RESUMEN
BACKGROUND & AIMS: Interleukin (IL)33/IL1F11 is an important mediator for the development of type 2 T-helper cell (Th2)-driven inflammatory disorders and has also been implicated in the pathogenesis of gastrointestinal (GI)-related cancers, including gastric carcinoma. We therefore sought to mechanistically determine IL33's potential role as a critical factor linking chronic inflammation and gastric carcinogenesis using gastritis-prone SAMP1/YitFc (SAMP) mice. METHODS: SAMP and (parental control) AKR mice were assessed for baseline gastritis and progression to metaplasia. Expression/localization of IL33 and its receptor, ST2/IL1R4, were characterized in corpus tissues, and activation and neutralization studies were both performed targeting the IL33/ST2 axis. Dissection of immune pathways leading to metaplasia was evaluated, including eosinophil depletion studies using anti-IL5/anti-CCR3 treatment. RESULTS: Progressive gastritis and, ultimately, intestinalized spasmolytic polypeptide-expressing metaplasia (SPEM) was detected in SAMP stomachs, which was absent in AKR but could be moderately induced with exogenous, recombinant IL33. Robust peripheral (bone marrow) expansion of eosinophils and local recruitment of both eosinophils and IL33-expressing M2 macrophages into corpus tissues were evident in SAMP. Interestingly, IL33 blockade did not affect bone marrow-derived expansion and local infiltration of eosinophils, but markedly decreased M2 macrophages and SPEM features, while eosinophil depletion caused a significant reduction in both local IL33-producing M2 macrophages and SPEM in SAMP. CONCLUSIONS: IL33 promotes metaplasia and the sequelae of eosinophil-dependent downstream infiltration of IL33-producing M2 macrophages leading to intestinalized SPEM in SAMP, suggesting that IL33 represents a critical link between chronic gastritis and intestinalizing metaplasia that may serve as a potential therapeutic target for preneoplastic conditions of the GI tract.
Asunto(s)
Gastritis/etiología , Gastritis/patología , Interleucina-33/fisiología , Neoplasias Gástricas/etiología , Neoplasias Gástricas/patología , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Eosinófilos , Mucosa Gástrica/patología , Metaplasia , RatonesRESUMEN
BACKGROUND: The molecular mechanisms of cellular changes responsible for diabetic gastroparesis, primarily seen in middle-aged women, still remain incompletely defined. We hypothesized that a decrease in the expression, dimerization, and post-translational modification of neuronal nitric oxide synthase alpha (nNOSα) is estrogen mediated and responsible for the gender-specific prevalence of this malady. METHODS: We induced diabetes by injecting male and female rats with streptozotocin. Male diabetic rats without gastroparesis were then injected with estrogen for 3 weeks and evaluated for gastroparesis development. Gastric tissues were analyzed for the elucidation of biochemical events associated with diabetes and gastroparetic dysfunction. RESULTS: Although male diabetic, gastroparetic (either streptozotocin- or estrogen-induced) rats exhibited similarity in disease pathology to that of females, the molecular mechanisms of development were different. Our results indicate that slow gastric emptying in both male diabetic, gastroparetic rat groups was not associated with the level of expression of nNOSα in gastric tissues. However, nNOSα dimerization, which reflects nNOSα activation, did decline slightly in the antrum of diabetic males with estrogen-induced gastroparesis, suggesting a possible estrogen role. Females with diabetic gastroparesis, in contrast, demonstrated significantly impaired levels and dimerization of nNOSα in the antrum and pylorus. Although the precise mechanism remains unknown, nNOSα dimerization impairment in female antrum is apparently associated with reduced phosphorylation of Ser(1416) in the activation loop of nNOSα. CONCLUSION: Taken together, these results demonstrate that gender and estrogens may be leading factors, through molecular changes involved in nitric oxide synthesis down-regulation, within the antrum and pylorus of female diabetic, gastroparetic rats.
Asunto(s)
Diabetes Mellitus Experimental , Estradiol/metabolismo , Gastroparesia/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Western Blotting , Dimerización , Femenino , Vaciamiento Gástrico , Gastroparesia/fisiopatología , Masculino , Fosforilación , Ratas , Factores SexualesRESUMEN
The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1beta and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K(+) on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K(+) was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K(+) has been suggested to trigger inflammasome activation, in neural cells, high extracellular K(+) activates caspase-1 probably through pannexin 1.
Asunto(s)
Astrocitos/fisiología , Conexinas/fisiología , Inflamación/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Neuroinmunomodulación/fisiología , Neuronas/fisiología , Animales , Astrocitos/citología , Caspasa 1/metabolismo , Muerte Celular/fisiología , Células Cultivadas , Corteza Cerebral/citología , Conexinas/genética , Conexinas/metabolismo , Silenciador del Gen , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Oocitos/fisiología , Técnicas de Placa-Clamp , Potasio/farmacología , Probenecid/farmacología , Ratas , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Xenopus laevisRESUMEN
The purinergic receptor P2X(7) is part of a complex signaling mechanism participating in a variety of physiological and pathological processes. Depending on the activation scheme, P2X(7) receptors in vivo are non-selective cation channels or form large pores that can mediate apoptotic cell death. Expression of P2X(7)R in Xenopus oocytes results exclusively in formation of a non-selective cation channel. However, here we show that co-expression of P2X(7)R with pannexin1 in oocytes leads to the complex response seen in many mammalian cells, including cell death with prolonged ATP application. While the cation channel activity is resistant to carbenoxolone treatment, this gap junction and hemichannel blocking drug suppressed the currents induced by ATP in pannexin1/P2X(7)R co-expressing cells. Thus, pannexin1 appears to be the molecular substrate for the permeabilization pore (or death receptor channel) recruited into the P2X(7)R signaling complex.
Asunto(s)
Conexinas/química , Receptores Purinérgicos P2/química , Adenosina Trifosfato/farmacología , Animales , Secuencia de Bases , Línea Celular , Conexinas/genética , Conexinas/metabolismo , Femenino , Humanos , Técnicas In Vitro , Canales Iónicos/metabolismo , Ratones , Complejos Multiproteicos , Proteínas del Tejido Nervioso , Oocitos/efectos de los fármacos , Oocitos/metabolismo , ARN Interferente Pequeño/genética , Ratas , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , XenopusRESUMEN
Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP-release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non-junctional membrane channels. Two leech innexins previously demonstrated in glia were expressed in frog oocytes. In addition to making gap junctions, innexins also formed non-junctional membrane channels with properties similar to those of pannexons. In addition, carbenoxolone reversibly blocked the loss of carboxyfluorescein dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins we assayed.
Asunto(s)
Conexinas/fisiología , Animales , Señalización del Calcio , Citoplasma/metabolismo , Uniones Comunicantes/metabolismo , Sanguijuelas/metabolismo , Modelos Biológicos , Neuroglía/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Xenopus/metabolismoRESUMEN
The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP-induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.
Asunto(s)
Adenosina Trifosfato/farmacología , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Conexinas/metabolismo , Receptores Purinérgicos P2/metabolismo , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Conexinas/genética , Femenino , Humanos , Fosfatos de Inositol/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Proteínas del Tejido Nervioso , Oocitos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , XenopusRESUMEN
Intercellular calcium wave propagation initiated by mechanical stress is a phenomenon found in nearly all cell types. The waves utilize two pathways: transfer of InsP3 directly from cell to cell through gap junction channels and release of ATP onto extracellular purinergic receptors. The conduit for ATP has remained elusive and both a vesicular and a channel mediated release have been considered. Here, we describe the properties of single pannexin 1 channels. They have a wide expression spectrum, they are of large conductance and permeant for ATP, and they are mechanosensitive. Hence, pannexins are candidates for the release of ATP to the extracellular space upon mechanical stress.
Asunto(s)
Uniones Comunicantes/fisiología , Canales Iónicos/fisiología , Oocitos/fisiología , Animales , Humanos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Proteínas Recombinantes/metabolismo , Transfección , XenopusRESUMEN
BACKGROUND: We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the "paradoxical" effects of TNF in preventing SAMP ileitis. METHODS: Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice. RESULTS: Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice. CONCLUSIONS: Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.
Asunto(s)
Bacterias , Ileítis/metabolismo , Ileítis/prevención & control , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Probióticos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Homeostasis/efectos de los fármacos , Ileítis/microbiología , Ileítis/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Permeabilidad/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
Pannexin 1 (Panx1), a member of the second gap junction protein family identified in vertebrates, appears to preferentially form non-junctional membrane channels. A candidate regulatory protein of Panx1 is the potassium channel subunit Kvbeta3, previously identified by bacterial two-hybrid strategies. Here, we report on the physical association of Panx1 with Kvbeta3 by immunoprecipitation when co-expressed in a neuroblastoma cell line (Neuro2A). Furthermore, in vivo co-expression of Panx1 and Kvbeta3 was shown to occur in murine hippocampus and cerebellum. Kvbeta3 is known to accelerate inactivation of otherwise slowly inactivating potassium channels under reducing conditions. We subsequently found that Panx1 channel currents exhibit a significant reduction when exposed to reducing agents, and that this effect is attenuated in the presence of Kvbeta3. Apparently, Kvbeta3 is involved in regulating the susceptibility of Panx1 channels to redox potential. Furthermore, the Panx1 channel blockers carbenoxolone and Probenecid were less effective in inhibiting Panx1 currents when Kvbeta3 was co-expressed. The influence of Kvbeta3 on Panx1 is the first example of modulation of Panx1 channel function(s) by interacting proteins, and suggests the physiological importance of sensing changes in redox potentials.
Asunto(s)
Conexinas/fisiología , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio con Entrada de Voltaje/fisiología , Animales , Química Encefálica , Línea Celular , Conexina 43/fisiología , Conexinas/análisis , Conexinas/química , Ratones , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/química , Oxidación-Reducción , Canales de Potasio con Entrada de Voltaje/análisis , Canales de Potasio con Entrada de Voltaje/química , Ratas , XenopusRESUMEN
Probenecid is a well-established drug for the treatment of gout and is thought to act on an organic anion transporter, thereby affecting uric acid excretion in the kidney by blocking urate reuptake. Probenecid also has been shown to affect ATP release, leading to the suggestion that ATP release involves an organic anion transporter. Other pharmacological evidence and the observation of dye uptake, however, suggest that the nonvesicular release of ATP is mediated by large membrane channels, with pannexin 1 being a prominent candidate. In the present study we show that probenecid inhibited currents mediated by pannexin 1 channels in the same concentration range as observed for inhibition of transport processes. Probenecid did not affect channels formed by connexins. Thus probenecid allows for discrimination between channels formed by connexins and pannexins.
Asunto(s)
Adenosina Trifosfato/metabolismo , Supresores de la Gota/farmacología , Canales Iónicos/antagonistas & inhibidores , Probenecid/farmacología , Animales , Conexinas/antagonistas & inhibidores , Conexinas/metabolismo , Relación Dosis-Respuesta a Droga , Canales Iónicos/genética , Canales Iónicos/metabolismo , Potenciales de la Membrana , Nitrobenzoatos/farmacología , Oocitos , Xenopus laevis , Proteína beta1 de Unión ComunicanteRESUMEN
Connexin mimetic peptides are widely used to assess the contribution of nonjunctional connexin channels in several processes, including ATP release. These peptides are derived from various connexin sequences and have been shown to attenuate processes downstream of the putative channel activity. Yet so far, no documentation of effects of peptides on connexin channels has been presented. We tested several connexin and pannexin mimetic peptides and observed attenuation of channel currents that is not compatible with sequence specific actions of the peptides. Connexin mimetic peptides inhibited pannexin channel currents but not the currents of the channel formed by connexins from which the sequence was derived. Pannexin mimetic peptides did inhibit pannexin channel currents but also the channels formed by connexin 46. The same pattern of effects was observed for dye transfer, except that the inhibition levels were more pronounced than for the currents. The channel inhibition by peptides shares commonalities with channel effects of polyethylene glycol (PEG), suggesting a steric block as a mechanism. PEG accessibility is in the size range expected for the pore of innexin gap junction channels, consistent with a functional relatedness of innexin and pannexin channels.
Asunto(s)
Conexinas/química , Uniones Comunicantes/química , Uniones Comunicantes/fisiología , Imitación Molecular , Péptidos/química , Animales , Calcio/fisiología , Conexina 43/química , Conexina 43/genética , Conexina 43/fisiología , Conexinas/genética , Conexinas/fisiología , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Conformación Molecular , Proteínas del Tejido Nervioso , Oocitos/fisiología , Técnicas de Placa-Clamp , Péptidos/genética , Péptidos/fisiología , Xenopus laevis , Proteína beta1 de Unión ComunicanteRESUMEN
Vertebrates express two families of gap junction proteins: the well characterized connexins and the recently discovered pannexins. The latter are related to invertebrate innexins. Here we present the hypothesis that pannexins, rather than providing a redundant system to gap junctions formed by connexins, exert a physiological role as nonjunctional membrane channels. Specifically, we propose that pannexins can serve as ATP release channels. This function presumptively is also performed by innexins in invertebrates, in addition to their traditional gap junction role.
Asunto(s)
Conexinas/fisiología , Uniones Comunicantes/fisiología , Animales , Humanos , Proteínas del Tejido NerviosoRESUMEN
ATP is a widely used extracellular signaling molecule. The mechanism of ATP release from cells is presently unresolved and may be either vesicular or channel-mediated. Erythrocytes release ATP in response to low oxygen or to shear stress. In the absence of vesicles, the release has to be through channels. Erythrocytes do not form gap junctions. Yet, here we show with immunohistochemical and electrophysiological data that erythrocytes express the gap junction protein pannexin 1. This protein, in addition to forming gap junction channels in paired oocytes, can also form a mechanosensitive and ATP-permeable channel in the nonjunctional plasma membrane. Consistent with a role of pannexin 1 as an ATP release channel, ATP release by erythrocytes was attenuated by the gap junction blocker carbenoxolone. Furthermore, under conditions of ATP release, erythrocytes took up fluorescent tracer molecules permeant to gap junction channels.