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1.
Traffic ; 13(8): 1072-82, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22510086

RESUMEN

In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane (BBM) to the subapical endosomes requires the minus-end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus-end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin-3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP-stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co-immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1a's role in BBM localization of sucrase-isomaltase.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Enterocitos/metabolismo , Cadenas Pesadas de Miosina/genética , Animales , Células CACO-2 , Cloruros , Colon/citología , Estimulación Eléctrica , Endocitosis , Enterocitos/citología , Exocitosis , Humanos , Intestino Delgado/citología , Transporte Iónico , Ratones , Ratones Noqueados , Microscopía Confocal , Microvellosidades/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Especificidad de Órganos , Vesículas Transportadoras/metabolismo
2.
Am J Physiol Cell Physiol ; 305(9): C981-96, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-23986201

RESUMEN

Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to colocalize with CFTR in intestinal CFTR high expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR(-/-) mice and polarized intestinal CaCo-2BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Coimmunoprecipitation studies examined V-ATPase interaction with CFTR. The pH-sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR colocalization in the apical domain of intestinal cells and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the brush border membrane. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR(-/-) intestine but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHi in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Enterocitos/metabolismo , ATPasas de Translocación de Protón Vacuolares/fisiología , Animales , Células CACO-2 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/metabolismo , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley
3.
Am J Physiol Gastrointest Liver Physiol ; 305(6): G453-65, 2013 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-23868408

RESUMEN

The CFTR High Expresser (CHE) cells express eightfold higher levels of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel compared with neighboring enterocytes and were first identified by our laboratory (Ameen et al., Gastroenterology 108: 1016, 1995). We used double-label immunofluorescence microscopy to further study these enigmatic epithelial cells in rat intestine in vivo or ex vivo. CHE cells were found in duodenum, most frequent in proximal jejunum, and absent in ileum and colon. CFTR abundance increased in CHE cells along the crypt-villus axis. The basolateral Na(+)K(+)Cl(-) cotransporter NKCC1, a key transporter involved in Cl(-) secretion, was detected at similar levels in CHE cells and neighboring enterocytes at steady state. Microvilli appeared shorter in CHE cells, with low levels of Myosin 1a, a villus enterocyte-specific motor that retains sucrase/isomaltase in the brush-border membrane (BBM). CHE cells lacked alkaline phosphatase and absorptive villus enterocyte BBM proteins, including Na(+)H(+) exchanger NHE3, Cl(-)/HCO3(-) exchanger SLC26A6 (putative anion exchanger 1), and sucrase/isomaltase. High levels of the vacuolar-ATPase proton pump were observed in the apical domain of CHE cells. Levels of the NHE regulatory factor NHERF1, Na-K-ATPase, and Syntaxin 3 were similar to that of neighboring enterocytes. cAMP or acetylcholine stimulation robustly increased apical CFTR and basolateral NKCC1 disproportionately in CHE cells relative to neighboring enterocytes. These data strongly argue for a specialized role of CHE cells in Cl(-)-mediated "high-volume" fluid secretion on the villi of the proximal small intestine.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/citología , Acetilcolina/farmacología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mucosa Intestinal/ultraestructura , Masculino , Microvellosidades/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratas , Ratas Sprague-Dawley , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo
4.
Am J Physiol Gastrointest Liver Physiol ; 305(3): G258-75, 2013 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-23744739

RESUMEN

The Brunner's glands of the proximal duodenum exert barrier functions through secretion of glycoproteins and antimicrobial peptides. However, ion transporter localization, function, and regulation in the glands are less clear. Mapping the subcellular distribution of transporters is an important step toward elucidating trafficking mechanisms of fluid transport in the gland. The present study examined 1) changes in the distribution of intestinal anion transporters and the aquaporin 5 (AQP5) water channel in rat Brunner's glands following second messenger activation and 2) anion transporter distribution in Brunner's glands from healthy and disease-affected human tissues. Cystic fibrosis transmembrane conductance regulator (CFTR), AQP5, sodium-potassium-coupled chloride cotransporter 1 (NKCC1), sodium-bicarbonate cotransporter (NBCe1), and the proton pump vacuolar ATPase (V-ATPase) were localized to distinct membrane domains and in endosomes at steady state. Carbachol and cAMP redistributed CFTR to the apical membrane. cAMP-dependent recruitment of CFTR to the apical membrane was accompanied by recruitment of AQP5 that was reversed by a PKA inhibitor. cAMP also induced apical trafficking of V-ATPase and redistribution of NKCC1 and NBCe1 to the basolateral membranes. The steady-state distribution of AQP5, CFTR, NBCe1, NKCC1, and V-ATPase in human Brunner's glands from healthy controls, cystic fibrosis, and celiac disease resembled that of rat; however, the distribution profiles were markedly attenuated in the disease-affected duodenum. These data support functional transport of chloride, bicarbonate, water, and protons by second messenger-regulated traffic in mammalian Brunner's glands under physiological and pathophysiological conditions.


Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Glándulas Duodenales/metabolismo , Agua/metabolismo , Animales , Proteínas de Transporte de Anión/análisis , Acuaporina 5/análisis , Acuaporina 5/metabolismo , Bicarbonatos/metabolismo , Transporte Biológico/efectos de los fármacos , Glándulas Duodenales/química , Glándulas Duodenales/patología , Carbacol/farmacología , Enfermedad Celíaca/metabolismo , Cloruros/metabolismo , AMP Cíclico/farmacología , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Duodeno/química , Duodeno/patología , Humanos , Masculino , Protones , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario/fisiología , ATPasas de Translocación de Protón Vacuolares/análisis
5.
Am J Physiol Gastrointest Liver Physiol ; 303(8): G937-50, 2012 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-22936272

RESUMEN

Changes in intestinal luminal pH affect mucosal ion transport. The aim of this study was to compare how luminal pH and specific second messengers modulate the membrane traffic of four major ion transporters (CFTR, NHE3, NKCC1, and NBCe1) in rat small intestine. Ligated duodenal, jejunal, and ileal segments were infused with acidic or alkaline saline, 8-Br-cAMP, or the calcium agonist carbachol in vivo for 20 min. Compared with untreated intestine, lumen pH was reduced after cAMP or carbachol and increased following HCO(3)(-)-saline. Following HCl-saline, lumen pH was restored to control pH levels. All four secretory stimuli resulted in brush-border membrane (BBM) recruitment of CFTR in crypts and villi. In villus enterocytes, CFTR recruitment was coincident with internalization of BBM NHE3 and basolateral membrane recruitment of the bicarbonate transporter NBCe1. Both cAMP and carbachol recruited NKCC1 to the basolateral membrane of enterocytes, while luminal acid or HCO(3)(-) retained NKCC1 in intracellular vesicles. Luminal acid resulted in robust recruitment of CFTR and NBCe1 to their respective enterocyte membrane domains in the upper third of the villi; luminal HCO(3)(-) induced similar membrane changes lower in the villi. These findings indicate that each stimulus promotes a specific transporter trafficking response along the crypt-villus axis. This is the first demonstration that physiologically relevant secretory stimuli exert their actions in villus enterocytes by membrane recruitment of CFTR and NBCe1 in tandem with NHE3 internalization.


Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Ácidos/farmacología , Bicarbonatos/farmacología , Carbacol/farmacología , Mucosa Intestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Masculino , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Simportadores de Sodio-Bicarbonato/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12
6.
Dig Dis Sci ; 57(11): 2826-45, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22923315

RESUMEN

BACKGROUND AND AIM: Lubiprostone is a chloride channel activator in clinical use for the treatment of chronic constipation, but the mechanisms of action of the drug are poorly understood. The aim of this study was to determine whether lubiprostone exerts secretory effects in the intestine by membrane trafficking of ion transporters and associated machinery. METHODS: Immunolabeling and quantitative fluorescence intensity were used to examine lubiprostone-induced trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), sodium/potassium-coupled chloride co-transporter 1 (NKCC1), electrogenic sodium/bicarbonate co-transporter 1 (NBCe1), down-regulated in adenoma (DRA), putative anion transporter 1 (PAT1), sodium/proton exchanger 3 (NHE3), Ca(2+) activated chloride channel 2 (ClC-2) serotonin and its transporter SERT, E prostanoid receptors EP4 and EP1, sodium/potassium ATPase (Na-K-ATPase) and protein kinase A (PKA). The effects of lubiprostone on mucus exocytosis in rat intestine and human rectosigmoid explants were also examined. RESULTS: Lubiprostone induced contraction of villi and proximal colonic plicae and membrane trafficking of transporters that was more pronounced in villus/surface cells compared to the crypt. Membrane trafficking was determined by: (1) increased membrane labeling for CFTR, PAT1, NKCC1, and NBCe1 and decreased membrane labeling for NHE3, DRA and ClC-2; (2) increased serotonin, SERT, EP4, EP1 and PKA labeling in enterochromaffin cells; (3) increased SERT, EP4, EP1, PKA and Na-K-ATPase in enterocytes; and (4) increased mucus exocytosis in goblet cells. CONCLUSION: These data suggest that lubiprostone can target serotonergic, EP4/PKA and EP1 signaling in surface/villus regions; stimulate membrane trafficking of CFTR/NBCe1/NKCC1 in villus epithelia and PAT1/NBCe1/NKCC1 in colonic surface epithelia; suppress NHE3/DRA trafficking and fluid absorption; and enhance mucus-mobilization and mucosal contractility.


Asunto(s)
Alprostadil/análogos & derivados , Exocitosis/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Moco/efectos de los fármacos , Moco/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Alprostadil/farmacología , Análisis de Varianza , Animales , Antiportadores/metabolismo , Biopsia , Canales de Cloruro CLC-2 , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Transporte Iónico , Lubiprostona , Proteínas de Transporte de Membrana/metabolismo , Transportadores de Anión Orgánico/metabolismo , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Subtipo EP1 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Transportadores de Sulfato
7.
J Biol Chem ; 285(22): 17177-87, 2010 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-20351096

RESUMEN

The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and alpha-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether alpha-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with alpha-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to alpha-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with alpha-AP-2. siRNA silencing of alpha-AP-2 in cells significantly reduced CFTR endocytosis, further supporting alpha-AP-2 as the direct binding partner for CFTR. alpha-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell's Waltzer myosin VI((sv/sv)) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI((sv/sv)) enterocytes. The findings support alpha-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.


Asunto(s)
Canales de Cloruro/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mucosa Intestinal/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Factor de Transcripción AP-2/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Proteínas Reguladoras de la Apoptosis , Endocitosis , Enterocitos/metabolismo , Masculino , Ratones , Ratones Noqueados , Microvellosidades/metabolismo , Modelos Biológicos , Ratas , Ratas Sprague-Dawley
8.
Am J Physiol Gastrointest Liver Physiol ; 300(1): G82-98, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21030607

RESUMEN

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na(+) bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na(+)/H(+) exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunner's glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl(-)-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl(-) secretion by goblet cells and Cl(-) and HCO(3)(-) secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Intestino Delgado/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Animales , Carbacol/farmacología , Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Masculino , Ratas , Intercambiador 3 de Sodio-Hidrógeno , Miembro 2 de la Familia de Transportadores de Soluto 12
9.
Drug Alcohol Depend ; 220: 108468, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33540349

RESUMEN

BACKGROUND: Public health has begun using social media forums such as Reddit to enhance surveillance and modernize interventions for young people. The current study's objective was to examine Reddit posts about the HBO series Euphoria to identify show themes that resonate with adolescent and young adult viewers in order to inform future social media interventions. METHODS: Reddit comments in the r/television community from June to August 2019 were downloaded. Following filtering, 725 comments were analyzed and coded using a codebook and ATLAS.ti. Coded comments were analyzed for themes relevant to Redditor substance use, reactions to Euphoria and the main character (Rue), and mental health concerns. RESULTS: During their discussion of the show, Redditors disclosed both personal recreational and prescription drug use, including substance use to cope with mental illness symptoms. There were approximately equal numbers of comments with positive and negative reactions to the show overall and to the main character, Rue. Redditors often found Euphoria's storyline and portrayed events to be relatable and realistic to the experience of young people who use drugs, as well as sometimes triggering. Overall, Redditors thought Rue accurately depicted an individual's struggle with a substance use disorder. CONCLUSIONS: This exploratory study highlights how television and social media can contribute to young peoples' understanding of substance use disorders and mental health. Findings could inform the design of social media interventions for adolescents and young adults on a variety of substance use issues, including stigma and the interconnectedness of substance use and mental health challenges.


Asunto(s)
Trastornos Mentales/psicología , Medios de Comunicación Sociales , Trastornos Relacionados con Sustancias/psicología , Televisión , Adaptación Psicológica , Adulto , Femenino , Humanos , Masculino , Salud Mental , Salud Pública , Adulto Joven
10.
Am J Physiol Cell Physiol ; 299(6): C1450-60, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20844248

RESUMEN

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2(BB)e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2(BB)e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.


Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Diarrea/metabolismo , Enterotoxinas/metabolismo , Exocitosis , Proteínas Qa-SNARE/metabolismo , Animales , Células CACO-2 , Diarrea/microbiología , Células HEK293 , Humanos , Yeyuno/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Qa-SNARE/análisis , Ratas , Ratas Sprague-Dawley
11.
Mol Imaging Biol ; 7(5): 342-50, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16240059

RESUMEN

PURPOSE: This study was conducted to test for possible circadian control of viral infection in live animals using bioluminescence imaging of a firefly luciferase transgene. METHODS: Transgenic mice expressing the firefly luciferase gene under the control of the promoter and enhancer of the human cytomegalovirus major immediate-early gene (CMV::luc) were examined through whole-animal imaging. Mice were crossed with HRS/J hairless albino mice to improve imaging of deep structures. RESULTS: Transgene expression in the extremities and head was elevated around dusk in mice maintained in cycles of light and dark. Signal was also elevated during the animal's night in mice maintained in extended darkness. The viral promoter was induced during the active phase of the circadian locomotor rhythm in several tissues. Both the acinar cells and islets expressed the transgene in dissociated pancreas cultures. CONCLUSIONS: These results suggest that viruses may exploit the circadian system for optimal timing of infection at particular phases in several tissue types.


Asunto(s)
Ritmo Circadiano/genética , Genes Virales/genética , Luciferasas de Luciérnaga/genética , Regiones Promotoras Genéticas/genética , Animales , Citomegalovirus/genética , Expresión Génica , Humanos , Luciferasas de Luciérnaga/metabolismo , Ratones , Ratones Transgénicos , Páncreas/citología , Páncreas/enzimología , Carrera
12.
BMC Physiol ; 3: 8, 2003 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-12927048

RESUMEN

BACKGROUND: Gene promoters fused to the firefly luciferase gene (luc) are useful for examining gene regulation in live transgenic mice and they provide unique views of functioning organs. The dynamics of gene expression in cells and tissues expressing luciferase can be observed by imaging this enzyme's bioluminescent oxidation of luciferin. Neural pathways involved in specific behaviors have been identified by localizing expression of immediate-early genes such as c-fos. A transgenic mouse line with luc controlled by the human c-fos promoter (fos::luc) has enabled gene expression imaging in brain slice cultures. To optimize imaging of immediate-early gene expression throughout intact mice, the present study examined fos::luc mice and a second transgenic mouse containing luc controlled by the human cytomegalovirus immediate-early gene 1 promoter and enhancer (CMV::luc). Because skin pigments and hair can significantly scatter light from underlying structures, the two transgenic lines were crossed with a hairless albino mouse (HRS/J) to explore which deep structures could be imaged. Furthermore, live anesthetized mice were compared with overdosed mice. RESULTS: Bioluminescence imaging of anesthetized mice over several weeks corresponded with expression patterns in mice imaged rapidly after a lethal overdose. Both fos::luc and CMV::luc mice showed quantifiable bright bioluminescence in ear, nose, paws, and tail whether they were anesthetized or overdosed. CMV::luc and fos::luc neonates had bioluminescence patterns similar to those of adults, although intensity was significantly higher in neonates. CMV::luc mice crossed with HRS/J mice had high expression in bone, claws, head, pancreas, and skeletal muscle, but less in extremities than haired CMV::luc mice. Imaging of brain bioluminescence through the neonatal skull was also practical. By imaging luciferin autofluorescence it was clear that substrate distribution did not restrict bioluminescence imaging to capillaries after injection. Luciferin treatment and anesthesia during imaging did not adversely affect circadian rhythms in locomotor activity. CONCLUSIONS: Imaging of gene expression patterns with luciferase can be extended from studies of live animals to rapid imaging of mice following a pentobarbital overdose before significant effects from postmortem changes occurs. Bioluminescent transgenic mice crossed with HRS/J mice are valuable for examining gene expression in deep tissues.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Luciferasas/genética , Animales , Escarabajos/enzimología , Cruzamientos Genéticos , Citomegalovirus/genética , Genes Virales/genética , Luciferasas/metabolismo , Ratones , Ratones Pelados , Ratones Transgénicos , Pentobarbital/administración & dosificación , Pentobarbital/envenenamiento , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Estructurales Virales/genética
13.
PLoS One ; 8(7): e69050, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23935921

RESUMEN

Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Cloruros/metabolismo , Toxina del Cólera/farmacología , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activación Enzimática , Humanos , Técnicas In Vitro , Mucosa Intestinal/efectos de los fármacos , Masculino , Ratones , Fosforilación , Ratas
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