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Colorectal cancer (CRC) is the third most common type of cancer worldwide. Its treatment options have had a limited impact on cancer remission prognosis. Therefore, there is an ongoing need to discover novel anti-cancer agents. Medicinal plants have gained recognition as a source of anti-cancer bioactive compounds. Recently, ethanolic extract of L. virginicum stems ameliorated dinitrobenzene sulfonic acid (DNBS)-induced colitis by modulating the intestinal immune response. However, no scientific study has demonstrated this potential cytotoxic impact on colon cancer cells. The objective of this study was to evaluate the cytotoxic effect of the methanolic extract of L. virginicum (ELv) on a human colorectal adenocarcinoma cell line (Caco-2) and to identify and quantify the phenolic compounds present in ELv extracts by liquid chromatography-mass spectrometry analysis. The cytotoxic activity was assessed using cell viability assays by reduction in the compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH). MTT and LDH assays revealed that the ELv decreases cell viability in the Caco-2 cell line in a concentration-dependent manner. Cell death was a result of DNA fragmentation and p53-mediated apoptosis. Eight phenolic acids and five flavonoids were identified and quantified in the stems. In conclusion, our findings demonstrate that the extract of L. virginicum possesses cytotoxic properties on Caco-2 cell line, suggesting that it could be a potential source of new drugs against CRC.
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Apoptosis , Supervivencia Celular , Lepidium , Metanol , Extractos Vegetales , Proteína p53 Supresora de Tumor , Humanos , Células CACO-2 , Extractos Vegetales/farmacología , Extractos Vegetales/química , Apoptosis/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Supervivencia Celular/efectos de los fármacos , Metanol/química , Lepidium/química , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/química , Fenoles/farmacología , Fenoles/químicaRESUMEN
Intestinal low-grade inflammation induced by a high-fat diet has been found to detonate chronic systemic inflammation, which is a hallmark of obesity, and precede the apparition of insulin resistance, a key factor for developing type 2 diabetes (T2D). Aberrant purinergic signaling pathways have been implicated in the pathogenesis of inflammatory bowel disease and other gastrointestinal diseases. However, their role in the gut inflammation associated with obesity and T2D remains unexplored. C57BL/6 J mice were fed a cafeteria diet for 21 weeks and received one injection of streptozotocin in their sixth week into the diet. The gene expression profile of purinergic signaling components in colon tissue was assessed by RT-qPCR. Compared to control mice, the treated group had a significant reduction in colonic length and mucosal and muscular layer thickness accompanied by increased NF-κB and IL-1ß mRNA expression. Furthermore, colonic P2X2, P2X7, and A3R gene expression levels were lower, while the P2Y2, NT5E, and ADA expression levels increased. In conclusion, these data suggest that these purinergic signaling components possibly play a role in intestinal low-grade inflammation associated with obesity and T2D and thus could represent a novel therapeutic target for the treatment of the metabolic complications related to these diseases.
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Previous studies have demonstrated that acute colonic inflammation leads to an increase in dorsal root ganglia (DRG) neuronal excitability. However, the signaling elements implicated in this hyperexcitability have yet to be fully unraveled. Extracellular adenosine 5'-triphosphate (ATP) is a well-recognized sensory signaling molecule that enhances the nociceptive response after inflammation through activation of P2X3 receptors, which are expressed mainly by peripheral sensory neurons. The aim of this study is to continue investigating how P2X3 affects neuronal hypersensitivity in an acute colitis animal model. To achieve this, DNBS (Dinitrobenzene sulfonic acid; 200 mg/kg) was intrarectally administered to C57BL/6 mice, and inflammation severity was assessed according to the following parameters: weight loss, macroscopic and microscopic scores. Perforated patch clamp technique was used to evaluate neuronal excitability via measuring changes in rheobase and action potential firing in T8-L1 DRG neurons. A-317491, a well-established potent and selective P2X3 receptor antagonist, served to dissect their contribution to recorded responses. Protein expression of P2X3 receptors in DRG was evaluated by western blotting and immunofluorescence. Four days post-DNBS administration, colons were processed for histological analyses of ulceration, crypt morphology, goblet cell density, and immune cell infiltration. DRG neurons from DNBS-treated mice were significantly more excitable compared with controls; these changes correlated with increased P2X3 receptor expression. Furthermore, TNF-α mRNA expression was also significantly higher in inflamed colons compared to controls. Incubation of control DRG neurons with TNF-α resulted in similar cell hyperexcitability as measured in DNBS-derived neurons. The selective P2X3 receptor antagonist, A-317491, blocked the TNF-α-induced effect. These results support the hypothesis that TNF-α enhances colon-innervating DRG neuron excitability via modulation of P2X3 receptor activity.
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Colitis , Ganglios Espinales , Adenosina Trifosfato , Animales , Inflamación , Ratones , Ratones Endogámicos C57BL , Antagonistas del Receptor Purinérgico P2X , Receptores Purinérgicos P2X3 , Células Receptoras Sensoriales , Factor de Necrosis Tumoral alfaRESUMEN
Background: Glucagon-like peptide-1 receptor (GLP-1R) agonists are powerful glycemia-lowering agents, which have systematically been shown to lower cardiovascular events and mortality. These beneficial effects were difficult to pinpoint within atherosclerotic plaque due to lack of particular specificity of such agonists to the vascular cells and an inadequate understanding of the GLP-1R expression in atherosclerosis. Here, we hypothesized that the direct engagement of the GLP-1R in atherosclerosis by targeted agonists will alleviate vascular inflammation and plaque burden, even at a very low dose. Methods: The expression of GLP-1 receptor (GLP-1R, Glp1r mRNA) in human lesions with pathologic intimal thickening, Apoe-/- mouse atheroma and cultured immune/non-immune cells was investigated using genetic lineage tracing, Southern blotting and validated antisera against human GLP-1R. Protease-resistant and "activatable" nanoparticles (NPs) carrying GLP-1R agonist liraglutide (GlpNP) were engineered and synthesized. Inclusion of gadolinium chelates into GlpNP allowed for imaging by MRI. Atherosclerotic Apoe-/- mice were treated intravenously with a single dose (30 µg/kg of liraglutide) or chronically (1 µg/kg, 6 weeks, 2x/week) with GlpNP, liraglutide or control NPs, followed by assessment of metabolic parameters, atheroma burden, inflammation and vascular function. Results: Humal plaque specimens expressed high levels of GLP-1R within the locus of de-differentiated smooth muscle cells that also expressed myeloid marker CD68. However, innate immune cells under a variety of conditions expressed very low levels of Glp1r, as seen in lineage tracing and Southern blotting experiments examining full-length open reading frame mRNA transcripts. Importantly, de-differentiated vascular smooth muscle cells demonstrated significant Glp1r expression levels, suggesting that these could represent the cells with predominant Glp1r-positivity in atherosclerosis. GlpNP resisted proteolysis and demonstrated biological activity including in vivo glycemia lowering at 30 µg/kg and in vitro cholesterol efflux. Activatable properties of GlpNP were confirmed in vitro by imaging cytometry and in vivo using whole organ imaging. GlpNP targeted CD11b+/CD11c+ cells in circulation and smooth muscle cells in aortic plaque in Apoe-/- mice when assessed by MRI and fluorescence imaging. At a very low dose of 1 µg/kg, previously known to have little effect on glycemia and weight loss, GlpNP delivered i.v. for six weeks reduced triglyceride-rich lipoproteins in plasma, plaque burden and plaque cholesterol without significant effects on weight, glycemia and plasma cholesterol levels. Conclusions: GlpNP improves atherosclerosis at weight-neutral doses as low as 1 µg/kg with the effects independent from the pancreas or the central nervous system. Our study underlines the importance of direct actions of GLP-1 analogs on atherosclerosis, involving cholesterol efflux and inflammation. Our findings are the first to suggest the therapeutic modulation of vascular targets by GlpNP, especially in the context of smooth muscle cell inflammation.
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Aterosclerosis , Receptor del Péptido 1 Similar al Glucagón , Placa Aterosclerótica , Animales , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/tratamiento farmacológico , Diferenciación Celular , Receptor del Péptido 1 Similar al Glucagón/agonistas , Inflamación/metabolismo , Liraglutida/farmacología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Músculo Liso/metabolismo , Placa Aterosclerótica/diagnóstico por imagen , Placa Aterosclerótica/tratamiento farmacológico , Proteolisis , ARN Mensajero/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Lepidium virginicum L. (Brassicaceae) is a plant widely used in traditional Mexican medicine as an expectorant, diuretic, and as a remedy to treat diarrhea and dysentery, infection-derived gastroenteritis. However, there is no scientific study that validates its clinical use as an anti-inflammatory in the intestine. AIM OF THE STUDY: This study aimed to investigate the anti-inflammatory properties of the ethanolic extract of Lepidium virginicum L. (ELv) in an animal model of inflammatory bowel disease (IBD)-like colitis. MATERIALS AND METHODS: The 2,4-dinitrobenzene sulfonic acid (DNBS) animal model of IBD was used. Colitis was induced by intrarectal instillation of 200 mg/kg of DNBS dissolved vehicle, 50% ethanol. Control rats only received the vehicle. Six hours posterior to DNBS administration, ELv (3, 30, or 100 mg/kg) was administered daily by gavage or intraperitoneal injection. The onset and course of the inflammatory response were monitored by assessing weight loss, stool consistency, and fecal blood. Colonic damage was evaluated by colon weight/length ratio, histopathology, colonic myeloperoxidase (MPO) activity, and gene expression of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), chemokine C-X-C motif ligand 1 (CXCL-1), and interleukin-6 (IL-6). RESULTS: Rats treated with DNBS displayed significant weight loss, diarrhea, fecal blood, colon shortening, a significant increase in immune cell infiltration and MPO activity, as well as increased proinflammatory cytokine expression. Intraperitoneal administration of ELv significantly reduced colon inflammation, whereas oral treatment proved to be ineffective. In fact, intraperitoneal ELv significantly attenuated the clinical manifestations of colitis, immune cell infiltration, MPO activity, and pro-inflammatory (CXCL-1, TNF-α, and IL-1ß) gene expression in a dose-dependent manner. CONCLUSION: Traditional medicine has employed ELv as a remedy for common infection-derived gastrointestinal symptoms; however, we hereby present the first published study validating its anti-inflammatory properties in the mitigation of DNBS-induced colitis.
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Antiinflamatorios/farmacología , Colitis/tratamiento farmacológico , Lepidium/química , Extractos Vegetales/farmacología , Animales , Antiinflamatorios/aislamiento & purificación , Colitis/genética , Colitis/fisiopatología , Dinitrofluorobenceno/análogos & derivados , Relación Dosis-Respuesta a Droga , Etanol/química , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/fisiopatología , Medicina Tradicional , Extractos Vegetales/administración & dosificación , Ratas , Ratas WistarRESUMEN
This study sought to examine the co-expression of the following purinergic receptor subunits: P2X1, P2X1del, P2X4, and P2X7 and characterize the P2X response in human monocyte-derived macrophages (MDMs). Single-cell RT-PCR shows the presence of P2X1, P2X1del, P2X4, and P2X7 mRNA in 40%, 5%, 20%, and 90% of human MDMs, respectively. Of the studied human MDMs, 25% co-expressed P2X1 and P2X7 mRNA; 5% co-expressed P2X4 and P2X7; and 15% co-expressed P2X1, P2X4, and P2X7 mRNA. In whole-cell patch clamp recordings of human MDMs, rapid application of ATP (0.01 mM) evoked fast current activation and two different desensitization kinetics: 1. a rapid desensitizing current antagonized by PPADS (1 µM), reminiscent of the P2X1 receptor's current; 2. a slow desensitizing current, insensitive to PPADS but potentiated by ivermectin (3 µM), similar to the P2X4 receptor's current. Application of 5 mM ATP induced three current modalities: 1. slow current activation with no desensitization, similar to the P2X7 receptor current, present in 69% of human macrophages and antagonized by A-804598 (0.1 µM); 2. fast current activation and fast desensitization, present in 15% of human MDMs; 3. fast activation current followed by biphasic desensitization, observed in 15% of human MDMs. Both rapid and biphasic desensitization kinetics resemble those observed for the recombinant human P2X1 receptor expressed in oocytes. These data demonstrate, for the first time, the co-expression of P2X1, P2X4, and P2X7 transcripts and confirm the presence of functional P2X1, P2X4, and P2X7 receptors in human macrophages.
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Macrófagos/metabolismo , Receptores Purinérgicos P2X1/biosíntesis , Receptores Purinérgicos P2X4/biosíntesis , Receptores Purinérgicos P2X7/biosíntesis , Adenosina Trifosfato/farmacología , Animales , Células Cultivadas , Femenino , Expresión Génica , Humanos , Macrófagos/efectos de los fármacos , Agonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X1/genética , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X7/genética , Xenopus laevisRESUMEN
P2X7 receptor/channels in the retinal microvasculature not only regulate vasomotor activity, but can also trigger cells in the capillaries to die. While it is known that this purinergic vasotoxicity is dependent on the transmembrane pores that form during P2X7 activation, events linking pore formation with cell death remain uncertain. To better understand this pathophysiological process, we used YO-PRO-1 uptake, dichlorofluorescein fluorescence, perforated-patch recordings, fura-2 imaging and trypan blue dye exclusion to assess the effects of the P2X7 agonist, benzoylbenzoyl-ATP (BzATP), on pore formation, oxidant production, ion channel activation, [Ca2+]i and cell viability. Experiments demonstrated that exposure of retinal microvessels to BzATP increases capillary cell oxidants via a mechanism dependent on pore formation and the enzyme, NADPH oxidase. Indicative that oxidation plays a key role in purinergic vasotoxicity, an inhibitor of this enzyme completely prevented BzATP-induced death. We further discovered that vasotoxicity was boosted 4-fold by a pathway involving the oxidation-driven activation of hyperpolarizing KATP channels and the resulting increase in calcium influx. Our findings revealed that the previously unappreciated pore/oxidant/KATP channel/Ca2+ pathway accounts for 75% of the capillary cell death triggered by sustained activation of P2X7 receptor/channels. Elucidation of this pathway is of potential therapeutic importance since purinergic vasotoxicity may play a role in sight-threatening disorders such as diabetic retinopathy.
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We, hereby, characterize the pharmacological effects of physiological concentrations of Zinc on native myenteric P2X receptors from guinea-pig small intestine and on P2X2 isoforms present in most myenteric neurons. This is the first study describing opposite effects of Zinc on these P2X receptors. It was not possible to determine whether both effects were concentration dependent, yet the inhibitory effect was mediated by competitive antagonism and was concentration dependent. The potentiating effect appears to be mediated by allosteric changes induced by Zinc on P2X myenteric channels, which is more frequently observed in myenteric neurons with low zinc concentrations. In P2X2-1 and P2X2-2 variants, the inhibitory effect is more common than in P2X myenteric channels. However, in the variants, the potentiatory effect is of equal magnitude as the inhibitory effect. Inhibitory and potentiatory effects are likely mediated by different binding sites that appear to be present on both P2X2 variants. In conclusion, in myenteric native P2X receptors, Zinc has quantitatively different pharmacological effects compared to those observed on homomeric channels: P2X2-1 and P2X2-2. Potentiatory and inhibitory Zinc effects upon these receptors are mediated by two different binding sites. All our data suggest that myenteric P2X receptors have a more complex pharmacology than those of the recombinant P2X2 receptors, which is likely related to other subunits known to be expressed in myenteric neurons. Because these dual effects occur at Zinc physiological concentrations, we suggest that they could be involved in physiological and pathological processes.
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Plexo Mientérico/efectos de los fármacos , Receptores Purinérgicos P2X2/metabolismo , Zinc/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Cobayas , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Masculino , Plexo Mientérico/metabolismo , Cultivo Primario de Células , XenopusRESUMEN
Here we describe a culture technique of cells dissociated from the external muscularis of the guinea pig small intestine, which allows us to maintain all the elements involved in the intestinal peristaltic reflex. After a few days in culture, these cells reorganize to form a small group of cells that permit the generation of pacemaker activity, spontaneous contractions, and the development of inhibitory and excitatory junction potentials in the petri dish, all elements involved in the peristaltic reflex. Therefore, these co-cultures are suitable to study the cellular and molecular aspects related to the development, maintenance, and modulation of motor intestinal functions.
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Técnicas de Cocultivo/métodos , Intestino Delgado/fisiología , Neuronas Motoras/citología , Miocitos del Músculo Liso/citología , Potenciales de Acción , Animales , Femenino , Cobayas , Intestino Delgado/citología , Masculino , Ratones Endogámicos C57BL , Contracción Muscular , Miocitos del Músculo Liso/fisiología , Peristaltismo , RatasRESUMEN
Over-activation of p38 is implicated in many cardiovascular diseases (CVDs), including myocardial infarction, hypertrophy, heart failure, and ischemic heart disease. Numerous therapeutic interventions for CVDs have been directed toward the inhibition of the p38 mitogen-activated protein kinase activation that contributes to the detrimental effect after ischemia/reperfusion (I/R) injuries. However, the efficacy of these treatments is far from ideal, as they lack specificity and are associated with high toxicity. Previously, we demonstrated that N-acetyl cysteine (NAC) pretreatment up-regulates DUSP4 expression in endothelial cells, regulating p38 and ERK1/2 activities, and thus providing a protective effect against oxidative stress. Here, endothelial cells under hypoxia/reoxygenation (H/R) insult and isolated heart I/R injury were used to investigate the role of DUSP4 in the modulation of the p38 pathway. In rat endothelial cells, DUSP4 is time-dependently degraded by H/R (0.25 ± 0.07-fold change of control after 2h H/R). Its degradation is closely associated with hyperphosphorylation of p38 (2.1 ± 0.36-fold change) and cell apoptosis, as indicated by the increase in cells immunopositive for cleaved caspase-3 (12.59 ± 3.38%) or TUNEL labeling (29.46 ± 3.75%). The inhibition of p38 kinase activity with 20 µM SB203580 during H/R prevents H/R-induced apoptosis, assessed via TUNEL (12.99 ± 1.89%). Conversely, DUSP4 gene silencing in endothelial cells augments their sensitivity to H/R-induced apoptosis (45.81 ± 5.23%). This sensitivity is diminished via the inhibition of p38 activity (total apoptotic cells drop to 17.47 ± 1.45%). Interestingly, DUSP4 gene silencing contributes to the increase in superoxide generation from cells. Isolated Langendorff-perfused mouse hearts were subjected to global I/R injury. DUSP4(-/-) hearts had significantly larger infarct size than WT. The increase in I/R-induced infarct in DUSP4(-/-) mice significantly correlates with reduced functional recovery (assessed by RPP%, LVDP%, HR%, and dP/dtmax) as well as lower CF% and a higher initial LVEDP. From immunoblotting analysis, it is evident that p38 is significantly overactivated in DUSP4(-/-) mice after I/R injury. The activation of cleaved caspase-3 is seen in both WT and DUSP4(-/-) I/R hearts. Infusion of a p38 inhibitor prior to ischemia and during the reperfusion improves both WT and DUSP4(-/-) cardiac function. Therefore, the identification of p38 kinase modulation by DUSP4 provides a novel therapeutic target for oxidant-induced diseases, especially myocardial infarction.
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Aorta/fisiología , Endotelio Vascular/fisiología , Corazón/fisiopatología , Isquemia Miocárdica/fisiopatología , Estrés Oxidativo , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Aorta/citología , Apoptosis , Western Blotting , Proliferación Celular , Células Cultivadas , Endotelio Vascular/citología , Regulación de la Expresión Génica , Técnicas para Inmunoenzimas , Ratones , Ratones Noqueados , Daño por Reperfusión Miocárdica/fisiopatología , Fosforilación , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Redox imbalance is a primary cause of endothelial dysfunction (ED). Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source in cells, can significantly regulate cell function via reversible protein thiol modification. N-acetylcysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. From HPLC analysis, NAC significantly increases both cellular GSH and tetrahydrobiopterin levels. Immunoblotting of endothelial NO synthase (eNOS) and DUSP4, a dual-specificity phosphatase with a cysteine as its active residue, revealed that both enzymes are upregulated by NAC. EPR spin trapping further demonstrated that NAC enhances NO generation from cells. Long-term exposure to Cd(2+) contributes to DUSP4 degradation and the uncontrolled activation of p38 and ERK1/2, leading to apoptosis. Treatment with NAC prevents DUSP4 degradation and protects cells against Cd(2+)-induced apoptosis. Moreover, the increased DUSP4 expression can redox-regulate the p38 and ERK1/2 pathways from hyperactivation, providing a survival mechanism against the toxicity of Cd(2+). DUSP4 gene knockdown further supports the hypothesis that DUSP4 is an antioxidant gene, critical in the modulation of eNOS expression, and thus protects against Cd(2+)-induced stress. Depletion of intracellular GSH by buthionine sulfoximine makes cells more susceptible to Cd(2+)-induced apoptosis. Pretreatment with NAC prevents p38 overactivation and thus protects the endothelium from this oxidative stress. Therefore, the identification of DUSP4 activation by NAC provides a novel target for future drug design.
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Acetilcisteína/farmacología , Fosfatasas de Especificidad Dual/metabolismo , Células Endoteliales/enzimología , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Estrés Oxidativo/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Cadmio/toxicidad , Bovinos , Procesos de Crecimiento Celular , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Citoprotección/efectos de los fármacos , Fosfatasas de Especificidad Dual/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Glutatión/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , ARN Interferente Pequeño/genética , Ratas , Regulación hacia ArribaRESUMEN
CONTEXT: High-density lipoprotein (HDL) particles perform numerous vascular-protective functions. Animal studies demonstrate that exposure to fine or ultrafine particulate matter (PM) can promote HDL dysfunction. However, the impact of PM on humans remains unknown. OBJECTIVE: We aimed to determine the effect of exposure to coarse concentrated ambient particles (CAP) on several metrics of HDL function in healthy humans. METHODS: Thirty-two adults (25.9 ± 6.6 years) were exposed to coarse CAP [76.2 ± 51.5 µg·m(-3)] in a rural location and filtered air (FA) for 2 h in a randomized double-blind crossover study. Venous blood collected 2- and 20-h post-exposures was measured for HDL-mediated efflux of [(3)H]-cholesterol from cells and 20-h exposures for HDL anti-oxidant capacity by a fluorescent assay and paraoxonase activity. The changes [median (first, third quartiles)] between exposures among 29 subjects with available results were compared by matched Wilcoxon tests. RESULTS: HDL-mediated cholesterol efflux capacity did not differ between exposures at either time point [16.60% (15.17, 19.19) 2-h post-CAP versus 17.56% (13.43, 20.98) post-FA, p = 0.768 and 14.90% (12.47, 19.15) 20-h post-CAP versus 17.75% (13.22, 23.95) post-FA, p = 0.216]. HOI [0.26 (0.24, 0.35) versus 0.28 (0.25, 0.40), p = 0.198] and paraoxonase activity [0.54 (0.39, 0.82) versus 0.60 µmol·min(-1 )ml plasma(-1) (0.40, 0.85), p = 0.137] did not differ 20-h post-CAP versus FA, respectively. CONCLUSIONS: Brief inhalation of coarse PM from a rural location did not acutely impair several facets of HDL functionality. Whether coarse PM derived from urban sites, fine particles or longer term PM exposures can promote HDL dysfunction warrant future investigations.
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Contaminantes Atmosféricos/toxicidad , Lipoproteínas HDL/sangre , Material Particulado/toxicidad , Adolescente , Adulto , Contaminación del Aire/efectos adversos , Animales , Arildialquilfosfatasa/sangre , Línea Celular Tumoral , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Tamaño de la Partícula , Población Rural , Adulto JovenRESUMEN
Leukotrienes have been implicated in the pathogenesis of degenerative diabetic retinopathy, with research focusing primarily on leukotriene B(4), with little attention devoted to the cysteinyl leukotrienes (cysLTs), which act through cysLT receptors (CysLT(1)R and CysLT(2)R). We demonstrate here the presence of CysLT(2)R in pericytes and endothelial cells of superficial retinal vasculature using an indirect assay by assessment of ß-galactosidase expression in CysLT(2)R-knockout (KO) mice. Retinal damage was induced in KO and wild-type (WT) mice using an established oxygen-induced retinopathy (OIR) model. CysLT(2)R expression following OIR was intensely up-regulated compared to sham-treated controls. Staining with Griffonia simplicifolia lectin revealed enhanced tissue damage (as assessed by vasoobliteration/vasoproliferation) in KO mice compared to WT controls, yet the opposite was true with respect to retinal edema. However, vascular endothelial growth factor receptor 1 (VEGFR1) transcripts were increased by OIR similarly with respect to genotype. Intravitreal application of exogenous cysLTs elicited greater vasculature leakage (assessed ex vivo) in eyes from WT mice compared to KO mice. While mRNA encoding enzymes for various components of the leukotriene cascade were detected in sham- and OIR-treated retinas, only prostaglandins and hydroxyeicosatetraenoic acids, but not leukotrienes, were detected in A23187-treated retina preparations. Together, these results implicate the CysLT(2)R in the progression of ischemic retinopathy.
Asunto(s)
Modelos Animales de Enfermedad , Papiledema/genética , Receptores de Leucotrienos/genética , Enfermedades de la Retina/genética , Neovascularización Retiniana/genética , Albúminas/metabolismo , Animales , Calcimicina/farmacología , Ionóforos de Calcio/farmacología , Permeabilidad Capilar/efectos de los fármacos , Cisteína/farmacología , Endotelio Vascular/metabolismo , Expresión Génica , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inmunohistoquímica , Leucotrienos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oxígeno , Papiledema/metabolismo , Pericitos/metabolismo , Prostaglandinas/metabolismo , Receptores de Leucotrienos/deficiencia , Retina/efectos de los fármacos , Retina/metabolismo , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/metabolismo , Neovascularización Retiniana/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed ß-galactosidase-ß-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be â¼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 µM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Ácidos Ciclohexanocarboxílicos/farmacología , Antagonistas de Leucotrieno/farmacología , Leucotrieno D4/antagonistas & inhibidores , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Ácidos Ftálicos/farmacología , Receptores de Leucotrienos/metabolismo , SRS-A/análogos & derivados , Animales , Arrestinas/análisis , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Oído/irrigación sanguínea , Humanos , Ratones , Ratones Transgénicos , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Peroxidasa/metabolismo , SRS-A/farmacología , beta-Arrestinas , beta-Galactosidasa/metabolismoRESUMEN
Cysteinyl leukotrienes (cysLTs: LTC4, LTD4, and LTE4) are pro-inflammatory lipid molecules synthesized from arachidonic acid. They exert their actions on at least two cysLT receptors (CysLT1R and CysLT2R). Endothelial expression and activation of these receptors is linked to vasoactive responses and to the promotion of vascular permeability. Here we track the expression pattern of CysLT2R in a loss-of-function murine model (CysLT2R-LacZ) to neurons of the myenteric and submucosal plexus in the small intestine, colonic myenteric plexus, dorsal root ganglia, and nodose ganglion. Cysteinyl leukotriene (LTC4/D4) stimulation of colonic submucosal venules elicited a greater permeability response in wild-type mice. In a dextran sulfate sodium-induced colon inflammation model, the disease activity index and colonic edema (measured by wet:dry weights and submucosal thickness) were significantly reduced in knockout (KO) mice compared to controls. Tumor necrosis factor-α levels in colon tissue were significantly lower in KO mice; however, myeloperoxidase activity was similar in both the KO and wild-type groups. Finally, patch-clamp recordings of basal neuronal activity of colonic-projecting nociceptive neurons from dorsal root ganglia (T9-13) revealed significantly higher excitability in KO neurons compared to wild type. These results suggest that a lack of neuronal expression of CysLT2R in the murine colonic myenteric plexus attenuates colitis disease progression via a reduction in inflammation-associated tissue edema and increases neuronal sensitivity to nociceptive stimuli.
Asunto(s)
Tracto Gastrointestinal/metabolismo , Receptores de Leucotrienos/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Permeabilidad Capilar/efectos de los fármacos , Colitis/complicaciones , Colitis/metabolismo , Colitis/patología , Colon/efectos de los fármacos , Colon/inervación , Colon/patología , Colon/fisiopatología , Cisteína/farmacología , Sulfato de Dextran , Edema/complicaciones , Edema/patología , Edema/fisiopatología , Extravasación de Materiales Terapéuticos y Diagnósticos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Ganglios Espinales/fisiopatología , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/fisiopatología , Mucosa Intestinal/irrigación sanguínea , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Leucotrienos/farmacología , Ratones , Ratones Noqueados , Receptores de Leucotrienos/deficiencia , Albúmina Sérica/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Vénulas/efectos de los fármacos , Vénulas/metabolismo , Vénulas/patología , Vénulas/fisiopatologíaRESUMEN
BACKGROUND: Ischemia and ischemia-reperfusion (I/R) are common clinical insults that disrupt the molecular structure of coronary vascular endothelial luminal membrane (VELM) that result in diverse microvasculature dysfunctions. However, the knowledge of the associated biochemical changes is meager. We hypothesized that ischemia and I/R-induced structural and functional VELM alterations result from biochemical changes. First, these changes need to be described and later the mechanisms behind be identified. METHODS: During control conditions, in isolated perfused rat hearts VELM proteins were labeled with biotin. The groups of hearts were: control (C), no flow ischemia (I; 25 min), and I/R (I; 25 min, reperfusion 30 min). The biotinylated luminal endothelial membrane proteins in these three different groups were examined by 2-D electrophoresis and identified. But, it must be kept in mind the proteins were biotin-labeled during control. RESULTS: A comparative analysis of the protein profiles under the 3 conditions following 2D gel electrophoresis showed differences in the molecular weight distribution such that MW(C) > MW(I) > MW(I/R). Similar analysis for isoelectric points (pH(i)) showed a shift toward more acidic pHi under ischemic conditions. Of 100 % proteins identified during control 66% and 88% changed their MW-pH(i) during ischemia and I/R respectively. Among these lost proteins there were 9 proteins identified as adhesins and G-protein coupled receptors. GENERAL SIGNIFICANCE: I and I/R insults alter MW-pH(i) of most luminal glycocalyx proteins due to the activation of nonspecific hydrolizing mechanisms; suspect metalloproteases and glycanases. This makes necessary the identification of hydrolyzing enzymes reponsible of multiple microvascular dysfunctions in order to maintain the integrity of vascular endothelial membrane. VELM must become a target of future therapeutics.
RESUMEN
Coronary blood flow applied to the endothelial lumen modulates parenchymal functions via paracrine effectors, but the mechanism of flow sensation is unknown. We and others have demonstrated that coronary endothelial luminal membrane (CELM) oligosaccharides and lectins are involved in flow detection, and we proposed that cardiac effects of coronary flow result from a reversible flow-modulated lectin-oligosaccharide interaction. Recently, glycosylated and amiloride-sensitive Na(+)/Ca(++) channels (ENaCs) have been proposed to be involved in the flow-induced endothelial responses. Because N-acetylglucosamine (GlcNac) is one of the main components of glycocalyx oligosaccharides (i.e., hyaluronan [-4GlcUAbeta1-3GlcNAcbeta1-](n)), the aim of this article is to isolate and define CELM GlcNac-binding lectins and determine their role in cardiac and vascular flow-induced effects. For this purpose, we synthesized a 460-kDa GlcNac polymer (GlcNac-Pol) with high affinity toward GlcNac-recognizing lectins. In the heart, intracoronary administration of GlcNac-Pol upon binding to CELM diminishes the flow-dependent positive inotropic and dromotropic effects. Furthermore, GlcNac-Pol was used as an affinity probe to isolate CELM GlcNac-Pol-recognizing lectins and at least 35 individual lectinic peptides were identified, one of them the beta-ENaC channel. Some of these lectins could participate in flow sensing and in GlcNac-Pol-induced effects. We also adopted a flow-responsive and well-accepted model of endothelial-parenchymal paracrine interaction: isolated blood vessels perfused at controlled flow rates. We established that flow-induced vasodilatation (FIV) is blocked by endothelial luminal membrane (ELM) bound GlcNac-Pol, nitro-l-arginine methyl ester and indomethacin, amiloride, and hyaluronidase. The effect of hyaluronidase was reversed by infusion of soluble hyaluronan. These results indicate that GlcNac-Pol inhibits FIV by competing and displacing intrinsic hyaluronan bound to a lectinic structure such as the amiloride-sensitive ENaC. Nitric oxide and prostaglandins are the putative paracrine mediators of FIV.
Asunto(s)
Acetilglucosamina/metabolismo , Circulación Coronaria/fisiología , Endotelio Vascular/fisiología , Lectinas/metabolismo , Miocardio/metabolismo , Animales , Cromatografía de Afinidad , Cobayas , Masculino , Contracción Miocárdica/fisiología , Vasodilatación/fisiologíaRESUMEN
Serotonin (5-HT)-containing enterochromaffin (EC) cells of the intestine transduce chemical and mechanical stimuli from the intestinal lumen by releasing 5-HT on to afferent nerve terminals. Dysfunctional mucosal 5-HT signaling has been implicated in heightened visceral sensitivity and altered motility in patients with inflammatory bowel disease and in animal models. Our aim was to characterize the release and uptake of 5-HT in the mouse dextran sulfate sodium (DSS; 5% wt/vol) model of colitis. We made electrochemical recordings and used an ELISA assay to determine mucosal 5-HT release and uptake in untreated mice and mice with DSS-induced colitis. Peak and steady-state 5-HT concentrations were measured before and during blockade of the serotonin reuptake transporter (SERT) with 1 microM fluoxetine. Electrochemical recordings showed that colons from DSS-treated mice had roughly twice the steady-state levels of extracellular 5-HT and compression-evoked 5-HT release compared with untreated mice. Fluoxetine doubled the compression-evoked and steady-state 5-HT levels in control and DSS mice. These data were supported by ELISA assays, which showed enhanced 5-HT release during colitis, by immunohistochemical analyses, which showed increases in EC cell numbers, and by real-time PCR, which identified a decrease in SERT mRNA expression in the mucosa during colitis. These data are the first to demonstrate 5-HT release close to its release site and near its site of action during DSS-colitis. We conclude that DSS-colitis increases 5-HT availability primarily by an increase in the numbers of EC cells and/or of content of 5-HT in these EC cells.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Serotonina/metabolismo , Animales , Recuento de Células , Colitis/inducido químicamente , Colitis/patología , Colon/efectos de los fármacos , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Electroquímica , Electrodos , Células Enterocromafines/metabolismo , Células Enterocromafines/patología , Ensayo de Inmunoadsorción Enzimática , Fluoxetina/farmacología , Expresión Génica/genética , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos , Estimulación Física , Serotonina/análisis , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
Diverse intracoronary agonists cause cardiac effects while acting on coronary endothelial luminal membrane (CELM) receptor. Our data show: a) the presence of AT(1)R in isolated CELM and in all cardiac cell types and b) sustained intracoronary infusions of Ang II-POL, a large sized molecule (approximately 15,000 kDa) confined to the vessel lumen that can only act on CELM's AT(1)R or Ang II (approximately 1 kDa); both exert the same maximum positive inotropic (PIE) and coronary constriction (CPP). The effects of these two agonists are blocked by Losartan and by Sar-POL; a large size antagonist (approximately 15,000 kDa) that acts only on CELM. Ang II effects are transient due to desensitization and cause tachyphylaxis to Ang II and toward Ang II-POL suggesting that both Ang II and Ang II-POL act on the same receptor group. In contrast, Ang II-POL effects are sustained and do not cause tachyphylaxis. The results show that intravascular Ang II and Ang II-POL act differentially by an unknown mechanism on CELM's AT(1)R and suggest that intravascular Ang II and Ang II-POL cause PIE and CCP by activation limited to CELM's AT(1)R through an unknown mechanism that is space-confined to the CELM's AT(1)R.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Receptor de Angiotensina Tipo 1/agonistas , Angiotensina II/farmacología , Animales , Vasos Coronarios/fisiología , Endotelio Vascular/fisiología , Losartán/farmacología , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/análisis , Receptor de Angiotensina Tipo 1/efectos de los fármacos , Saralasina/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
The coronary endothelial luminal membrane (CELM) glycocalyx has diverse molecules involved in blood flow signal transduction. Evidence suggests that some of these structures may be lectinic. To test this, we synthesized two monosaccharide polymers (Mon-Pols) made of Mannose (Man-Pol) or Galactose (Gal-Pol) covalently coupled to Dextran (70 kDa) and used them as lectin affinity probes. In situ intracoronary infusion of both polymers resulted in CELM-binding but only Man-Pol caused a reduction in flow-induced positive inotropism and dromotropism. To demonstrate that our lectinic probes could bind to CELM lectins, a representative CELM protein fraction was isolated via intracoronary infusion of a cationic silica colloid and either Mannose- or Galactose-binding lectins were purified from the CELM protein fraction using the corresponding Mon-Pol affinity chromatography resin. Resin-bound CELM proteins were eluted with the corresponding monosaccharide. 2D-SDS-PAGE (pH 4-7) revealed 9 Mannose- and approximately 100 Galactose-selective CELM lectins. In summary, the CELM glycocalyx contains Mannose- and Galactose-binding lectins that may be involved in translating coronary flow into a cardiac parenchymal response.