RESUMEN
Divalent metal-ion transporter-1 (DMT1), the principal mechanism by which nonheme iron is taken up at the intestinal brush border, is energized by the H(+)-electrochemical potential gradient. The provenance of the H(+) gradient in vivo is unknown, so we have explored a role for brush-border Na(+)/H(+) exchanger (NHE) isoforms by examining iron homeostasis and intestinal iron handling in mice lacking NHE2 or NHE3. We observed modestly depleted liver iron stores in NHE2-null (NHE2(-/-)) mice stressed on a low-iron diet but no change in hematological or blood iron variables or the expression of genes associated with iron metabolism compared with wild-type mice. Ablation of NHE3 strongly depleted liver iron stores, regardless of diet. We observed decreases in blood iron variables but no overt anemia in NHE3-null (NHE3(-/-)) mice on a low-iron diet. Intestinal expression of DMT1, the apical surface ferrireductase cytochrome b reductase-1, and the basolateral iron exporter ferroportin was upregulated in NHE3(-/-) mice, and expression of liver Hamp1 (hepcidin) was suppressed compared with wild-type mice. Absorption of (59)Fe from an oral dose was substantially impaired in NHE3(-/-) compared with wild-type mice. Our data point to an important role for NHE3 in generating the H(+) gradient that drives DMT1-mediated iron uptake at the intestinal brush border.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Hierro/metabolismo , Microvellosidades/fisiología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Transporte Biológico , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Oocitos , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Desequilibrio Hidroelectrolítico , XenopusRESUMEN
KEY POINTS: An in vitro approach to study gastric development is primary mouse-derived epithelium cultured as three-dimensional spheroids known as organoids. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Organoids maintained in co-culture with immortalized stomach mesenchymal cells express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. We report the use of these models for studies of epithelial cell biology and cell damage and repair. ABSTRACT: Studies of gastric function and disease have been limited by the lack of extended primary cultures of the epithelium. An in vitro approach to study gastric development is primary mouse-derived antral epithelium cultured as three-dimensional spheroids known as organoids. There have been no reports on the use of organoids for gastric function. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Both models were generated from single glands dissociated from whole fundic tissue and grown in basement membrane matrix (Matrigel) and organoid growth medium. Model 1 enriches for a stem cell-like niche via simple passage of the organoids. Maintained in Matrigel and growth medium, proliferating organoids expressed high levels of stem cell markers CD44 and Lgr5. Model 2 is a system of gastric organoids co-cultured with immortalized stomach mesenchymal cells (ISMCs). Organoids maintained in co-culture with ISMCs express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. Thus, we report the use of these models for studies of epithelial cell biology and cell damage and repair.
Asunto(s)
Técnicas de Cocultivo/métodos , Células Epiteliales/citología , Mucosa Gástrica/citología , Organoides/citología , Células Madre/citología , Animales , Diferenciación Celular , RatonesRESUMEN
The majority of antibiotic-induced diarrhea is caused by Clostridium difficile (C. difficile). Hospitalizations for C. difficile infection (CDI) have tripled in the last decade, emphasizing the need to better understand how the organism colonizes the intestine and maintain infection. The mucus provides an interface for bacterial-host interactions and changes in intestinal mucus have been linked host health. To assess mucus production and composition in healthy and CDI patients, the main mucins MUC1 and MUC2 and mucus oligosaccharides were examined. Compared with healthy subjects, CDI patients demonstrated decreased MUC2 with no changes in surface MUC1. Although MUC1 did not change at the level of the epithelia, MUC1 was the primary constituent of secreted mucus in CDI patients. CDI mucus also exhibited decreased N-acetylgalactosamine (GalNAc), increased N-acetylglucosamine (GlcNAc), and increased terminal galactose residues. Increased galactose in CDI specimens is of particular interest since terminal galactose sugars are known as C. difficile toxin A receptor in animals. In vitro, C. difficile is capable of metabolizing fucose, mannose, galactose, GlcNAc, and GalNAc for growth under healthy stool conditions (low Na(+) concentration, pH 6.0). Injection of C. difficile into human intestinal organoids (HIOs) demonstrated that C. difficile alone is sufficient to reduce MUC2 production but is not capable of altering host mucus oligosaccharide composition. We also demonstrate that C. difficile binds preferentially to mucus extracted from CDI patients compared with healthy subjects. Our results provide insight into a mechanism of C. difficile colonization and may provide novel target(s) for the development of alternative therapeutic agents.
Asunto(s)
Clostridioides difficile/metabolismo , Colon/metabolismo , Colon/microbiología , Enterocolitis Seudomembranosa/metabolismo , Enterocolitis Seudomembranosa/microbiología , Moco/metabolismo , Acetilgalactosamina/metabolismo , Acetilglucosamina/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Clostridioides difficile/crecimiento & desarrollo , Clostridioides difficile/aislamiento & purificación , Heces/microbiología , Femenino , Galactosa/análogos & derivados , Galactosa/metabolismo , Interacciones Huésped-Patógeno , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Masculino , Persona de Mediana Edad , Mucina-1/metabolismo , Mucina 2/metabolismo , Organoides , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/microbiologíaRESUMEN
Clostridium difficile infection (CDI) is principally responsible for hospital acquired, antibiotic-induced diarrhea and colitis and represents a significant financial burden on our healthcare system. Little is known about C. difficile proliferation requirements, and a better understanding of these parameters is critical for development of new therapeutic targets. In cell lines, C. difficile toxin B has been shown to inhibit Na(+)/H(+) exchanger 3 (NHE3) and loss of NHE3 in mice results in an altered intestinal environment coupled with a transformed gut microbiota composition. However, this has yet to be established in vivo in humans. We hypothesize that C. difficile toxin inhibits NHE3, resulting in alteration of the intestinal environment and gut microbiota. Our results demonstrate that CDI patient biopsy specimens have decreased NHE3 expression and CDI stool has elevated Na(+) and is more alkaline compared with stool from healthy individuals. CDI stool microbiota have increased Bacteroidetes and Proteobacteria and decreased Firmicutes phyla compared with healthy subjects. In vitro, C. difficile grows optimally in the presence of elevated Na(+) and alkaline pH, conditions that correlate to changes observed in CDI patients. To confirm that inhibition of NHE3 was specific to C. difficile, human intestinal organoids (HIOs) were injected with C. difficile or healthy and CDI stool supernatant. Injection of C. difficile and CDI stool decreased NHE3 mRNA and protein expression compared with healthy stool and control HIOs. Together these data demonstrate that C. difficile inhibits NHE3 in vivo, which creates an altered environment favored by C. difficile.
Asunto(s)
Clostridioides difficile/crecimiento & desarrollo , Colon/metabolismo , Colon/microbiología , Enterocolitis Seudomembranosa/metabolismo , Enterocolitis Seudomembranosa/microbiología , Microbiota , Intercambiadores de Sodio-Hidrógeno/metabolismo , Adulto , Anciano , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Clostridioides difficile/aislamiento & purificación , Clostridioides difficile/metabolismo , Regulación hacia Abajo , Heces/microbiología , Femenino , Interacciones Huésped-Patógeno , Humanos , Concentración de Iones de Hidrógeno , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Masculino , Persona de Mediana Edad , Organoides , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/microbiología , ARN Mensajero/metabolismo , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)). DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and (54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Absorción Intestinal/fisiología , Hierro/metabolismo , Manganeso/metabolismo , Anemia Hipocrómica/genética , Anemia Hipocrómica/patología , Animales , Proteínas de Transporte de Catión/genética , Transportador de Cobre 1 , Regulación de la Expresión Génica/fisiología , Homeostasis/fisiología , Ratones , Ratones Noqueados , Zinc/metabolismoRESUMEN
BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) promote gastric cancer in response to gastritis. In culture, BM-MSCs are prone to mutation with continued passage but it is unknown whether a similar process occurs in vivo in response to gastritis. AIM: The purpose of this study was to identify the role of chronic gastritis in the transformation of BM-MSCs leading to an activated cancer-promoting phenotype. METHODS: Age matched C57BL/6 (BL/6) and gastrin deficient (GKO) mice were used for isolation of stomach, serum and mesenchymal stem cells (MSCs) at 3 and 6 months of age. MSC activation was assessed by growth curve analysis, fluorescence-activated cell sorting and xenograft assays. To allow for the isolation of bone marrow-derived stromal cells and assay in response to chronic gastritis, IRG/Vav-1(Cre) mice that expressed both enhanced green fluorescent protein-expressing hematopoietic cells and red fluorescent protein-expressing stromal cells were generated. In a parabiosis experiment, IRG/Vav-1(Cre) mice were paired to either an uninfected Vav-1(Cre) littermate or a BL/6 mouse inoculated with Helicobacter pylori. RESULTS: GKO mice displayed severe atrophic gastritis accompanied by elevated gastric tissue and circulating transforming growth factor beta (TGFß) by 3 months of age. Compared to BM-MSCs isolated from uninflamed BL/6 mice, BM-MSCs isolated from GKO mice displayed an increased proliferative rate and elevated phosphorylated-Smad3 suggesting active TGFß signaling. In xenograft assays, mice injected with BM-MSCs from 6-month-old GKO animals displayed tumor growth. RFP+ stromal cells were rapidly recruited to the gastric mucosa of H. pylori parabionts and exhibited changes in gene expression. CONCLUSIONS: Gastritis promotes the in vivo activation of BM-MSCs to a phenotype reminiscent of a cancer-promoting cell.
Asunto(s)
Transformación Celular Neoplásica , Mucosa Gástrica/patología , Gastritis Atrófica/patología , Células Madre Mesenquimatosas/patología , Fenotipo , Animales , Biomarcadores/metabolismo , Proliferación Celular , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Gastrinas/deficiencia , Gastritis Atrófica/metabolismo , Gastritis Atrófica/microbiología , Proteínas Hedgehog/metabolismo , Infecciones por Helicobacter/patología , Helicobacter pylori , Immunoblotting , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Parabiosis , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Changes in the intestinal microbiota have been linked to diabetes, obesity, inflammatory bowel disease, and Clostridium difficile (C. difficile)-associated disease. Despite this, it remains unclear how the intestinal environment, set by ion transport, affects luminal and mucosa-associated bacterial composition. Na(+)/H(+)-exchanger isoform 3 (NHE3), a target of C. difficile toxin B, plays an integral role in intestinal Na(+) absorption. Thus the NHE3-deficient mouse model was chosen to examine the effect of pH and ion composition on bacterial growth. We hypothesized that ion transport-induced change in the intestinal environment would lead to alteration of the microbiota. Region-specific changes in ion composition and pH correlated with region-specific alteration of luminal and mucosal-associated bacteria with general decreases in Firmicutes and increases in Bacteroidetes members. Bacteroides thetaiotaomicron (B. thetaiotaomicron) increased in NHE3(-/-) terminal ileum and was examined in vitro to determine whether altered Na(+) was sufficient to affect growth. Increased in vitro growth of B. thetaiotaomicron occurred in 43 mM Na(+) correlating with the NHE3(-/-) mouse terminal ileum [Na(+)]. NHE3(-/-) terminal ileum displayed increased fut2 mRNA and fucosylation correlating with B. thetaiotaomicron growth. Inoculation of B. thetaiotaomicron in wild-type and NHE3(-/-) terminal ileum organoids displayed increased fut2 and fucosylation, indicating that B. thetaiotaomicron alone is sufficient for the increased fucosylation seen in vivo. These data demonstrate that loss of NHE3 alters the intestinal environment, leading to region-specific changes in bacteria, and shed light on the growth requirements of some gut microbiota members, which is vital for creating better treatments of complex diseases with an altered gut microbiota.
Asunto(s)
Bacteroides/clasificación , Bacteroides/crecimiento & desarrollo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Medios de Cultivo , Concentración de Iones de Hidrógeno , Intestinos/microbiología , Intestinos/fisiología , Ratones , Sodio , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
BACKGROUND: With the rise of antibiotic resistance, new alternatives are being sought to effectively modulate the characteristics of gut microbiota to obtain pathogen resistance without the use of antibiotics. In the past, an oligosaccharide derivative of carrots, galursan HF 7K (GHF7K), has been used clinically in Austria and recently in the fowl-industry to promote health. This study examined the potential role of GHF7K as a prebiotic to alter the gut microbiota in mice. METHODS: Mice were fed either a control diet (CT) or a diet containing 2% GHF7K in the water and chow for 2 weeks, and weight, food and water consumption, gut microbiota and ion composition of the intestinal fluid were examined. RESULTS: Dietary supplement of GHF7K did not alter mouse weight or daily food consumption. Additionally, no changes were observed in the total number of luminal or mucosa-associated bacteria populations in GHF7K-fed mice. GHF7K supplementation significantly altered the composition of luminal, and to a less extent, mucosa-associated bacterial populations at the level of the phyla, with region-specific differences. Similar to antibiotic use, Proteobacteria number was increased in the ileum and colon of GHF7K-fed mice, with no changes in the number of beneficial Lactobacillus and Bifidobacterium genera of phylum Firmicutes. Corresponding with the altered gut microbiota, changes in the ion composition of the intestinal fluid were observed. An increased Cl(-) concentration was observed in the duodenum and jejunum, while the Na(+) concentration was increased in the cecum of GHF7K-fed mice. Decreases were observed in the K(+) concentration in the cecum and distal colon. CONCLUSIONS: Dietary supplement of GHF7K is capable of altering the gut microbiota, which correlates to changes in the intestinal environment. These data suggest that GHF7K dietary supplement can purposefully be used to alter the gut microbiota, and thus could potentially represent an alternative approach to prophylactic antibiotic use.
Asunto(s)
Intestinos/efectos de los fármacos , Microbiota/efectos de los fármacos , Ácido Pipemídico/administración & dosificación , Prebióticos , Animales , Antibacterianos/administración & dosificación , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Peso Corporal , Suplementos Dietéticos , Intestinos/microbiología , RatonesRESUMEN
BACKGROUND: The mechanisms bacteria use to proliferate and alter the normal bacterial composition remain unknown. The ability to link changes in the intestinal micro-environment, such as ion composition and pH, to bacterial proliferation is clinically advantageous for diseases that involve an altered gut microbiota, such as Inflammatory Bowel Disease, obesity and diabetes. In human and mouse intestine, the apical Na(+)/H(+) exchangers NHE2 and NHE3 affect luminal Na(+), water, and pH. Loss of NHE2 results in acidic luminal pH. Since acid resistance systems in gram-positive bacteria are well documented, we hypothesize that gram-positive bacteria would increase in representation in the acidic NHE2(-/-) intestine. METHODS: Intestinal ion composition was measured by fame photometry and chloridometry and pH measured electrochemically. DNA extracted from intestinal flushes or from mucosal scrapings was analyzed by qRT-PCR to examine luminal and mucosa-associated bacterial populations. Epithelial mucus oligosaccharide patterns were examined by histology with FIT-C labeled lectins . RESULTS: Although total luminal and mucosa-associated bacteria were unchanged in NHE2(-/-) intestine, gram-positive bacterial phyla were increased in the mucosa-associated bacterial population in a region-specific manner. The genera Clostridium and Lactobacillus were increased in the cecum and colon which corresponded to changes in NHE2(-/-) mucus oligosaccharide composition of mannose, N-acetyglucosamine, N-acetygalactosamine and galactose. CONCLUSIONS: Together these data indicate that changes in ion transport induce region-specific bacterial changes, which alter host mucus oligosaccharide patterns. These host-bacterial interactions provide a possible mechanism of niche-development and shed insight on how certain groups proliferate in changing environments and maintain their proliferation by altering the host.
Asunto(s)
Ácidos/metabolismo , Iones/aislamiento & purificación , Membrana Mucosa/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Humanos , Concentración de Iones de Hidrógeno , Iones/química , Ratones , Microbiota/efectos de los fármacos , Membrana Mucosa/microbiología , Moco/efectos de los fármacos , Moco/metabolismo , Oligosacáridos/metabolismo , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
In secretory epithelial cells, the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl(-) secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-beta-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle "pinchase" dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, approximately 80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and approximately 40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway.
Asunto(s)
Clatrina/fisiología , Endocitosis/efectos de los fármacos , Simportadores de Cloruro de Sodio-Potasio/fisiología , Acetato de Tetradecanoilforbol/farmacología , Animales , Línea Celular , Perros , Glucosa/farmacología , Inmunohistoquímica , Riñón/citología , Riñón/efectos de los fármacos , Riñón/fisiología , Microscopía Fluorescente , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
The early events in an intestinal ischemic episode have been difficult to evaluate. Using in vivo microscopy we have analyzed in real-time the effects of short (15 min) and long (40-50 min) ischemia with subsequent reperfusion (IR), evaluating structure, integrity, and functioning of the mouse jejunal mucosa while monitoring blood flow by confocal microscopy. IR was imposed by inflation/deflation of a vascular occluder, and blood flow was monitored and confirmed with scanning confocal imaging. After short ischemia, villus tip cells revealed a rapid increase (23%) in the intracellular NAD(P)H concentration (confocal autofluorescence microscopy), and the pH-sensitive probe BCECF showed a biphasic response of the intracellular pH (pH(i)), quickly alkalinizing from the resting value of 6.8 +/- 0.1 to 7.1 +/- 0.1 but then strongly acidifying to 6.3 +/- 0.1. Upon reperfusion, values returned toward control. In contrast, results were heterogeneous after long IR. During long ischemia, one-third of the epithelial cells remained viable with reversible changes upon reperfusion, but remaining cells lost membrane integrity (Lucifer Yellow uptake, LY) and had membrane blebs during ischemia. These effects became more pronounced as the reperfusion interval progressed when cells exhibited more severely affected NAD(P)H and pH(i) values, larger blebs, and more LY uptake and eventually were shed from the villus. Results from stereo microscopy suggest that these irreversible effects of IR may have occurred as a result of incomplete restorations of local blood flow, especially at the antimesenteric side of the intestine. We conclude that the adverse effects of short ischemia on the jejunum epithelium are fully reversible during the reperfusion interval. However, after long ischemia, reperfusion cannot restore normal structure and functioning of a majority of cells, which deteriorate further. Our results provide a basis for defining the cellular events that cause tissue to transit from reversible to irreversible damage during IR.
Asunto(s)
Mucosa Intestinal/irrigación sanguínea , Yeyuno/irrigación sanguínea , Daño por Reperfusión/fisiopatología , Circulación Esplácnica , Animales , Biomarcadores/metabolismo , Forma de la Célula , Modelos Animales de Enfermedad , Concentración de Iones de Hidrógeno , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Yeyuno/metabolismo , Yeyuno/patología , Masculino , Ratones , Ratones Endogámicos ICR , Microcirculación , Microscopía Confocal , Microscopía Fluorescente , NADP/metabolismo , Permeabilidad , Recuperación de la Función , Flujo Sanguíneo Regional , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Factores de TiempoRESUMEN
Basolateral ammonium produces an inhibition of Cl- secretion the magnitude of which is dependent on the NH4+ to K+ concentration ratio. Inhibition is maximal at a mole fraction ratio of 0.25 K+ to NH4+. This anomalous mole fraction effect is due to effects on the basolateral K+ channel as well as Na(+)-K(+)-2Cl- cotransporter. However, only Cl- loading, not K+ loading, appears affected in an anomalous mole fraction manner. Transepithelial current is only slightly inhibited relative to equilmolar K+ by NH4+. As in other systems, both Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- can act in Na(+)-NH4(+)-ATPase and Na(+)-NH4(+)-2Cl- transport modes. NH4+ conducts through most K+ channels and thus likely through the apical K+ channel present in native crypt cells. This suggests that, similar to the kidney, colonic secretory cells have the capacity to secrete NH4+ when in a K(+)-secreting mode with elevated basolateral NH4+ levels.
Asunto(s)
Colon/citología , Colon/metabolismo , Canales Iónicos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Animales , Humanos , Concentración de Iones de Hidrógeno , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Although colonic lumen NH(4)(+) levels are high, 15-44 mM normal range in humans, relatively few studies have addressed the transport mechanisms for NH(4)(+). More extensive studies have elucidated the transport of NH(4)(+) in the kidney collecting duct, which involves a number of transporter processes also present in the distal colon. Similar to NH(4)(+) secretion in the renal collecting duct, we show that the distal colon secretory model, T84 cell line, has the capacity to secrete NH(4)(+) and maintain an apical-to-basolateral NH(4)(+) gradient. NH(4)(+) transport in the secretory direction was supported by basolateral NH(4)(+) loading on NKCC1, Na(+)-K(+)-ATPase, and the NH(4)(+) transporter, RhBG. NH(4)(+) was transported on NKCC1 in T84 cells nearly as well as K(+) as determined by bumetanide-sensitive (86)Rb-uptake. (86)Rb-uptake and ouabain-sensitive current measurement indicated that NH(4)(+) is transported by Na(+)-K(+)-ATPase in these cells to an equal extent as K(+). T84 cells expressed mRNA for the basolateral NH(4)(+) transporter RhBG and the apical NH(4)(+) transporter RhCG. Net NH(4)(+) transport in the secretory direction determined by (14)C-methylammonium (MA) uptake and flux occurred in T84 cells suggesting functional RhG protein activity. The occurrence of NH(4)(+) transport in the secretory direction within a colonic crypt cell model likely serves to minimize net absorption of NH(4)(+) because of surface cell NH(4)(+) absorption. These findings suggest that we rethink the present limited understanding of NH(4)(+) handling by the distal colon as being due solely to passive absorption.
Asunto(s)
Proteínas Sanguíneas/fisiología , Colon/citología , Colon/metabolismo , Glicoproteínas de Membrana/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Simportadores de Cloruro de Sodio-Potasio/fisiología , Adenosina Trifosfatasas/metabolismo , Transporte Biológico Activo/fisiología , Línea Celular , AMP Cíclico/farmacología , ADN Complementario/genética , Humanos , Concentración de Iones de Hidrógeno , Metilaminas/metabolismo , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Radioisótopos de Rubidio , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
The role of Slc26a6 (PAT1) on apical Cl-/HCO3- exchange and bicarbonate secretion in pancreatic duct cells was investigated using Slc26a6 null and wild-type (WT) mice. Apical Cl-/HCO3- exchange activity was measured with the pH-sensitive dye BCECF in microperfused interlobular ducts. The HCO3(-)-influx mode of apical [Cl-]i/[HCO3-]o exchange (where brackets denote concentration and subscripts i and o denote intra- and extracellular, respectively) was dramatically upregulated in Slc26a6 null mice (P < 0.01 vs. WT), whereas the HCO3(-)-efflux mode of apical [Cl-]o/[HCO3-]i exchange was decreased in Slc26a6 null mice (P < 0.05 vs. WT), suggesting the unidirectionality of the Slc26a6-mediated HCO3- transport. Fluid secretory rate in interlobular ducts were comparable in WT and Slc26a6 null mice (P > 0.05). In addition, when pancreatic juice was collected from whole animal in basal and secretin-stimulated conditions, neither juice volume nor its pH showed differences between WT and Slc26a6 null mice. Semiquantitative RT-PCR demonstrated more than fivefold upregulation in Slc26a3 (DRA) expression in Slc26a6 knockout pancreas. In conclusion, these results point to the role of Slc26a6 in HCO3- efflux at the apical membrane and also suggest the presence of a robust Slc26a3 compensatory upregulation, which can replace the function of Slc26a6 in pancreatic ducts.
Asunto(s)
Antiportadores/deficiencia , Antiportadores/genética , Antiportadores/fisiología , Antiportadores de Cloruro-Bicarbonato/fisiología , AMP Cíclico/farmacología , Conductos Pancreáticos/fisiología , Animales , Bicarbonatos/metabolismo , Ratones , Ratones Noqueados , Conductos Pancreáticos/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Secretina/farmacología , Transportadores de SulfatoRESUMEN
SLC26A7 is a Cl(-)/HCO(3)(-) exchanger that is expressed on the basolateral membrane and in the cytoplasm of two distinct acid-secreting epithelial cells: The A-intercalated cells in the kidney outer medullary collecting duct and the gastric parietal cells. The intracellular localization of SLC26A7 suggests the possibility of trafficking between cell membrane and intracellular compartments. For testing this hypothesis, full-length human SLC26A7 cDNA was fused with green fluorescence protein and transiently expressed in MDCK epithelial cells. In monolayer cells in isotonic medium, SLC26A7 showed punctate distribution throughout the cytoplasm. However, in medium that was made hypertonic for 16 h, SLC26A7 was detected predominantly in the plasma membrane. The presence of mitogen-activated protein kinase inhibitors blocked the trafficking of SLC26A7 to the plasma membrane. Double-labeling studies demonstrated the localization of SLC26A7 to the transferrin receptor-positive endosomes. A chimera that was composed of the amino terminal fragment of SLC26A7 and the carboxyl terminal fragment of SLC26A1, and a C-terminal-truncated SLC26A7 were retained in the cytoplasm in hypertonicity. In separate studies, SLC26A7 showed predominant localization in plasma membrane in potassium-depleted isotonic medium (0.5 or 2 mEq/L KCl) versus cytoplasmic distribution in normal potassium isotonic medium (4 mEq/L). It is concluded that SLC26A7 is present in endosomes, and its targeting to the basolateral membrane is increased in hypertonicity and potassium depletion. The trafficking to the cell surface suggests novel functional upregulation of SLC26A7 in states that are associated with hypokalemia or increased medullary tonicity. Additional studies are needed to ascertain the role of SLC26A7 in enhanced bicarbonate absorption in outer medullary collecting duct in hypokalemia and in acid-base regulation in conditions that are associated with increased medullary tonicity.
Asunto(s)
Antiportadores/metabolismo , Endosomas/metabolismo , Túbulos Renales Colectores/metabolismo , Animales , Antiportadores/química , Antiportadores/genética , Secuencia de Bases , Línea Celular , Antiportadores de Cloruro-Bicarbonato/metabolismo , ADN/genética , Perros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Soluciones Hipertónicas , Hipopotasemia/metabolismo , Técnicas In Vitro , Médula Renal/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo , Transportadores de SulfatoRESUMEN
Mutations in the SLC26A3 (DRA (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26a3. slc26a3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26a3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26a3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26a3-null mice. We conclude that slc26a3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26a3 regulates colonic crypt proliferation. Deletion of slc26a3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters.
Asunto(s)
Antiportadores/deficiencia , Cloruros/metabolismo , Colon/metabolismo , Colon/patología , Animales , Antiportadores/genética , Antiportadores/fisiología , Secuencia de Bases , Proliferación Celular , Cartilla de ADN/genética , Femenino , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Transporte Iónico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Transportadores de Sulfato , Regulación hacia ArribaRESUMEN
Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is due to the action of NH(4)(+) on an apical anion exchanger.
Asunto(s)
Bicarbonatos/metabolismo , Colon/metabolismo , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/farmacología , Compuestos de Amonio Cuaternario/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Antiportadores/efectos de los fármacos , Antiportadores/metabolismo , Northern Blotting , Western Blotting , Carbacol/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Línea Celular , Canales de Cloruro/antagonistas & inhibidores , Colon/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Proteínas de Transporte de Membrana/metabolismo , Agonistas Muscarínicos/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estilbenos/farmacología , Transportadores de SulfatoRESUMEN
The kidney Na(+):HCO(3)(-) cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and S427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH(2) terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant S427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-S427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R and S427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.
Asunto(s)
Polaridad Celular/fisiología , Riñón/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Acidosis Tubular Renal/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Perros , Regulación de la Expresión Génica/fisiología , Riñón/citología , Túbulos Renales Proximales , Mutación Missense , Oocitos , Transporte de Proteínas/genética , Xenopus laevisRESUMEN
Ischemia is the central pathogenic factor underlying a spectrum of intestinal disorders. The study of the cellular signaling responses to ischemic stress in nonepithelial cells has progressed substantially in the previous several years, but little is known about the response in epithelial cells. Unique features of the epithelial response to ischemic stress suggest differential regulation with regards to signaling. The PKC family of proteins has been implicated in ischemic stress in nonepithelial systems. The role of PKC isoforms in chemical ischemia in intestinal epithelial cells is evaluated in this study. Additionally, the phosphorylation of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS) is also studied. Chemical ischemia resulted in the transient activation of only the isoform PKC-epsilon as detected by translocation employing the subcellular fractionation technique. The pharmacological agonists phorbol 12-myristate 13-acetate and carbachol also led to the translocation of PKC-epsilon. By immunofluoresence, MARCKS is noted to be located at the lateral membrane under control conditions. In response to carbachol, MARCKS translocates to the cytosol, indicating its phosphorylation, which is additionally confirmed biochemically. Consistent with this observation, carbachol induces the translocation of PKC-epsilon to proximity with MARCKS at the lateral membrane. In response to chemical ischemia, MARCKS fails to translocate and phosphorylation does not increase. Additionally, the translocation of PKC-epsilon is not to the lateral membrane but rather basally. The data suggest that the differential translocation of PKC-epsilon in response to pharmacological agonists versus ischemic stress may lead to different effects on downstream targets.
Asunto(s)
Intestinos/citología , Intestinos/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa C/farmacología , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Células Epiteliales/fisiología , Humanos , Hipoxia , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fosforilación , Proteína Quinasa C/metabolismo , Proteína Quinasa C-epsilon , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
A significant amount of ammonium (NH4+) is absorbed by the colon. The nature of NH4+ effects on transport and NH4+ transport itself in colonic epithelium is poorly understood. The goal of this study was to elucidate the effects of NH4+ on cAMP-stimulated Cl- secretion in the colonic cell line T84. In HEPES-buffered solutions, application of basolateral NH4+ resulted in a reduced level of Cl- secretory current. The effect of NH4+ appears to occur by at least three mechanisms: 1) basolateral membrane depolarization, 2) a competitive effect with K+, and 3) a long-term (>20 min) increase in transepithelial resistance (TER). The competitive effect with K+ exhibits anomalous mole fraction behavior. Transepithelial current relative to that in 10 mM basolateral K+ was inhibited 15% by 10 mM NH4+ alone and by 30% with a mixture of 2 mM K+ and 8 mM NH4+. A mole fraction mix of 2 mM K+:8 mM NH4+ produced a greater inhibition of basolateral membrane K+ current than pure K+ or NH4+ alone. Similar anomalous behavior was also observed for inhibition of bumetanide-sensitive 36Cl- uptake, e.g., Na+-K+-2Cl- -cotransporter (NKCC-1). No anomalous effect was observed on Na+-K+-ATPase current. Both NKCC-1 and Na+-K+-ATPase activity were elevated in 10 mM NH4+ with respect to 10 mM K+. The effect on TER did not exhibit anomalous mole fraction behavior. The overall effect of basolateral NH4+ on cAMP-stimulated transport is dependent on the [K+]o /[NH4+]o ratio at the basolateral membrane, where o is outside of the cell.