular calcium release, especially from endoplasmic reticulum, by binding to the InsP3 receptor (InsP3R). Various PIs and the enzymes, such as phosphatidylinositol synthase and phosphatidylinositol 4-kinase, necessary for their turnover have been characterized in Apicomplexa, a large phylum of mostly commensal organisms that also includes several clinically relevant parasites. However, InsP3Rs have not been identified in genomes of apicomplexans, despite evidence that these parasites produce InsP3 that mediates intracellular Ca2+ signaling. CONCLUSION: Evidence to supporting IP3-dependent signaling cascades in apicomplexans suggests that they may harbor a primitive or non-canonical InsP3R. Understanding these pathways may be informative about early branching eukaryotes, where such signaling pathways also diverge from animal systems, thus identifying potential novel and essential targets for therapeutic intervention.
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Apicomplexa/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Transducción de Señal , Animales , Sistemas de Mensajero SecundarioRESUMEN
KEY POINTS: Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP3 ) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca2+ ]i ) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca2+ -mobilizing hormones resulting in more sustained and prolonged [Ca2+ ]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP3 ) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol-fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone-stimulated PLC activity, indicating calcium-dependent PLCs are not upregulated by alcohol. We propose that the liver 'adapts' to chronic alcohol exposure by increasing hormone-dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.
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Consumo de Bebidas Alcohólicas/metabolismo , Alcoholismo/metabolismo , Señalización del Calcio , Hepatocitos/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Calcio/metabolismo , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratas Sprague-Dawley , Vasopresinas/farmacologíaRESUMEN
How Ca(2+) oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca(2+) oscillations report signal strength via frequency, whereas Ca(2+) spike amplitude and wave velocity remain constant. IP3 uncaging also triggers oscillatory Ca(2+) release, but, in contrast to hormones, Ca(2+) spike amplitude, width, and wave velocity were dependent on [IP3] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP3 uncaging are driven by the biphasic regulation of the IP3 receptor by Ca(2+), and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca(2+) did not perturb Ca(2+) oscillations elicited by IP3 uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca(2+) influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca(2+) oscillations but had no effect on Ca(2+) increases induced by uncaging IP3. Importantly, PKC activation and inhibition differentially affected Ca(2+) spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP3 metabolism to attenuate IP3 levels and suppress the generation of Ca(2+) oscillations. Inhibition of PKC relieves negative feedback regulation of IP3 accumulation and, thereby, shifts Ca(2+) oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca(2+) wave velocity, whereas responses to IP3 uncaging are enhanced. The ability to assess Ca(2+) responses in the absence of PLC activity indicates that IP3 receptor modulation by PKC regulates Ca(2+) release and wave velocity.
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Señalización del Calcio , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Proteína Quinasa C/metabolismo , Animales , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Hepatocitos/metabolismo , Hormonas/química , Hormonas/metabolismo , Humanos , Inositol/química , Inositol/metabolismo , Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteína Quinasa C/química , Ratas , Transducción de SeñalRESUMEN
Receptor-mediated oscillations in cytosolic Ca(2+) concentration ([Ca(2+)]i) could originate either directly from an autonomous Ca(2+) feedback oscillator at the inositol 1,4,5-trisphosphate (IP3) receptor or as a secondary consequence of IP3 oscillations driven by Ca(2+) feedback on IP3 metabolism. It is challenging to discriminate these alternatives, because IP3 fluctuations could drive Ca(2+) oscillations or could just be a secondary response to the [Ca(2+)]i spikes. To investigate this problem, we constructed a recombinant IP3 buffer using type-I IP3 receptor ligand-binding domain fused to GFP (GFP-LBD), which buffers IP3 in the physiological range. This IP3 buffer slows hormone-induced [IP3] dynamics without changing steady-state [IP3]. GFP-LBD perturbed [Ca(2+)]i oscillations in a dose-dependent manner: it decreased both the rate of [Ca(2+)]i rise and the speed of Ca(2+) wave propagation and, at high levels, abolished [Ca(2+)]i oscillations completely. These data, together with computational modeling, demonstrate that IP3 dynamics play a fundamental role in generating [Ca(2+)]i oscillations and waves.
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Señalización del Calcio/efectos de los fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Vasopresinas/farmacología , Animales , Células COS , Calcio/farmacología , Chlorocebus aethiops , Simulación por Computador , Proteínas Fluorescentes Verdes/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ligandos , Modelos Biológicos , Estructura Terciaria de Proteína , RatasRESUMEN
A major role of the liver is to integrate multiple signals to maintain normal blood glucose levels. The balance between glucose storage and mobilization is primarily regulated by the counteracting effects of insulin and glucagon. However, numerous signals converge in the liver to ensure energy demand matches the physiological status of the organism. Many circulating hormones regulate glycogenolysis, gluconeogenesis and mitochondrial metabolism by calcium-dependent signaling mechanisms that manifest as cytosolic Ca(2+) oscillations. Stimulus-strength is encoded in the Ca(2+) oscillation frequency, and also by the range of intercellular Ca(2+) wave propagation in the intact liver. In this article, we describe how Ca(2+) oscillations and waves can regulate glucose output and oxidative metabolism in the intact liver; how multiple stimuli are decoded though Ca(2+) signaling at the organ level, and the implications of Ca(2+) signal dysregulation in diseases such as metabolic syndrome and non-alcoholic fatty liver disease.
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Calcio/metabolismo , Glucosa/metabolismo , Hígado/metabolismo , Animales , Señalización del Calcio , Hígado Graso/metabolismo , Hígado Graso/patología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/patología , Mitocondrias/metabolismoRESUMEN
Acidocalcisomes are acidic calcium stores rich in polyphosphate and found in a diverse range of organisms. The mechanism of Ca(2+) release from these organelles was unknown. Here we present evidence that Trypanosoma brucei acidocalcisomes possess an inositol 1,4,5-trisphosphate receptor (TbIP(3)R) for Ca(2+) release. Localization studies in cell lines expressing TbIP(3)R in its endogenous locus fused to an epitope tag revealed its partial colocalization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes. IP(3) was able to stimulate Ca(2+) release from a chicken B-lymphocyte cell line in which the genes for all three vertebrate IP(3)Rs have been stably ablated (DT40-3KO) and that were stably expressing TbIP(3)R, providing evidence of its function. IP(3) was also able to release Ca(2+) from permeabilized trypanosomes or isolated acidocalcisomes and photolytic release of IP(3) in intact trypanosomes loaded with Fluo-4 elicited a transient Ca(2+) increase in their cytosol. Ablation of TbIP(3)R by RNA interference caused a significant reduction of IP(3)-mediated Ca(2+) release in trypanosomes and resulted in defects in growth in culture and infectivity in mice. Taken together, the data provide evidence of the presence of a functional IP(3)R as a Ca(2+) release channel in acidocalcisomes of trypanosomes and suggest that a Ca(2+) signaling pathway that involves acidocalcisomes is required for growth and establishment of infection.
Asunto(s)
Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Orgánulos/metabolismo , Trypanosoma brucei brucei/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Mutación , Orgánulos/efectos de los fármacos , Interferencia de ARN , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis/parasitología , Tripanosomiasis/terapiaRESUMEN
Circulating hormones stimulate the phospholipase Cß (PLC)/Ca(2+) influx pathway to regulate numerous cell functions, including vascular tone. It was proposed previously that Ca(2+)-independent phospholipase A(2) (iPLA(2))-dependent store-operated Ca(2+) influx channels mediate hormone-induced contractions in isolated arteries, because bromoenol lactone (BEL), a potent irreversible inhibitor of iPLA(2), inhibited such contractions. However, the effects of BEL on other channels implicated in mediating hormone-induced vessel contractions, specifically voltage-gated Ca(2+) (Ca(V)1.2) and transient receptor potential canonical (TRPC) channels, have not been defined clearly. Using isometric tension measurements, we found that thapsigargin-induced contractions were â¼34% of those evoked by phenylephrine or KCl. BEL completely inhibited not only thapsigargin- but also phenylephrine- and KCl-induced ring contractions, suggesting that Ca(V)1.2 and receptor-operated TRPC channels also may be sensitive to BEL. Therefore, we investigated the effects of BEL on heterologously expressed Ca(V)1.2 and TRPC channels in human embryonic kidney cells, a model system that allows probing of individual protein function without interference from other signaling elements of native cells. We found that low micromolar concentrations of BEL inhibited Ca(V)1.2, TRPC5, TRPC6, and heteromeric TRPC1-TRPC5 channels in an iPLA(2)-independent manner. BEL also attenuated PLC activity, suggesting that the compound may inhibit TRPC channel activity in part by interfering with an initial PLC-dependent step required for TRPC channel activation. Conversely, BEL did not affect endogenous voltage-gated K(+) channels in human embryonic kidney cells. Our findings support the hypothesis that iPLA(2)-dependent store-operated Ca(2+) influx channels and iPLA(2)-independent hormone-operated TRPC channels can serve as smooth muscle depolarization triggers to activate Ca(V)1.2 channels and to regulate vascular tone.
Asunto(s)
Canales de Calcio/metabolismo , Naftalenos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Pironas/farmacología , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Conducta Adictiva/fisiopatología , Carbazoles/farmacología , Cocaína/efectos adversos , Cocaína/farmacología , Condicionamiento Operante/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Inhibidores de Captación de Dopamina/efectos adversos , Inhibidores de Captación de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Fosfolipasas/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores sigma/análisis , Receptores sigma/antagonistas & inhibidores , Receptores sigma/efectos de los fármacos , Autoadministración , Porcinos , Tapsigargina/farmacologíaRESUMEN
IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.