RESUMO
Previous observations made in human and mouse colons suggest that reelin protects the colon from pathology. In this study, we evaluated reelin expression during the transition from either colitis or precancerous lesions to colon cancer and tried to elucidate reelin regulation under these transition processes. Samples of healthy and pathological colons from humans and mice treated with either azoxymethane/dextran sulfate sodium (DSS) or azoxymethane alone were used. The relative abundances of reelin, DNMT-1 and ApoER2 mRNAs were determined by PCR in the colon samples cited above and in the tissue adjacent to mouse colon polyps and adenocarcinomas. In both, humans and mice, reelin mRNA abundance increased significantly in ulcerative colitis and slightly in polyps and decreased in adenomas and adenocarcinomas. Reelin expression was higher in the tissue adjacent to the colon adenocarcinoma and lower in the lesion itself. The reelin expression changes may result, at least in part, from those in DNMT-1 and appear to be independent of ApoER2. Lack of reelin downregulated p-Akt and p53 in healthy colon and prevented their increases in the inflamed colon, whereas it increased GSK-3ß in DSS-untreated mice. In conclusion, reelin mRNA abundance depends on the severity of the colon pathology, and its upregulation in response to initial injuries might prevent the beginning of colon cancer, whereas reelin repression favors it. Increased p53 expression and activation may be involved in this protection. We also propose that changes in colon reelin abundance could be used to predict colon pathology progression.
RESUMO
Neuroinflammation underlies neurodegenerative diseases. Herein, we test whether acute colon inflammation activates microglia and astrocytes, induces neuroinflammation, disturbs neuron intrinsic electrical properties in the primary motor cortex, and alters motor behaviors. We used a rat model of acute colon inflammation induced by dextran sulfate sodium. Inflammatory mediators and microglial activation were assessed in the primary motor cortex by PCR and immunofluorescence assays. Electrophysiological properties of the motor cortex neurons were determined by whole-cell patch-clamp recordings. Motor behaviors were examined using open-field and rotarod tests. We show that the primary motor cortex of rats with acute colon inflammation exhibited microglial and astrocyte activation and increased mRNA abundance of interleukin-6, tumor necrosis factor-alpha, and both inducible and neuronal nitric oxide synthases. These changes were accompanied by a reduction in resting membrane potential and rheobase and increased input resistance and action potential frequency, indicating motor neuron hyperexcitability. In addition, locomotion and motor coordination were impaired. In conclusion, acute colon inflammation induces motor cortex microglial and astrocyte activation and inflammation, which led to neurons' hyperexcitability and reduced motor coordination performance. The described disturbances resembled some of the early features found in amyotrophic lateral sclerosis patients and animal models, suggesting that colon inflammation might be a risk factor for developing this disease.
Assuntos
Colite , Córtex Motor , Animais , Colite/induzido quimicamente , Colite/patologia , Humanos , Inflamação/patologia , Córtex Motor/patologia , Neurônios Motores/patologia , Doenças Neuroinflamatórias , RatosRESUMO
We reported that Disabled-2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E-cadherin. Dab2 depletion in Caco-2 cells led to E-cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium-induced colitis increased Dab2/E-cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E-cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E-cadherin delocalization. Epithelial Dab2 decrement preceded that of E-cadherin. We suggest that Dab2, by inhibiting E-cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/genética , Proteínas Reguladoras de Apoptose/genética , Caderinas/genética , Neoplasias do Colo/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Adenocarcinoma/patologia , Idoso , Animais , Células CACO-2 , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/patologia , Sulfato de Dextrana/toxicidade , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Pólipos/genética , Pólipos/patologia , RatosRESUMO
Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200 and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junction assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endocitose , Junções Intercelulares/metabolismo , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células CACO-2 , Comunicação Celular/genética , Células Cultivadas , Endocitose/genética , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Isoformas de Proteínas , Ratos , Ratos Wistar , Proteína Reelina , Distribuição TecidualRESUMO
We previously reported that reelin, an extracellular matrix protein first known for its key role in neuronal migration, reduces the susceptibility to dextran sulphate sodium (DSS)-colitis. The aim of the current study was to determine whether reelin protects from colorectal cancer and how reelin defends from colon pathology. In the colon of wild-type and of mice lacking reelin (reeler mice) we have analysed the: i) epithelium cell renewal processes, ii) morphology, iii) Sox9, Cdx2, Smad5, Cyclin D1, IL-6 and IFNγ mRNA abundance in DSS-treated and untreated mice, and iv) development of azoxymethane/DSS-induced colorectal cancer, using histological and real time-PCR methodologies. The reeler mutation increases colitis-associated tumorigenesis, with increased tumours number and size. It also impairs the intestinal barrier because it reduces cell proliferation, migration, differentiation and apoptosis; decreases the number and maturation of goblet cells, and expands the intercellular space of the desmosomes. The intestinal barrier impairment might explain the increased susceptibility to colon pathology exhibited by the reeler mice and is at least mediated by the down-regulation of Sox9 and Cdx2. In response to DSS-colitis, the reeler colon increases the mRNA abundance of IL-6, Smad5 and Cyclin D1 and decreases that of IFNγ, conditions that might result in the increased colitis-associated tumorigenesis found in the reeler mice. In conclusion, the results highlight a role for reelin in maintaining intestinal epithelial cell homeostasis and providing resistance against colon pathology.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Colite/metabolismo , Colo/metabolismo , Enterócitos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/biossíntese , Serina Endopeptidases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colite/induzido quimicamente , Colite/patologia , Colo/patologia , Sulfato de Dextrana/toxicidade , Enterócitos/patologia , Feminino , Masculino , Camundongos , Proteína ReelinaRESUMO
Reelin is an extracellular matrix protein first known for its key role in neuronal migration. Studies in rodent small intestine suggested that reelin protects the organism from intestinal pathology. Here we determined in mice colon, by real time-PCR and immunological assays, the expression of the reelin signalling system; its response to dextran sulphate sodium (DSS) and the response of wild-type and reeler mice to DSS-treatment. DNA methylation was determined by bisulfite modification and sequencing of genomic DNA. In the colon mucosa reelin expression is restricted to the myofibroblasts, whereas both epithelial cells and myofibroblasts express reelin receptors (ApoER2 and VLDLR) and its effector protein Dab1. The muscle layer also expresses reelin. DSS-treatment reduces reelin expression in the muscle but it is activated in the mucosa. Activation of mucosal reelin is greater in magnitude and is delayed until after the activation of the myofibroblasts marker, α-SMA. This indicates that the DSS-induced reelin up-regulation results from changes in the reelin gene expression rather than from myofibroblasts proliferation. DSS-treatment does not modify Sp1 or Tbr1 mRNA abundance, but increases that of TGF-ß1 and ApoER2, decreases that of CASK and DNMT1 and it also decreases the reelin promoter methylation. Finally, the reeler mice exhibit higher inflammatory scores than wild-type mice, indicating that the mutation increases the susceptibility to DSS-colitis. In summary, this data are the first to demonstrate that mouse distal colon increases reelin production in response to DSS-colitis via a DNMT1-dependent hypo-methylation of the gene promoter region and that reelin provides protection against colitis.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Colite/genética , Colo/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas do Tecido Nervoso/genética , Serina Endopeptidases/genética , Regulação para Cima , Doença Aguda , Animais , Colite/induzido quimicamente , Colite/patologia , Colo/patologia , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Sulfato de Dextrana , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Regiões Promotoras Genéticas , Proteína ReelinaRESUMO
The low renal excretion of betaine indicates that the kidney efficiently reabsorbs the betaine filtered by the glomeruli but the mechanisms involved in such a process have been scarcely investigated. We have detected concentrative and non-concentrative betaine transport activity in brush-border membrane vesicles (BBMV) from rat renal cortex and medulla. The concentrative system is the Sodium/Imino-acid Transporter 1 (SIT1) because it is Na+- and Cl--dependent, electrogenic and is inhibited by an anti-SIT1 antibody. Its apparent affinity constant for betaine, Kt, is 1.1±0.5 mM and its maximal transport velocity, Vmax, 0.5±0.1 nmol betaine/mg protein/s. Inhibitors of the Na+/Cl-/betaine uptake are L-proline (75%) and cold betaine, L-carnitine and choline (40-60%). Neither creatine, TEA, taurine, ß-alanine, GABA nor glycine significantly inhibited Na+/Cl-/betaine uptake. The non-concentrative betaine transport system is Na+- and H+-independent, electroneutral, with a Kt for betaine of 47±7 µM and a Vmax of 7.8±1 pmol betaine/mg protein/s. Its transport activity is nearly abolished by betaine, followed by L-carnitine (70-80%) and proline (40-50%), but a difference from the Na+/Cl-/betaine transport is that it is inhibited by TEA (approx. 50%) and unaffected by choline. The underlying carrier functions as an antiporter linking betaine entry into the BBMV with the efflux of either L-carnitine or betaine, an exchange unaffected by the anti-SIT1 antibody. As far as we know this is the first work reporting that betaine crosses the apical membrane of rat renal epithelium by SIT1 and by a Na+- and H+-independent transport system.
Assuntos
Betaína/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico Ativo , Células Cultivadas , Masculino , Taxa de Depuração Metabólica , Ratos , Ratos WistarRESUMO
BACKGROUND INFORMATION: The myofibroblasts placed underneath the epithelium of the rodent small intestine express reelin, and the reelin absence modifies both the morphology and the cell renewal processes of the crypt-villus unit. In the developing central nervous system, the reelin effects are mediated by the disabled-1 (Dab1) protein. The present work explores whether Dab1 mediates the reelin control of the crypt-villus unit dynamics by examining in the mouse small intestine the consequences of the absence of (i) Dab1 (scrambler mutation) on crypt-villus unit cell renewal processes and morphology and (ii) reelin (reeler mutation) on the intestinal expression of Dab1. RESULTS: The effects of the scrambler mutation on the crypt-villus unit renewal processes are remarkably similar to those caused by the lack of reelin. Thus, both mutations significantly reduce epithelial cell proliferation, migration and apoptosis, and the number of Paneth cells; affect the morphology of the villus, and expand the intercellular space of the adherens junctions and desmosomes. The Western blot assays reveal that the Dab1 isoform present in the enterocytes has a molecular weight of â¼63 kDa and that in the brain of â¼82 kDa. They also reveal that the absence of reelin increases Dab1 abundance in both brain and enterocytes. CONCLUSIONS: All together, the current findings link reelin with Dab1 and suggest that Dab1 functions downstream of reelin action on the homeostasis of the crypt-villus unit.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Animais , Apoptose , Movimento Celular , Proliferação de Células , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína ReelinaRESUMO
We previously proposed that Dab2 participates in the endocytosis of milk macromolecules in rat small intestine. Here we investigate the receptors that may mediate this endocytosis by studying the effects of age and diet on megalin, VLDLR, and ApoER2 expression, and that of age on the expression of cubilin and amnionless. Of megalin, VLDLR and ApoER2, only the megalin expression pattern resembles that of Dab2 previously reported. Thus the mRNA and protein levels of megalin and Dab2 are high in the intestine of the suckling rat, down-regulated by age and up-regulated by milk diet, mainly in the ileum. Neither age nor diet affect ApoER2 mRNA levels. The effect of age on VLDLR mRNA levels depends on the epithelial cell tested but they are down-regulated by milk diet. In the suckling rat, the intestinal expressions of both cubilin and amnionless are similar to that of megalin and megalin, cubilin, amnionless and Dab2 co-localize at the microvilli and in the apical endocytic apparatus. Co-localization of Dab2 with ApoER2 and VLDLR at the microvilli and in the apical endocytic apparatus is also observed. This is the first report showing intestinal co-localization of: megalin/cubilin/amnionless/Dab2, VLDLR/Dab2 and ApoER2/Dab2. We conclude that the megalin/cubilin/amnionless/Dab2 complex/es participate in intestinal processes, mainly during the lactation period and that Dab2 may act as an adaptor in intestinal processes mediated by ApoER2 and VLDLR.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Lactentes/metabolismo , Animais Lactentes/fisiologia , Endocitose/genética , Feminino , Intestino Delgado/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Lactação/genética , Lactação/metabolismo , Microvilosidades/ultraestrutura , RNA Mensageiro/metabolismo , Ratos , Receptores de LDL/metabolismoRESUMO
Intestinal myofibroblasts secrete substances that control organogenesis and wound repair of the intestine. The myofibroblasts of the rat small intestine express reelin and the present work explores whether reelin regulates crypt-villus unit homeostasis using normal mice and mice with the reelin gene disrupted (reeler). The results reveal that mouse small intestine expresses reelin, its receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VldlR) and the reelin effector protein Disabled-1 (Dab1) and that reelin expression is restricted to myofibroblasts. The absence of reelin significantly reduces epithelial cell proliferation, migration, and apoptosis and the number of Paneth cells. These effects are observed during the suckling, weaning, and adult periods. The number of Goblet cells is increased in the 2-month-old reeler mice. The absence of reelin also expands the extracellular space of the adherens junctions and desmosomes without significantly affecting either the tight-junction structure or the epithelial paracellular permeability. In conclusion, this is the first in vivo work showing that the absence of reelin alters intestinal epithelium homeostasis.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Homeostase/fisiologia , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Miofibroblastos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Animais , Células Cultivadas , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes Neurológicos , Receptores de LDL/metabolismo , Proteína ReelinaRESUMO
Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Epitélio/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Intestino Grosso/crescimento & desenvolvimento , Rim/crescimento & desenvolvimento , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Epitélio/embriologia , Epitélio/metabolismo , Íleo/embriologia , Íleo/crescimento & desenvolvimento , Íleo/metabolismo , Intestino Grosso/embriologia , Intestino Grosso/metabolismo , Jejuno/embriologia , Jejuno/crescimento & desenvolvimento , Jejuno/metabolismo , Rim/embriologia , Rim/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Oral mannose therapy is used to treat congenital disorders of glycosylation caused by a deficiency in phosphomannose isomerase. The segmental distribution and ontogenic regulation of D-mannose transport, phosphomannose isomerase, and phosphomannose mutase is investigated in the small intestine of fetuses, newborn, suckling, 1-month-old, and adult rats. The small intestine transports D-mannose by both Na(+)-dependent and Na(+)-independent transport mechanisms. The activities of both systems normalized to intestinal weight peak at birth and thereafter they decreased. In all the ages tested, the activity of the Na(+)-independent mechanism was higher than that of the Na(+)/mannose transport system. At birth, the Na(+)-independent D-mannose transport in the ileum was significantly higher than that in jejunum. Phosphomannose isomerase activity and mRNA levels increased at 1 month, and the values in the ileum were lower than in jejunum. Phosphomannose mutase activity in jejunum increased during the early stages of life, and it decreased at 1 month old, as does the amount of mannose incorporated into glycoproteins, whereas in the ileum, they were not affected by age. The phosphomannose isomerase/phosphomannose mutase activity ratio decreased at birth and during the suckling period, and increased at 1 month old. In conclusion, intestinal D-mannose transport activity and metabolism were affected by ontogeny and intestinal segment.
Assuntos
Transporte Biológico/fisiologia , Intestino Delgado/metabolismo , Manose/metabolismo , Animais , Transporte Biológico/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Manose-6-Fosfato Isomerase/genética , Manose-6-Fosfato Isomerase/metabolismo , Modelos Biológicos , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Reação em Cadeia da Polimerase , RatosRESUMO
The kidney synthesizes L-carnitine and reabsorbs it via the Na(+)/L-carnitine cotransporter OCTN2. This study investigates the ontogeny of OCTN2, gamma-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and gamma-butyrobetaine hydroxylase (BBH) in rat kidneys. Foetuses, newborn, suckling, weaning and adult rats were used. The apical membranes of foetal and newborn rat kidneys express OCTN2 transport activity, which is up-regulated by age. Maturation significantly increased the V(max) of this transport system without changing the apparent K(t), which excludes a maturation-related expression of different transporter isoforms. Northern analysis showed a 3.7kb transcript for OCTN2 in all the ages tested. Northern and RT-PCR assays revealed that maturation increased renal expression of OCTN2 mRNA. Foetuses express TMABA-DH mRNA and this expression increased during postnatal life. BBH mRNA, however, was detected during the suckling period onwards and its abundance was not changed significantly by maturation. This study reports for the first time that, in rat kidneys: (i) an apical OCTN2 transporter is active in rat foetuses, (ii) ontogeny up-regulates OCTN2 activity by increasing the density and/or turnover of the transporters, (iii) the maturation-related changes in OCTN2 are in part mediated by transcriptional mechanism(s) and (iv) the expression of both, TMABA-DH and BBH mRNA is ontogenically regulated. Some of these results were published as an abstract (García-Delgado et al., 2003).
Assuntos
Aldeído Oxirredutases/metabolismo , Regulação da Expressão Gênica/genética , Rim/enzimologia , Proteínas de Transporte de Cátions Orgânicos/metabolismo , gama-Butirobetaína Dioxigenase/metabolismo , Envelhecimento/fisiologia , Aldeído Oxirredutases/genética , Animais , Carnitina/metabolismo , Cinética , Proteínas de Transporte de Cátions Orgânicos/genética , RNA Mensageiro/genética , Ratos , Ratos Wistar , Membro 5 da Família 22 de Carreadores de Soluto , gama-Butirobetaína Dioxigenase/genéticaRESUMO
Aquaporins (AQPS) are transmembrane water channels poorly investigated in birds. Using degenerated primers and RT-PCR, we identified in kidney and gastrointestinal tract of Hubbard chickens (Gallus gallus) three fragments, corresponding to ck-AQP2, ck-AQP4, and ck-AQP5 mRNAs. Comparison of nucleotide ck-AQPs sequences to their rat and human orthologues revealed an overall identity of 75-90%. Expression in the renal and gastrointestinal systems of the three ck-AQPs mRNA was analysed by Northern assays. Transcript of ck-AQP2 was only identified in kidney. ck-AQP4 mRNA was highly expressed in brain, and to a lesser extent in kidney and stomach. ck-AQP5 mRNA was found in jejunum and ileum, and to a lesser extent in colon and lung. In situ hybridisation showed ck-AQP5 mRNA in the crypt cells of jejunum, ileum and colon, whereas it was absent from the cells lining the villi. Levels of ck-AQP5 mRNA (analyzed by Northern and in situ hybridisation assays) and protein (analysed by immunohistochemistry) decreased from the jejunum to the colon. This work confirmed the presence of AQPs in chicken, and showed that chicken and mammalian AQPs share a high degree of similarity in nucleotide sequence and tissue distribution.
Assuntos
Aquaporina 5/fisiologia , Aquaporinas/metabolismo , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Sequência de Aminoácidos , Animais , Aquaporina 5/metabolismo , Sequência de Bases , Northern Blotting , Galinhas , Clonagem Molecular , Colo/metabolismo , DNA Complementar/metabolismo , Mucosa Gástrica/metabolismo , Trato Gastrointestinal/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Jejuno/metabolismo , Rim/metabolismo , Dados de Sequência Molecular , RNA/metabolismo , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
D-mannose transport and metabolism has been studied in enterocytes isolated from chicken small intestine. In the presence of Na(+), the mannose taken up by the cells either remains free, is phosphorylated, is catabolized to H(2)O, or becomes part of membrane components. The mannose remaining free in the cytosol is released when the cells are transferred to an ice bath. The Na(+)-dependent D-mannose transport is electrogenic and inhibited by ouabain and dinitrophenol; its substrate specificity differs from SGLT-1 transporter. The Glut2 transporter inhibitors phloretin and cytochalasin B added following 30-min mannose uptake reduced the previously accumulated D-mannose, whereas these two agents increased the cell to external medium 3-O-methyl-glucose (3-OMG) concentration ratio. D-mannose efflux rate from preloaded D-[2-(3)H]-mannose enterocytes is Na(+)-independent. Phloretin did not affect D-mannose efflux rate, whereas it inhibited that of 3-OMG. Neither mannose uptake nor efflux rate were affected by fructose. It is concluded that part of the mannose taken up by the enterocytes is rapidly metabolized and that enterocytes have two D- mannose transport systems: one is concentrative and Na(+)-dependent and the other is Na(+)-independent and passive.
Assuntos
Enterócitos/metabolismo , Manose/metabolismo , Animais , Transporte Biológico , Galinhas , Citocalasina B/farmacologia , Enterócitos/enzimologia , Técnicas In Vitro , Íons , Cinética , Manose-6-Fosfato Isomerase , Floretina/farmacologia , FosforilaçãoRESUMO
The kidney efficiently salvages creatine from the urine; however, the mechanism(s) that mediates renal creatine reabsorption has not been investigated. This study characterizes the creatine transport mechanism in brush-border membrane vesicles isolated from the rat renal cortex. An osmolality plot revealed that creatine is transported into an osmotically active space and that it is also bound to the membranes. An inwardly directed NaCl gradient stimulated creatine uptake and the time course of uptake exhibited an overshoot phenomenon, which indicates the presence of an active process for creatine in these membranes. The uptake of creatine showed an absolute requirement for both Na(+) and Cl(-). The NaCl gradient-dependent creatine uptake was stimulated by a valinomycin-induced, inside-negative, K(+)-diffusion potential, which suggests that the uptake process is electrogenic. Stoichiometric analyses indicated a probable couple ratio of 2 Na(+):1 Cl(-):1 creatine molecule. The kinetic study showed that creatine is transported by a high-affinity system (K(m) of 15 microM). Creatine uptake was inhibited by a 100-fold excess of various compounds with the following potency order: cold creatine = guanidinopropionic acid > nipecotic acid > gamma-aminobutyric acid (GABA) = beta-alanine = betaine, whereas carnitine, glycine, taurine, and choline were without effect. This pattern of inhibition differs from that observed for GABA uptake (unlabeled GABA = GPA > beta-alanine > nipecotic acid >> creatine). The conclusion drawn was that the apical membrane of the renal cortical tubules contains an active, high-affinity, electrogenic, 2 Na(+)/1 Cl(-)/creatine cotransporter.