RESUMEN
Organ-organ crosstalk is involved in homeostasis. Gastrointestinal symptoms are common in patients with renal failure. The aim of this study was to elucidate the relationship between gastrointestinal motility and gastrointestinal symptoms in chronic kidney disease. We performed studies in C57BL/6 mice with chronic kidney disease after 5/6 nephrectomy. Gastrointestinal motility was evaluated by assessing the ex vivo responses of ileum and distal colon strips to electrical field stimulation. Feces were collected from mice, and the composition of the gut microbiota was analyzed using 16S ribosomal RNA sequencing. Mice with chronic kidney disease after 5/6 nephrectomy showed a decreased amount of stool, and this constipation was correlated with a suppressed contraction response in ileum motility and decreased relaxation response in distal colon motility. Spermine, one of the uremic toxins, inhibited the contraction response in ileum motility, but four types of uremic toxins showed no effect on the relaxation response in distal colon motility. The 5/6 nephrectomy procedure disturbed the balance of the gut microbiota in the mice. The motility dysregulation and constipation were resolved by antibiotic treatments. The expression levels of interleukin 6, tumor necrosis factor-α, and iNOS in 5/6 nephrectomy mice were increased in the distal colon but not in the ileum. In addition, macrophage infiltration in 5/6 nephrectomy mice was increased in the distal colon but not in the ileum. We found that 5/6 nephrectomy altered gastrointestinal motility and caused constipation by changing the gut microbiota and causing colonic inflammation. These findings indicate that renal failure was remarkably associated with gastrointestinal dysregulation.
Asunto(s)
Microbioma Gastrointestinal/fisiología , Motilidad Gastrointestinal/fisiología , Nefrectomía , Insuficiencia Renal Crónica/microbiología , Animales , Colon/microbiología , Colon/patología , Colon/cirugía , Tracto Gastrointestinal/microbiología , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Nefrectomía/métodos , Insuficiencia Renal Crónica/cirugíaRESUMEN
Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.
Asunto(s)
Muerte Celular , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Mitocondrias/patología , Óxido Nítrico/metabolismo , Estrés Oxidativo , Agregado de Proteínas , Factor Inductor de la Apoptosis/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Humanos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad MitocondrialRESUMEN
From the immunological point of view, macrophages are required to maintain metabolic homeostasis. Recently, there has been an increased focus on the influence of macrophage phenotypes in adipose tissue on the maintenance of metabolic homeostasis in healthy conditions because dysregulated metabolic homeostasis causes metabolic syndrome. This review notes several types of inflammatory and anti-inflammatory mediators in metabolic homeostasis. M1 macrophage polarization mediates inflammation, whereas M2 macrophage polarization mediates anti-inflammation. Fatty acids and their related factors mediate both inflammatory and anti-inflammatory responses. Saturated fatty acids and polyunsaturated fatty acids mediate inflammation, whereas marine-derived n-3 fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid, mediate anti-inflammation. In this review, we discuss the current understanding of the crosstalk between fatty acids and inflammation in macrophages and their influence on metabolic homeostasis.
Asunto(s)
Ácidos Grasos/metabolismo , Homeostasis/fisiología , Inflamación/metabolismo , Macrófagos/metabolismo , Tejido Adiposo/metabolismo , Animales , Humanos , Obesidad/metabolismoRESUMEN
Interleukin (IL)-19 is a member of the IL-10 family of interleukins and is an immuno-modulatory cytokine produced by the main macrophages. The gastrointestinal tissues of IL-19 knockout mice show exacerbated experimental colitis mediated by the innate immune system and T cells. There is an increasing focus on the interaction and relationship of IL-19 with the function of T cells. Contact hypersensitivity (CHS) is T cell-mediated cutaneous inflammation. Therefore, we asked whether IL-19 causes CHS. We investigated the immunological role of IL-19 in CHS induced by 1-fluoro-2,4-dinitrofluorobenzene as a hapten. IL-19 was highly expressed in skin exposed to the hapten, and ear swelling was increased in IL-19 knockout mice. The exacerbation of the CHS response in IL-19 knockout mice correlated with increased levels of IL-17 and IL-6, but no alterations were noted in the production of interferon (IFN)γ and IL-4 in the T cells of the lymph nodes. In addition to the effect on T cell response, IL-19 knockout mice increased production of inflammatory cytokines. These results show that IL-19 suppressed hapten-dependent skin inflammation in the elicitation phase of CHS.
Asunto(s)
Dermatitis por Contacto/metabolismo , Interleucinas/agonistas , Ganglios Linfáticos/metabolismo , Piel/metabolismo , Animales , Células Cultivadas , Dermatitis por Contacto/sangre , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Dinitrofluorobenceno/análogos & derivados , Dinitrofluorobenceno/toxicidad , Oído , Regulación de la Expresión Génica/efectos de los fármacos , Haptenos/toxicidad , Inmunidad Innata/efectos de los fármacos , Inmunohistoquímica , Interleucina-10 , Interleucina-17/agonistas , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-6/agonistas , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucinas/sangre , Interleucinas/genética , Interleucinas/metabolismo , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ratones Endogámicos BALB C , Ratones Noqueados , ARN Mensajero/metabolismo , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/patologíaRESUMEN
Synthesis of poly(ADP-ribose) (PAR) is catalyzed by PAR polymerase-1 (PARP-1) in neurons. PARP1 plays a role in various types of brain damage in neurodegenerative disorders. In neurons, overactivation of PARP-1 during oxidative stress induces robust PAR formation, which depletes nicotinamide adenine dinucleotide levels and leads to cell death. However, the role of the newly-formed PAR in neurodegenerative disorders remains elusive. We hypothesized that the effects of PAR could occur in the extracellular space after it is leaked from damaged neurons. Here we report that extracellular PAR (EC-PAR) functions as a neuroprotective molecule by inducing the synthesis of glial cell line-derived neurotrophic factor (GDNF) in astrocytes during neuronal cell death, both in vitro and in vivo. In primary rat astrocytes, exogenous treatment with EC-PAR produced GDNF but not other neurotrophic factors. The effect was concentration-dependent and did not affect cell viability in rat C6 astrocytoma cells. Topical injection of EC-PAR into rat striatum upregulated GDNF levels in activated astrocytes and improved pathogenic rotation behavior in a unilateral 6-hydroxydopamine model of Parkinson disease in rats. These findings indicate that EC-PAR acts as a neurotrophic enhancer by upregulating GDNF levels. This effect protects the remaining neurons following oxidative stress-induced brain damage, such as that seen with Parkinson disease.
Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Animales , Astrocitos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Modelos Animales de Enfermedad , Espacio Extracelular/metabolismo , Técnicas In Vitro , Factores de Crecimiento Nervioso/metabolismo , Enfermedad de Parkinson/terapia , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-ß peptide (Aß). Numerous studies have established Aß amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aß amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aß40 with small amounts of GAPDH aggregates significantly enhanced Aß40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aß40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aß40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aß40 co-incubated with GAPDH aggregates exhibited Aß40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aß co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aß aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aß amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Péptidos beta-Amiloides/biosíntesis , Animales , Humanos , Ratones , Ratones Transgénicos , Microscopía de Fuerza Atómica , Mitocondrias/fisiología , Células PC12 , RatasRESUMEN
In addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke.
Asunto(s)
Encéfalo/patología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/irrigación sanguínea , Encéfalo/enzimología , Encéfalo/metabolismo , Línea Celular , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Núcleo Celular/patología , Activación Enzimática , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Humanos , Infarto de la Arteria Cerebral Media/enzimología , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Nitrocompuestos/análisis , Nitrocompuestos/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/análisis , Ratas , Ratas WistarRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays roles in both energy maintenance, and stress signaling by forming a protein complex with seven in absentia homolog 1 (Siah1). Mechanisms to coordinate its glycolytic and stress cascades are likely to be very important for survival and homeostatic control of any living organism. Here we report that apoptosis signal-regulating kinase 1 (ASK1), a representative stress kinase, interacts with both GAPDH and Siah1 and is likely able to phosphorylate Siah1 at specific amino acid residues (Thr-70/Thr-74 and Thr-235/Thr-239). Phosphorylation of Siah1 by ASK1 triggers GAPDH-Siah1 stress signaling and activates a key downstream target, p300 acetyltransferase in the nucleus. This novel mechanism, together with the established S-nitrosylation/oxidation of GAPDH at Cys-150, provides evidence of how the stress signaling involving GAPDH is finely regulated. In addition, the present results imply crosstalk between the ASK1 and GAPDH-Siah1 stress cascades.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Regulación de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Células HEK293 , Humanos , Peróxido de Hidrógeno/farmacología , MAP Quinasa Quinasa Quinasa 5/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Estrés Oxidativo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.
Asunto(s)
Cisteína/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Óxido Nítrico/farmacología , Dominio Catalítico/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisteína/genética , Relación Dosis-Respuesta a Droga , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Agregado de Proteínas/efectos de los fármacos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
In gastric smooth muscles, the released Ca2+ activates the contractile proteins and Ca2+ taken up from the cytosol cause relaxation. The Na+/Ca2+ exchanger (NCX) is an antiporter membrane protein that controls Ca2+ influx and efflux across the membrane. However, the possible relation of NCX in gastric fundus motility is largely unknown. Here, we investigated electric field stimulation (EFS)-induced relaxations in the circular muscles of the gastric fundus in smooth muscle-specific NCX1 transgenic mice (Tg). EFS caused a bi-phasic response, transient and sustained relaxation. The sustained relaxation prolonged for an extended period after the end of the stimulus. EFS-induced transient relaxation and sustained relaxation were greater in Tg than in wild-type mice (WT). Disruption of nitric oxide component by N-nitro-l-arginine, EFS-induced transient and sustained relaxations caused still marked in Tg compared to WT. Inhibition of PACAP by antagonist, EFS-induced sustained relaxation in Tg was not seen, similar to WT. Nevertheless, transient relaxation remained more pronounced in Tg than in WT. Next, we examined responses to NO and PACAP in smooth muscles. The magnitudes of NOR-1, which generates NO, and PACAP-induced relaxations were greater in Tg than in WT. In this study, we demonstrate that NCX1 regulates gastric fundus motility.
Asunto(s)
Fundus Gástrico/fisiología , Intercambiador de Sodio-Calcio/biosíntesis , Animales , Estimulación Eléctrica , Fundus Gástrico/metabolismo , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Relajación Muscular/fisiología , Músculo Liso/metabolismo , Músculo Liso/fisiologíaRESUMEN
The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Oligopéptidos/farmacología , Agregado de Proteínas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Dominio Catalítico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/metabolismo , Neuronas/patología , Oligopéptidos/síntesis química , Oxidación-Reducción , Estrés Oxidativo , Células PC12 , Unión Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Recent reports indicate that interruption of acetylcholine release by intrastriatal injection of botulinum neurotoxin type A (BoNT/A) in a rat Parkinson's disease model reduces pathogenic behavior without adverse side effects such as memory dysfunction. Current knowledge suggests that BoNT/A subtype 1 (BoNT/A1) and BoNT/A subtype 2 (BoNT/A2) exert different effects. In the present study, we compared the effects of BoNT/A1 and BoNT/A2 on rotation behavior and in vivo cleavage of presynaptic protein SNAP-25 in a rat unilateral 6-hydroxydopamine-induced Parkinson's disease model. BoNT/A2 more effectively reduced pathogenic behavior by efficiently cleaving SNAP-25 in the striatum compared with that of BoNT/A1. Our results suggest that BoNT/A2 has greater clinical therapeutic value for treating subjects with Parkinson's disease compared to that of BoNT/A1.
Asunto(s)
Toxinas Botulínicas Tipo A/administración & dosificación , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Animales , Toxinas Botulínicas Tipo A/efectos adversos , Colina O-Acetiltransferasa/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Oxidopamina/farmacología , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/fisiopatología , Proteolisis , Ratas , Rotación , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
Na(+)/Ca(2+) exchanger 1 (NCX1) is a plasma membrane transporter involved in regulating intracellular Ca(2+) concentrations. NCX1 is critical for Ca(2+) regulation in cardiac muscle, vascular smooth muscle and nerve fibers. However, little is known about the physiological role of NCX1 in gastrointestinal motility. To determine the role of NCX1 in gastrointestinal tissues, we examined electric field stimulation (EFS)-induced responses in the longitudinal smooth muscle of the distal colon in smooth muscle-specific NCX1 transgenic mice (Tg). Tg show that NCX1 protein was overexpressed in the distal colon at a level twofold greater than that of endogenous NCX1. We found that the amplitudes of EFS-induced relaxation that persisted during EFS were greater in Tg than in wild-type mice (WT). Under the nonadrenergic, noncholinergic condition, the EFS-induced relaxation in Tg was also greater than that in WT. Inhibition of NO synthase, CO synthase, soluble guanylate cyclase (sGC), and protein kinase G (PKG) all attenuated the enhanced relaxation in Tg, demonstrating the importance of NCX1 in NO/sGC/PKG signaling. The action of NOR-1, an NO donor, induced enhanced relaxation in Tg compared with that in WT. Unlike NOR-1, pituitary adenylate cyclase-activating peptide and vasoactive intestinal peptide induced a similar relaxation in Tg compared with that in WT. In this study, we demonstrate that NCX1 plays an important role in smooth muscle motility in the mouse distal colon.
Asunto(s)
Colon/fisiología , Intercambiador de Sodio-Calcio/fisiología , Animales , Motilidad Gastrointestinal/fisiología , Hidroxilaminas/farmacología , Ratones Endogámicos C57BL , Ratones Transgénicos , Relajación Muscular , Donantes de Óxido Nítrico/farmacología , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
The gastrointestinal tract is composed of outer longitudinal muscle layers and inner circular muscle layers. Nitric oxide (NO), carbon monoxide (CO), and ATP play major roles as non-adrenergic non-cholinergic (NANC) inhibitory neurotransmitters in the longitudinal muscle of the mouse distal colon, whereas it is unclear which NANC inhibitory neurotransmitters are in its circular muscle. We investigated the electric field stimulation (EFS)-induced relaxations in the circular smooth muscle of the distal colon under NANC conditions. In the experiments in which N(ω)-nitro-L-arginine, an inhibitor of NO synthase, was added, the EFS-induced relaxation decreased in a concentration-dependent manner and finally vanished. In contrast, CO, purinergic receptor ligands, and peptidergic substances do not play major roles as NANC neurotransmitters in the circular muscle of the mouse distal colon. ODQ, an inhibitor of soluble guanylate cyclase, strongly attenuated EFS-induced relaxation. Ryanodine, a Ca(2+) release modulator at the sarcoplasmic reticulum, strongly attenuated EFS-induced relaxation as well. Relaxation induced by NOR-1, which generates NO, was inhibited by ODQ and ryanodine. Next, we performed experiments that simultaneously measured tension and the cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt). NOR-1 decreased the tension and [Ca(2+)]cyt levels in the circular muscle. ODQ and ryanodine strongly attenuated the NOR-1-induced change in both tension and [Ca(2+)]cyt levels. In this study, we demonstrate that NO functions as a NANC inhibitory neurotransmitter in the circular muscle obtained from the mouse distal colon.
Asunto(s)
Colon/fisiología , Neurotransmisores/fisiología , Óxido Nítrico/fisiología , Animales , Calcio/fisiología , Estimulación Eléctrica , Hidroxilaminas/farmacología , Técnicas In Vitro , Masculino , Ratones Endogámicos C57BL , Relajación Muscular , Músculo Liso/fisiologíaRESUMEN
The Naâº/Ca²âº exchanger (NCX) is a plasma membrane transporter involved in regulating intracellular Ca²âº concentrations. NCX is critical for Ca²âº regulation in cardiac muscle, vascular smooth muscle, and nerve fibers. To determine the role of NCX1 and NCX2 in gastrointestinal tissues, we examined electric field stimulation (EFS)-induced responses in the longitudinal smooth muscle of the distal colon in NCX1 and NCX2 double-heterozygote knockoutmice (Double HET). We found that the amplitudes of EFS-induced relaxation that persisted during EFS were greater in Double HET than in wild-type mice (WT). Under the non-adrenergic, non-cholinergic (NANC) condition, EFS-induced relaxation in Double HET was similar in amplitude to that of WT. In the experiments in which l-NNA was added under NANC conditions following the EFS, the magnitudes of EFS-induced relaxation were smaller in Double HET than those in WT. In addition, an NCX inhibitor, SN-6, enhanced EFS-induced relaxation but did not affect EFS-induced relaxation under NANC condition, as in Double HET. Moreover, the magnitudes of relaxation induced by NOR-1, which generates NO, were greater in Double HET compared with WT. Similarly, SN-6 potentiated the magnitudes of NOR-1-induced relaxation. In this study, we demonstrate that NCX regulate colonic motility by altering the sensitivity of the inhibitory component.
Asunto(s)
Calcio/metabolismo , Colon/fisiología , Motilidad Gastrointestinal/genética , Heterocigoto , Relajación Muscular/genética , Músculo Liso/fisiología , Óxido Nítrico/metabolismo , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/fisiología , Animales , Benzoatos/farmacología , Compuestos de Bencilo/farmacología , Colon/efectos de los fármacos , Colon/metabolismo , Estimulación Eléctrica , Motilidad Gastrointestinal/fisiología , Imidazoles/farmacología , Técnicas In Vitro , Ratones , Ratones Noqueados , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Óxido Nítrico/fisiología , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Tiazolidinas/farmacologíaRESUMEN
The cytokine, interleukin (IL)-19, is a member of the IL-10 family that includes IL-20, IL-22, IL-24, and IL-26. Recent studies have shown that IL-19 is produced by keratinocytes, epithelial cells, macrophages, and B-cells. Little is known about the exact biological role of IL-19 in immunological regulation, although there is an increasing body of data demonstrating that IL-19 is associated with the development of Th2 responses and the pathogenesis of psoriasis. In this review, I shall attempt to discuss current knowledge about the role of IL-19 on macrophages and the potential role in inflammatory bowel disease.
RESUMEN
The cytokine, interleukin (IL)-19, is a member of the IL-10 family that includes IL-20, IL-22, IL-24, and IL-26. Recent studies have shown that IL-19 is produced by keratinocytes, epithelial cells, macrophages, and B-cells. Little is known about the exact biological role of IL-19 in immunological regulation, although there is an increasing body of data demonstrating that IL-19 is associated with the development of Th2 responses and the pathogenesis of psoriasis. In this review, I shall attempt to discuss current knowledge about the role of IL-19 on macrophages and the potential role in inflammatory bowel disease.
Asunto(s)
Colon/inmunología , Inmunidad Innata , Factores Inmunológicos/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Interleucinas/inmunología , Macrófagos/inmunología , Animales , Linfocitos B/inmunología , Humanos , Psoriasis/inmunología , Psoriasis/fisiopatologíaRESUMEN
Peroxisome proliferator-activated receptor α (PPAR-α) is a ligand-activated transcription factor that exerts strong effects on metabolic pathways. Our aim was to elucidate the effect of clofibrate, a PPAR-α agonist, on the longitudinal muscle of the mouse distal colon. We initially found that clofibrate induced a relaxation response in this muscle. Notably, the PPAR-α antagonists GW9662 and T0070907 did not attenuate this clofibrate-induced relaxation. The structurally related PPAR-α agonists fenofibrate and bezafibrate induced relaxation in the distal colon as effectively as clofibrate. In contrast, wy-14643, which activates PPAR-α more selectively than clofibrate, had no effect. Furthermore, clofibrate-induced relaxation was not affected by N-nitro-L-arginine, an NO synthase inhibitor, 1H-[1,2,4]-oxadiazolo-[4,3- a]quinoxaline-1-one, a soluble guanylate cyclase inhibitor, or H89, a protein kinase A inhibitor. Tetrodotoxin, an Na⺠channel blocker, and glibenclamide, apamin, charybdotoxin and XE991, various K⺠channel blockers, had no effect on clofibrate-induced relaxation. Importantly, clofibrate induced a relaxation response that was not accompanied by any alteration in the cytoplasmic Ca²âº concentration in the longitudinal muscle of the mouse distal colon. Moreover, calyculin A, a myosin light-chain phosphatase (MLCP) inhibitor, attenuated clofibrate-induced relaxation. Our findings indicate that clofibrate relaxes the longitudinal smooth muscle of the mouse distal colon by regulating MLCP activity.
Asunto(s)
Anticolesterolemiantes/farmacología , Calcio/fisiología , Clofibrato/farmacología , Colon/fisiología , Relajación Muscular/fisiología , Músculo Liso/fisiología , Anilidas/farmacología , Animales , Anticolesterolemiantes/metabolismo , Benzamidas/farmacología , Clofibrato/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Guanilato Ciclasa/antagonistas & inhibidores , Masculino , Toxinas Marinas , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/fisiología , Fosfatasa de Miosina de Cadena Ligera/antagonistas & inhibidores , Óxido Nítrico Sintasa/antagonistas & inhibidores , Oxazoles/farmacología , PPAR alfa/agonistas , PPAR alfa/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Bloqueadores de los Canales de Sodio/farmacología , Guanilil Ciclasa SolubleRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(2) is a classic glycolytic enzyme that also mediates cell death by its nuclear translocation under oxidative stress. Meanwhile, we previously presented that oxidative stress induced disulfide-bonded GAPDH aggregation in vitro. Here, we propose that GAPDH aggregate formation might participate in oxidative stress-induced cell death both in vitro and in vivo. We show that human GAPDH amyloid-like aggregate formation depends on the active site cysteine-152 (Cys-152) in vitro. In SH-SY5Y neuroblastoma, treatment with dopamine decreases the cell viability concentration-dependently (IC(50) = 202 microM). Low concentrations of dopamine (50-100 microM) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200-300 microM)-induced cell death. Doxycycline-inducible overexpression of wild-type GAPDH in SH-SY5Y, but not the Cys-152-substituted mutant (C152A-GAPDH), accelerates cell death accompanying both endogenous and exogenous GAPDH aggregate formation in response to high concentrations of dopamine. Deprenyl, a blocker of GAPDH nuclear translocation, fails to inhibit the aggregation both in vitro and in cells but reduced cell death in SH-SY5Y treated with only a low concentration of dopamine (100 microM). These results suggest that GAPDH participates in oxidative stress-induced cell death via an alternative mechanism in which aggregation but not nuclear translocation of GAPDH plays a role. Moreover, we observe endogenous GAPDH aggregate formation in nigra-striatum dopaminergic neurons after methamphetamine treatment in mice. In transgenic mice overexpressing wild-type GAPDH, increased dopaminergic neuron loss and GAPDH aggregate formation are observed. These data suggest a critical role of GAPDH aggregates in oxidative stress-induced brain damage.
Asunto(s)
Muerte Celular , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Dominio Catalítico , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Cisteína/química , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Glucólisis , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Transgénicos , Modelos Biológicos , Mutación , Neuronas/metabolismo , Estrés OxidativoRESUMEN
The present study was designed to identify the inhibitory neurotransmitters mediating nonadrenergic noncholinergic relaxation in the longitudinal muscle of C57/BL mouse distal colon. Relaxation induced by electrical field stimulation (EFS) was recorded isotonically in the presence of atropine and guanethidine. Cyclic guanosine-3',5'-monophosphate (cyclic GMP) content was measured by radioimmunoassay. EFS-induced relaxation was inhibited by nitro-L-arginine (L-NNA) and Sn (IV) protoporphyrin dichloride IX (SnPP-IX), a nitric oxide (NO) and carbon monoxide (CO) synthase inhibitor, respectively. A combination of both inhibitors produced an additive effect. ODQ, a soluble guanylate cyclase inhibitor, inhibited EFS-induced relaxation. NOR-1, a NO donor, and carbon monoxide-releasing molecule-2 (CORM-2), a CO donor, treatment relaxed the distal colon and increased cyclic GMP content. The effects of NOR-1 and CORM-2 were inhibited by ODQ. KT5823, a cyclic GMP-dependent protein kinase inhibitor, inhibited EFS-induced relaxation. EFS-induced relaxation in the presence of KT5823 was further inhibited by L-NNA, but not by SnPP-IX. In addition, KT5823 inhibited CORM-2-induced relaxation, but not NOR-1-induced relaxation. H89, a cyclic AMP-dependent protein kinase inhibitor, inhibited EFS-induced relaxation, and EFS-induced relaxation in the presence of H89 was further inhibited by L-NNA. These results suggested that NO and CO function as inhibitory neurotransmitters in the longitudinal muscle of C57BL mouse distal colon.