RESUMO
Two experiments were conducted to investigate the kinetics of phosphorus (P) absorption and expressions of type IIb sodium-dependent phosphate cotransporter (NaP-IIb), inorganic phosphate transporters 1 and 2 (PiT-1 and PiT-2) in primary cultured duodenal epithelial cells of chick embryos. In experiment 1, the P absorptions across duodenal epithelial cell monolayers at different incubation time points (0, 10, 20, 40, 60, 80, 100 and 120 min) were compared. In experiment 2, the kinetics of P absorption was performed at 40 min after incubation of duodenal epithelial cells with the media containing 0, 0.75, 1.5, 3.0, 6.0, 12.0, 24.0 and 48.0 mmol P/L as KH2 PO4 , and the mRNA and protein expression levels of NaP-IIb, PiT-1 and PiT-2 in duodenal epithelial cells with the media containing 0, 6.0 and 48.0 mmol P/L were determined at 87 min after incubation. The results from experiment 1 showed that the P absorption increased linearly (p < .0001) from 0 to 80 min and the fastest increase occurred at 40 min; the asymptotic model was shown to have the best fit degree, and the optimal incubation time for saturable P absorption was determined to be 87 min. The kinetic curves of P absorption from experiment 2 demonstrated that P absorption was a mixed process of a non-saturable diffusion plus a saturable carrier-mediated transport across the duodenal epithelial cells. The high P concentration (48.0 mmol/L) decreased (p < .05) NaP-IIb and PiT-1 mRNA and protein levels and increased (p < .0001) PiT-2 mRNA level. These results indicated that the P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos was a mixed process of a non-saturable diffusion plus a saturable carrier-mediated transport and could be restricted by reducing the NaP-IIb and PiT-1 expressions while increasing the PiT-2 expression at a high P concentration.
Assuntos
Duodeno/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/citologia , Proteínas de Transporte de Fosfato/metabolismo , Fósforo/farmacocinética , Animais , Transporte Biológico , Embrião de Galinha , Proteínas de Transporte de Fosfato/genéticaRESUMO
Using DNA comet assay we found that polysaccharides from Tussilago farfara L. reduced the intensity of polychemotherapy-induced apoptosis and DNA damage in bone marrow cells and small intestinal epithelium of C57Bl/6 mice, which attested to genoprotective properties of these polysaccharides.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/antagonistas & inibidores , Células da Medula Óssea/efeitos dos fármacos , DNA/química , Mucosa Intestinal/efeitos dos fármacos , Polissacarídeos/farmacologia , Tussilago/química , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Cisplatino/antagonistas & inibidores , Cisplatino/toxicidade , Ensaio Cometa , DNA/metabolismo , Duodeno/citologia , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Feminino , Injeções Intraperitoneais , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Irinotecano/antagonistas & inibidores , Irinotecano/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Paclitaxel/antagonistas & inibidores , Paclitaxel/toxicidade , Extratos Vegetais/química , Polissacarídeos/isolamento & purificaçãoRESUMO
BACKGROUND: Iron (Fe) deficiency anemia remains the largest nutritional deficiency disorder worldwide. How the gut acquires iron from nano Fe(III), especially at the apical surface, is incompletely understood. OBJECTIVE: We developed a novel Fe supplement consisting of nanoparticulate tartrate-modified Fe(III) poly oxo-hydroxide [here termed nano Fe(III)], which mimics the Fe oxide core of ferritin and effectively treats iron deficiency anemia in rats. METHODS: We determined transfer to the systemic circulation of nano Fe(III) in iron-deficient and iron-sufficient outbread Swiss mouse strain (CD1) mice with use of (59)Fe-labeled material. Iron deficiency was induced before starting the Fe-supplementation period through reduction of Fe concentrations in the rodent diet. A control group of iron-sufficient mice were fed a diet with adequate Fe concentrations throughout the study. Furthermore, we conducted a hemoglobin repletion study in which iron-deficient CD1 mice were fed for 7 d a diet supplemented with ferrous sulfate (FeSO4) or nano Fe(III). Finally, we further probed the mechanism of cellular acquisition of nano Fe(III) by assessing ferritin formation, as a measure of Fe uptake and utilization, in HuTu 80 duodenal cancer cells with targeted inhibition of divalent metal transporter 1 (DMT1) and duodenal cytochrome b (DCYTB) before exposure to the supplemented iron sources. Differences in gene expression were assessed by quantitative polymerase chain reaction. RESULTS: Absorption (means ± SEMs) of nano Fe(III) was significantly increased in iron-deficient mice (58 ± 19%) compared to iron-sufficient mice (18 ± 17%) (P = 0.0001). Supplementation of the diet with nano Fe(III) or FeSO4 significantly increased hemoglobin concentrations in iron-deficient mice (170 ± 20 g/L, P = 0.01 and 180 ± 20 g/L, P = 0.002, respectively). Hepatic hepcidin mRNA expression reflected the nonheme-iron concentrations of the liver and was also comparable for both nano Fe(III)- and FeSO4-supplemented groups, as were iron concentrations in the spleen and duodenum. Silencing of the solute carrier family 11 (proton-coupled divalent metal ion transporter), member 2 (Slc11a2) gene (DMT1) significantly inhibited ferritin formation from FeSO4 (P = 0.005) but had no effect on uptake and utilization of nano Fe(III). Inhibiting DCYTB with an antibody also had no effect on uptake and utilization of nano Fe(III) but significantly inhibited ferritin formation from ferric nitrilotriacetate chelate (Fe-NTA) (P = 0.04). Similarly, cellular ferritin formation from nano Fe(III) was unaffected by the Fe(II) chelator ferrozine, which significantly inhibited uptake and utilization from FeSO4 (P = 0.009) and Fe-NTA (P = 0.005). CONCLUSIONS: Our data strongly support direct nano Fe(III) uptake by enterocytes as an efficient mechanism of dietary iron acquisition, which may complement the known Fe(II)/DMT1 uptake pathway.
Assuntos
Duodeno/citologia , Duodeno/efeitos dos fármacos , Ferritinas/administração & dosagem , Nanopartículas/química , Anemia Ferropriva/tratamento farmacológico , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Suplementos Nutricionais , Duodeno/metabolismo , Enterócitos/metabolismo , Compostos Férricos/metabolismo , Ferritinas/farmacocinética , Compostos Ferrosos/administração & dosagem , Compostos Ferrosos/farmacocinética , Hemoglobinas , Hepcidinas/genética , Hepcidinas/metabolismo , Ferro da Dieta/administração & dosagem , Ferro da Dieta/farmacocinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Leucine is well known to regulate protein metabolism in muscle. We recently reported that enteral leucine infusion decreased proteasome activity in human duodenal mucosa and enhanced intestinal cell proliferation, but its effects on gut proteome remain unknown. Therefore, we aimed to assess the effects of an enteral leucine infusion on the whole proteome of duodenal mucosa. In this work, 5 healthy volunteers received for 5h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g kg(-1) h(-1)) or maltodextrins supplemented with leucine (0.035 g kg(-1) h(-1)). At the end of infusion, endoscopic duodenal biopsy samples were collected and analyzed by 2D-PAGE. Eleven protein spots were differentially and significantly (P<0.05) expressed in response to the leucine-supplemented maltodextrins compared with maltodextrins alone. Forty percent of identified proteins by mass spectrometry were located in mitochondria. Four proteins were involved in lipid metabolism: HADHA, ACADVL and CPT2 expressions were reduced, whereas FABP1 expression was increased. In addition, the expression of DHA kinase involved in glycerol metabolism was also downregulated. Finally, leucine supplementation altered the duodenal mucosal proteome by regulating the expression of several enzymes mainly involved in lipid metabolism. These results suggest that leucine supplementation may slowdown fatty acid beta-oxidation in human duodenal mucosa.
Assuntos
Duodeno/metabolismo , Ácidos Graxos/metabolismo , Mucosa Intestinal/metabolismo , Leucina/administração & dosagem , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteoma/metabolismo , Adolescente , Adulto , Duodeno/citologia , Eletroforese em Gel Bidimensional , Feminino , Humanos , Mucosa Intestinal/citologia , Metabolismo dos Lipídeos/fisiologia , Masculino , Oxirredução/efeitos dos fármacosRESUMO
Enteroendocrine cells have a critical role in regulation of appetite and energy balance. I-cells are a subtype of enteroendocrine cells localized in duodenum that release cholecystokinin in response to ingested fat and amino-acids. Despite their potentially pivotal role in nutrient sensing and feeding behaviour, native I-cells have previously been difficult to isolate and study. Here we describe a robust protocol for the isolation and characterization of native duodenal I-cells and additionally, using semi-quantitative RT-PCR we determined that mouse duodenal I-cells contain mRNA transcripts encoding key fatty acid and endocannabinoid receptors including the long chain fatty acid receptors GPR40/FFAR1, GPR120/O3FAR1; short chain fatty acid receptors GPR41/FFAR3 and GPR43/FFAR2; the oleoylethanolamide receptor GPR119 and the classic endocannabinoid receptor CB1. These data suggest that I-cells sense a wide range of gut lumen nutrients and also have the capacity to respond to signals of fatty-acid derivatives or endocannabinoid peptides.
Assuntos
Duodeno/metabolismo , Endocanabinoides/metabolismo , Células Enteroendócrinas/metabolismo , Ácidos Graxos/metabolismo , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G/genética , Animais , DNA Complementar , Duodeno/citologia , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Receptores da Colecistocinina/genética , Receptores da Colecistocinina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
BACKGROUND & AIMS: Long-chain fatty acid receptors G-protein-coupled receptor 40 (GPR40) (FFAR1) and GPR120 have been implicated in the chemosensation of dietary fats. I cells in the intestine secrete cholecystokinin (CCK), a peptide hormone that stimulates digestion of fat and protein, but these cells are rare and hard to identify. We sought to determine whether dietary fat-induced secretion of CCK is directly mediated by GPR40 expressed on I cells. METHODS: We used fluorescence-activated cell sorting to isolate a pure population of I cells from duodenal mucosa in transgenic mice that expressed green fluorescent protein under the control of the CCK promoter (CCK-enhanced green fluorescent protein [eGFP] bacterial artificial chromosome mice). CCK-eGFP cells were evaluated for GPR40 expression by quantitative reverse transcription polymerase chain reaction and immunostaining. GPR40(-/-) mice were bred with CCK-eGFP mice to evaluate functional relevance of GPR40 on long-chain fatty acid-stimulated increases in [Ca(2+)]i and CCK secretion in isolated CCK-eGFP cells. Plasma levels of CCK after olive oil gavage were compared between GPR40(+/+) and GPR40(-/-) mice. RESULTS: Cells that expressed eGFP also expressed GPR40; expression of GPR40 was 100-fold greater than that of cells that did not express eGFP. In vitro, linoleic, oleic, and linolenic acids increased [Ca(2+)]i; linolenic acid increased CCK secretion by 53% in isolated GPR40(+/+) cells that expressed eGFP. In contrast, in GPR40(-/-) that expressed eGFP, [Ca(2+)]i response to linoleic acid was reduced by 50% and there was no significant CCK secretion in response to linolenic acid. In mice, olive oil gavage significantly increased plasma levels of CCK compared with pregavage levels: 5.7-fold in GPR40(+/+) mice and 3.1-fold in GPR40(-/-) mice. CONCLUSIONS: Long-chain fatty acid receptor GPR40 induces secretion of CCK by I cells in response to dietary fat.
Assuntos
Colecistocinina/metabolismo , Duodeno/metabolismo , Células Enteroendócrinas/metabolismo , Ácidos Graxos/metabolismo , Mucosa Intestinal/metabolismo , Óleos de Plantas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Separação Celular/métodos , Colecistocinina/genética , Cromossomos Artificiais Bacterianos , Duodeno/citologia , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Imuno-Histoquímica , Mucosa Intestinal/citologia , Intubação Gastrointestinal , Ácido Linoleico/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ácido Oleico/metabolismo , Azeite de Oliva , Óleos de Plantas/administração & dosagem , Regiões Promotoras Genéticas , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Ácido alfa-Linolênico/metabolismoRESUMO
We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate the neuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.
Assuntos
Ácido Ascórbico/farmacologia , Diabetes Mellitus Experimental , Di-Hidrolipoamida Desidrogenase/metabolismo , Duodeno/efeitos dos fármacos , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/enzimologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Animais , Ácido Ascórbico/administração & dosagem , Peso Corporal/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Suplementos Nutricionais , Duodeno/citologia , Duodeno/enzimologia , Masculino , Ratos , Ratos WistarRESUMO
We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate theneuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6 ) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.(AU)
Assuntos
Animais , Masculino , Ratos , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/farmacologia , Ratos Wistar , Diabetes Mellitus Experimental , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Duodeno/citologia , Duodeno/efeitos dos fármacos , Duodeno/enzimologia , Di-Hidrolipoamida Desidrogenase/metabolismo , Peso Corporal/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Suplementos Nutricionais , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/enzimologiaRESUMO
We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate theneuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6 ) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.
Assuntos
Animais , Masculino , Ratos , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/farmacologia , Diabetes Mellitus Experimental , Di-Hidrolipoamida Desidrogenase/metabolismo , Duodeno/citologia , Duodeno , Duodeno/enzimologia , Neurônios , Neurônios/enzimologia , Ratos Wistar , Suplementos Nutricionais , Membrana Celular , Peso Corporal , Plexo Mientérico , Plexo Mientérico/enzimologiaRESUMO
Lesions of the ventromedial hypothalamus (VMH) result in obesity and enhanced cellular proliferation in various organs, including the pancreas, gastrointestinal tract, and liver. Previous studies have suggested that vagal hyperactivity, rather than overeating, induces the peripheral cell proliferation in VMH-lesioned rats. The goal of the present study was to investigate the mechanism of peripheral cell proliferation in VMH-lesion-induced obesity by infusing rats with the acetylcholine agonist, carbachol, and then measuring cellular proliferation in the pancreas and duodenum using immunohistochemistry. The ventromedial hypothalamus was bilaterally lesioned in five rats. In other rats, the bilateral vagus nerves were ligated (vagotomized), and saline or carbachol was continuously administered by an osmotic minipump (n = 5 in each group). Three days later, rats were killed, and cell proliferation was assessed in the pancreas and the duodenum using immunohistochemistry for proliferating cell nuclear antigen (PCNA). Additionally, cellular proliferation in the duodenum was more precisely examined by assessing incorporation of 5-bromo-2'-deoxyuridine (BrdU). Cellular proliferation was higher in rats that received carbachol infusions and in rats with VMH-lesions when compared with control rats (P < 0.05, respectively). The pancreatic PCNA-expressing cells were predominantly identified as the B-cells of the islets of Langerhans. These data demonstrate that carbachol infusion can induce pancreatic and duodenal cell proliferation to a degree that was comparable to that in vagal hyperactivity induced by VMH lesions.
Assuntos
Carbacol , Proliferação de Células/efeitos dos fármacos , Agonistas Colinérgicos , Hipotálamo , Obesidade/metabolismo , Nervo Vago , Animais , Peso Corporal , Carbacol/administração & dosagem , Carbacol/farmacologia , Agonistas Colinérgicos/administração & dosagem , Agonistas Colinérgicos/farmacologia , Modelos Animais de Doenças , Duodeno/citologia , Duodeno/metabolismo , Ingestão de Alimentos , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Vagotomia , Nervo Vago/efeitos dos fármacos , Nervo Vago/fisiologiaRESUMO
BACKGROUND: Even though coffee is not considered to be responsible for development of peptic ulcer, it may, however, prolong its healing by increasing acidity of gastric content. In our former work we observed a profound increase in sucrose permeability (above normal values) in healthy volunteers regularly drinking coffee for years. In literature, many factors affecting sucrose permeability have been described so far. None of them, however, studied the effect of coffee. SUBJECTS, MATERIALS AND METHODS: 10 young asymptomatic habitual coffee drinkers were included in the study. The probands underwent SaLM test twice--first time without coffee restriction and second time after 48-hour coffee abstinence. The ingested SaLM solution comprised sucrose (25.0 g), lactulose (10.0 g), mannitol (2.0 g), xylose (2.0 g) and water (up to 100 ml). Urine was collected for five hours and the samples were analysed using gas chromatography. Results were compared with those of 8 young healthy volunteers not drinking coffee. Permeability for sucrose was significantly higher in the group of habitual coffee drinkers in comparison with non-coffee drinkers (p < 0.01). After 48-hour coffee abstinence sucrose excretion decreased significantly (p < 0.05) to a level not differing from that of non-coffee drinkers (p = 0.54). CONCLUSIONS: Our results indicate that coffee may damage gastroduodenal mucosa in habitual coffee drinkers. In a time period of 48 hours the gastroduodenal mucosa is capable of a significant regeneration.
Assuntos
Café/efeitos adversos , Duodeno/fisiologia , Mucosa Gástrica/fisiologia , Mucosa Intestinal/fisiologia , Adulto , Duodeno/citologia , Feminino , Humanos , Masculino , Permeabilidade , Sacarose/urinaRESUMO
Dietary supplementation with fish oil that contains omega-3 polyunsaturated fatty acids has been shown to enhance bone density as well as duodenal calcium uptake in rats. The latter process is supported by membrane ATPases. The present in vitro study was undertaken to test the effect of omega-3 fatty acids on ATPase activity in isolated basolateral membranes from rat duodenal enterocytes. Ca-ATPase in calmodulin-stripped membranes was activated in a biphasic manner by docosahexanoic acid (DHA) (10-30 microg/ml) but not by eicosapentanoic acid (EPA). This effect was blocked partially by 0.5 microM calphostin (a protein kinase C blocker). DHA inhibited Na,K-ATPase (-49% of basal activity, [DHA]=30 microg/ml, P <0.01). This effect could be reversed partially by 50 microM genistein, a tyrosine kinase blocker. EPA also inhibited Na,K-ATPase: (-47% of basal activity, [EPA]=30 microg/ml, P <0.01), this effect was partially reversed by 100 microM indomethacin, a cyclo-oxygenase blocker. Omega-3 fatty acids are thus involved in multiple signalling effects that effect ATPases in BLM.
Assuntos
Apirase/metabolismo , Cálcio/metabolismo , Duodeno/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Absorção/efeitos dos fármacos , Animais , Apirase/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Relação Dose-Resposta a Droga , Duodeno/citologia , Duodeno/efeitos dos fármacos , Duodeno/enzimologia , Enterócitos/efeitos dos fármacos , Enterócitos/enzimologia , Enterócitos/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , Naftalenos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , RatosRESUMO
The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17beta-estradiol (17beta-E2), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary Ca2+ on the expression of the duodenal Ca2+ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17beta-E2 increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17beta-E2. Treatment with 17beta-E2 upregulated mRNA levels of duodenal TRPV6 in these 1alpha-OHase knockout mice, which was accompanied by increased serum Ca2+ concentrations from 1.69 +/- 0.10 to 2.03 +/- 0.12 mM (P < 0.05). In addition, high dietary Ca2+ intake normalized serum Ca2+ in these mice and upregulated expression of genes encoding the duodenal Ca2+ transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1alpha-OHase knockout mice, but supplementation with 1,25(OH)2D3 upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17beta-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 only.
Assuntos
Calcitriol/genética , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Mucosa Intestinal/metabolismo , Animais , Calcitriol/farmacologia , Cálcio/metabolismo , Cálcio da Dieta/farmacologia , Duodeno/citologia , Duodeno/metabolismo , Estradiol/sangue , Estradiol/farmacologia , Feminino , Expressão Gênica/fisiologia , Mucosa Intestinal/citologia , Camundongos , Camundongos Knockout , Ovariectomia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Ratos Wistar , Canais de Cátion TRPVRESUMO
The allograft inflammatory factor (AIF-1/daintain) is a hormone-like peptide produced by activated monocytic cells in a variety of traumatic, inflammatory and degenerative lesions. Gut-derived AIF-1 has been shown to modulate insulin production and to attenuate autoimmune diabetes. As the localization of this gastrointestinal peptide in the porcine duodenum is not known and the pig is a convenient model for the study of nutritional modulation of the mucosal immune compartment, we have localized expression of AIF-1 by immunohistology in the duodenum of either malnourished (energy and protein supply 50% of demands, n = 5) or optimally fed pigs (n = 5). AIF-1 macrophages were predominantly located at the villus tip. The number of positively stained cells per high-power field was significantly (P < or = 0.001) higher in the malnourished pigs (74.6 +/- 2.44; least square means +/- SEM) compared to optimally fed pigs (32.56 +/- 1.99). It is likely that the effect in malnourished pigs can be explained by a more pronounced antigen contact of macrophages due to loss of epithelial integrity. Thus, AIF-1 is a novel marker for the study of the nutritional regulation of the mucosal immune system of the pig. AIF-1 expression in the duodenum was further validated by polymerase chain reaction and sequencing. Surprisingly, we detected a slight deviation from the original sequence (probably representing an allelic variation) and an AIF-1 splice variant, previously not known to occur in pigs.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Mucosa Intestinal/metabolismo , Distúrbios Nutricionais/veterinária , Doenças dos Suínos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , DNA Complementar/química , Duodeno/citologia , Duodeno/imunologia , Duodeno/metabolismo , Imunidade nas Mucosas , Imuno-Histoquímica/veterinária , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Dados de Sequência Molecular , Distúrbios Nutricionais/imunologia , Distúrbios Nutricionais/metabolismo , Reação em Cadeia da Polimerase/veterinária , Homologia de Sequência de Aminoácidos , Suínos , Doenças dos Suínos/imunologiaRESUMO
Duodenal ion transport processes are supported by ATPase enzymes in basolateral membranes of the enterocyte. In vivo studies have shown that long term n-6 poly-unsaturated fatty acid (PUFA) supplementation in rats causes increases in intestinal Ca absorption, coupled with a higher total calcium balance and bone calcium content. The present in vitro study was undertaken to test the effect of arachidonic acid (AA), a highly unsaturated (and thus physiologically potent) member of the n-6 PUFA family, on ATPases in enterocyte basolateral membranes isolated with a sorbitol density gradient procedure. This paper presents results which show that AA inhibits Na+,K+-ATPase in a dose-dependent manner (-67% of basal activity at a concentration of 30 microg/ml, P < 0.005) but that this effect is not mediated by protein kinase C, as shown by the use of the protein kinase C blocker calphostin (0.5 microM). Indomethacin (IDM) at 0.1 mM, a cyclo-oxygenase blocker, could also not reverse the inhibitory effect of AA on Na+,K+-ATPase. Ca2+-ATPase, on the other hand, is not affected significantly (-10%, P > 0.05) by arachidonic acid at 30 microg/ml.
Assuntos
Ácido Araquidônico/farmacologia , ATPases Transportadoras de Cálcio/metabolismo , Duodeno/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Enterócitos/enzimologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Ácido Araquidônico/antagonistas & inibidores , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Duodeno/citologia , Duodeno/enzimologia , Indometacina/farmacologia , Masculino , Naftalenos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de TempoRESUMO
Recently our studies have demonstrated that the central nervous system regulates in part mucosal cell growth and apoptosis in the rat small intestine. Ornithine decarboxylase (ODC) activity is a key enzyme for polyamine synthesis which plays an important role for the intestinal mucosal growth. We have demonstrated that the increase of ODC activity in the duodenum just before the dark period is abolished by truncal vagotomy and that the infusion of 2-deoxy-D-glucose into the third cerebroventricle activates ODC activity in the small intestine. Epithelial homeostasis is balanced by regulation of cell proliferation and cell death. Our preliminary data showed that intestinal mucosal apoptosis decreased in the ventromedial-hypothalamus-lesioned rat. These results indicate that the central nervous system, in addition to local factors, is related to regulation of mucosal homeostasis in the intestinal mucosa.
Assuntos
Apoptose , Sistema Nervoso Central/fisiologia , Mucosa Intestinal/citologia , Ornitina Descarboxilase/metabolismo , Animais , Divisão Celular , Ritmo Circadiano , Desoxiglucose/farmacologia , Duodeno/citologia , Duodeno/imunologia , Homeostase , Hipotálamo/patologia , Hipotálamo/fisiologia , Mucosa Intestinal/inervação , Fígado/fisiologia , Masculino , Ratos , Nervo Vago/fisiologiaRESUMO
Ten calves were used to elucidate the ultrastructure of enterocytes before and 24 h after colostral intake. Tissue samples were obtained from duodenum, jejunum (5 locations) and ileum. Protein A-gold technique was applied to immunoelectron-microscopically demonstrate colostral IgA. The prominent feature of the precolostral enterocytes are intracytoplasmic vacuoles. The frequency of vacuoles increases from cranial jejunum to ileum and from the villi bases to the tips. The appearance of absorptive vacuoles after colostral administration correlates with the incidence of precolostral empty vacuoles. Bovine IgA was detected in absorptive vacuoles and within the intestinal lumen of postcolostral calves. In addition to a diffuse IgA labelling of most vacuoles, a few corresponding enterocytic vacuoles labelled inhomogenously or negatively. This study demonstrates morphologically that the main site of colostral absorption is the middle-to-caudal region of the small intestine. Immunoelectron microscopy of IgA labelling provides indications of a selective IgA absorption in addition to pinocytosis.
Assuntos
Animais Recém-Nascidos/anatomia & histologia , Bovinos/anatomia & histologia , Duodeno/citologia , Íleo/citologia , Jejuno/citologia , Animais , Animais Recém-Nascidos/metabolismo , Animais Recém-Nascidos/fisiologia , Bovinos/metabolismo , Bovinos/fisiologia , Colostro/imunologia , Colostro/metabolismo , Duodeno/metabolismo , Duodeno/ultraestrutura , Íleo/metabolismo , Íleo/ultraestrutura , Imunoglobulina A/análise , Imunoglobulina A/metabolismo , Absorção Intestinal/fisiologia , Jejuno/metabolismo , Jejuno/ultraestrutura , Microscopia Eletrônica/veterinária , Microscopia Eletrônica de Varredura/veterinária , Microscopia Imunoeletrônica/veterinária , Fatores de TempoRESUMO
The intestinal absorption of calcium includes at least three definable steps; transfer across the microvillar membrane, movement through the cytosolic compartment, and energy-dependent extrusion into the lamina propria, Tracing the movement of calcium through the epithelium has been hampered by lack of suitable techniques and, in this study, advantage was taken of ion microscopy in conjunction with cryosectioning and use of the stable isotope 44Ca to visualize calcium in transit during the absorptive process. The effect of vitamin D, required for optimal calcium absorption, was investigated. Twenty millimolar 44Ca was injected into the duodenal lumen in situ of vitamin D-deficient and vitamin D-replete chickens. At 2.5, 5.0, and 20.0 min after injection, duodenal tissue was obtained and processed for ion microscopic imaging. At 2.5 min. 44Ca was seen to be concentrated in the region subjacent to the microvillar membrane in tissue from both groups. At 5.0 and 20.0 min, a similar pattern of localization was evident in D-deficient tissues. In D-replete tissues, the distribution of 44Ca became more homogenous, indicating that vitamin D increased the rate of transfer of Ca2+ from the apical to the basolateral membrane, a function previously ascribed to the vitamin D-induced calcium-binding protein (28-kDa calbindin-D). Quantitative aspects of the calcium absorptive process were determined in parallel experiments with the radionuclide 47Ca. Complementary information on the localization of the naturally occurring isotopes of calcium (40Ca) and potassium (39K) is also described.
Assuntos
Cálcio/metabolismo , Duodeno/metabolismo , Absorção Intestinal , Deficiência de Vitamina D/metabolismo , Animais , Transporte Biológico , Isótopos de Cálcio , Radioisótopos de Cálcio , Galinhas , Duodeno/citologia , Cinética , Masculino , Músculo Liso/citologia , Músculo Liso/metabolismo , Valores de ReferênciaRESUMO
Intestinal absorption of colostral lymphoid cells was studied in 23 piglets of four sows (sows A, B, C and D). From the colostrum and blood of the sows the lymphoid cells were isolated with Ficoll-Paque and labelled with technetium (Na99mTcO4). In the 7th hour after birth, 5-ml volumes of the cell suspensions were injected, following laparotomy, directly into the stomach (piglets of sow A) or into the jejunum (piglets of sow B), whereas piglets of sows C and D received the suspensions through a naso-oesophageal tube. Cryostat sections of duodenum, jejunum and lymph node samples of piglets killed by bleeding 8 h after the treatment were examined by autoradiography. It was found that lymphoid cells present in the colostrum of a piglet's own mother were absorbed from the digestive tract and, via the lymphatic vessels, were transported to the mesenteric lymph nodes. Electron microscopy revealed that absorption took place intercellularly. Colostral cells of sows other than a piglet's own mother were observed only in the epithelial layer of the mucous membrane. The lymphoid cells isolated from the sows' blood and heat-treated colostral lymphoid cells were not absorbed. The results indicate that in the pig, an animal having an epitheliochorial placenta, the colostral lymphoid cells are absorbed from the digestive tract and, hence, they can confer an active cellular immunity on the newborn piglets.