RESUMEN
Abstract Diabetic-metabolic syndrome (MetS-D) has a high prevalence worldwide, in which an association with the rupture of the intestinal epithelium barrier function (IEBF) has been pointed out, but the functional and morphological properties are still not well understood. This study aimed to evaluate the impact of acute hyperglycemia diabetes on intestinal tight junction proteins, metabolic failure, intestinal ion and water transports, and IEBF parameters. Diabetes was induced in male Rattus norvegicus (200-310 g) with 0.5 mL of streptozotocin (70 mg/kg). Glycemic and clinical parameters were evaluated every 7 days, and intestinal parameters were evaluated on the 14th day. The MetS-D animals showed a clinical pattern of hyperglycemia, with increases in the area of villi and crypts, lactulose:mannitol ratio, myeloperoxidase (MPO) activity, and intestinal tissue concentrations of malondialdehyde (MDA), but showed a reduction in reduced glutathione (GSH) when these parameters were compared to the control. The MetS-D group had increased secretion of Na+, K+, Cl-, and water compared to the control group in ileal tissue. Furthermore, we observed a reduction in mRNA transcript of claudin-2, claudin-15, and NHE3 and increases of SGLT-1 and ZO-1 in the MetS-D group. These results showed that MetS-D triggered intestinal tissue inflammation, oxidative stress, complex alterations in gene regulatory protein transcriptions of intestinal transporters and tight junctions, damaging the IEBF and causing hydroelectrolyte secretion.
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Strenuous exercise triggers deleterious effects on the intestinal epithelium, but their mechanisms are still uncertain. Here, we investigated whether a prolonged training and an additional exhaustive training protocol alter intestinal permeability and the putative effect of alanyl-glutamine (AG) pretreatment in this condition. Rats were allocated into 5 different groups: 1) sedentary; 2 and 3) trained (50 min per day, 5 days per week for 12 weeks) with or without 6 weeks oral (1.5 g/kg) AG supplementation; 4 and 5) trained and subjected to an additional exhaustive test protocol with or without oral AG supplementation. Venous blood samples were collected to determine gasometrical indices at the end of the 12-week protocol or after exhaustive test. Lactate and glucose levels were determined before, during, and after the exhaustive test. Ileum tissue collected after all experimental procedures was used for gene expression analysis of Zonula occludens 1 (ZO-1), occludin, claudin-2, and oligopeptide transporter 1 (PepT-1). Intestinal permeability was assessed by urinary lactulose/mannitol test collected after the 12-week protocol or the exhaustive test. The exhaustive test decreased pH and base excess and increased pCO2. Training sessions delayed exhaustion time and reduced the changes in blood glucose and lactate levels. Trained rats exhibited upregulation of PEPT-1, ZO-1, and occludin mRNA, which were partially protected by AG. Exhaustive exercise induced intestinal paracellular leakage associated with the upregulation of claudin-2, a phenomenon protected by AG treatment. Thus, AG partially prevented intestinal training adaptations but also blocked paracellular leakage during exhaustive exercise involving claudin-2 and occludin gene expression.
Asunto(s)
Animales , Masculino , Ratas , Permeabilidad/efectos de los fármacos , Condicionamiento Físico Animal/fisiología , Dipéptidos/administración & dosificación , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/fisiopatología , Ratas Wistar , Modelos AnimalesRESUMEN
Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.
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Humanos , Movimiento Celular/fisiología , Proteínas de Unión al GTP rho/fisiología , Factores de Virulencia/genética , Células Epiteliales/microbiología , Escherichia coli Enteropatógena/patogenicidad , Sistemas de Secreción Tipo III/fisiología , Western Blotting , Apoptosis , Factores de Virulencia/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Citometría de FlujoRESUMEN
Undernutrition represents a major public health challenge for middle- and low-income countries. This study aimed to evaluate whether a multideficient Northeast Brazil regional basic diet (RBD) induces acute morphological and functional changes in the ileum of mice. Swiss mice (∼25 g) were allocated into two groups: i) control mice were fed a standard diet and II) undernourished mice were fed the RBD. After 7 days, mice were killed and the ileum collected for evaluation of electrophysiological parameters (Ussing chambers), transcription (RT-qPCR) and protein expression (western blotting) of intestinal transporters and tight junctions. Body weight gain was significantly decreased in the undernourished group, which also showed decreased crypt depth but no alterations in villus height. Electrophysiology measurements showed a reduced basal short circuit current (Isc) in the undernourished group, with no differences in transepithelial resistance. Specific substrate-evoked Isc related to affinity and efficacy (glutamine and alanyl-glutamine) were not different between groups, except for the maximum Isc (efficacy) induced by glucose. Transcription of Sglt1 and Pept1 was significantly higher in the undernourished group, while SN-2 transcription was decreased. No changes were found in transcription of CAT-1 and CFTR, while claudin-2 and occludin transcriptions were significantly increased in the undernourished group. Despite mRNA changes, SGLT-1, PEPT-1, claudin-2 and occludin protein expression showed no difference between groups. These results demonstrate early effects of the RBD on mice, which include reduced body weight and crypt depth in the absence of significant alterations to villus morphology, intestinal transporters and tight junction expression.
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Animales , Masculino , Conejos , Desnutrición/fisiopatología , Desnutrición/metabolismo , Crecimiento/fisiología , Íleon/anatomía & histología , Íleon/metabolismo , Fenómenos Fisiológicos Nutricionales de los Animales , Factores de Tiempo , Peso Corporal , Ingestión de Energía/fisiología , ARN Mensajero , Immunoblotting , Enfermedad Aguda , Transporte Iónico/fisiología , Desnutrición/complicaciones , Modelos Animales de Enfermedad , Absorción Intestinal/fisiologíaRESUMEN
Apolipoprotein E (APOE=gene, apoE=protein) is a known factor regulating the inflammatory response that may have regenerative effects during tissue recovery from injury. We investigated whether apoE deficiency reduces the healing effect of alanyl-glutamine (Ala-Gln) treatment, a recognized gut-trophic nutrient, during tissue recovery after 5-FU-induced intestinal mucositis. APOE-knockout (APOE-/-) and wild-type (APOE+/+) C57BL6J male and female mice (N=86) were given either Ala-Gln (100 mM) or phosphate buffered saline (PBS) by gavage 3 days before and 5 days after a 5-fluorouracil (5-FU) challenge (450 mg/kg, via intraperitoneal injection). Mouse body weight was monitored daily. The 5-FU cytotoxic effect was evaluated by leukometry. Intestinal villus height, villus/crypt ratio, and villin expression were monitored to assess recovery of the intestinal absorptive surface area. Crypt length, mitotic, apoptotic, and necrotic crypt indexes, and quantitative real-time PCR for insulin-like growth factor-1 (IGF-1) and B-cell lymphoma 2 (Bcl-2) intestinal mRNA transcripts were used to evaluate intestinal epithelial cell turnover. 5-FU challenge caused significant weight loss and leukopenia (P<0.001) in both mouse strains, which was not improved by Ala-Gln. Villus blunting, crypt hyperplasia, and reduced villus/crypt ratio (P<0.05) were found in all 5-FU-challenged mice but not in PBS controls. Ala-Gln improved villus/crypt ratio, crypt length and mitotic index in all challenged mice, compared with PBS controls. Ala-Gln improved villus height only in APOE-/- mice. Crypt cell apoptosis and necrotic scores were increased in all mice challenged by 5-FU, compared with untreated controls. Those scores were significantly lower in Ala-Gln-treated APOE+/+ mice than in controls. Bcl-2 and IGF-1 mRNA transcripts were reduced only in the APOE-/--challenged mice. Altogether our findings suggest APOE-independent Ala-Gln regenerative effects after 5-FU challenge.
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Animales , Femenino , Masculino , Antimetabolitos Antineoplásicos/efectos adversos , Apolipoproteínas E/deficiencia , Dipéptidos/farmacología , Fluorouracilo/efectos adversos , Mucosa Intestinal/efectos de los fármacos , Mucositis/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Peso Corporal , Dipéptidos/uso terapéutico , Factor I del Crecimiento Similar a la Insulina/análisis , Mucosa Intestinal/patología , Recuento de Leucocitos , Linfoma de Células B , Mitosis/efectos de los fármacos , Mucositis/inducido químicamente , Mucositis/patología , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Factores de Tiempo , Resultado del TratamientoRESUMEN
The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.
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Animales , Toxinas Bacterianas/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Hormonas Gastrointestinales/genética , Guanilato Ciclasa/fisiología , Péptidos Natriuréticos/genética , Equilibrio Hidroelectrolítico/fisiología , Adenilil Ciclasas/fisiología , Toxinas Bacterianas/aislamiento & purificación , Evolución Molecular , Enterotoxinas/aislamiento & purificación , Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Predicción , Guanilato Ciclasa/uso terapéutico , Mamíferos/fisiología , Péptidos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.
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Animales , Masculino , Ratas , Canales de Calcio/efectos de los fármacos , Carbacol/farmacología , Piperazinas/farmacología , Sulfonas/farmacología , Canales Catiónicos TRPC/efectos de los fármacos , Tráquea/efectos de los fármacos , Vasodilatadores/farmacología , Canales de Calcio/metabolismo , Carbacol/antagonistas & inhibidores , Expresión Génica , Lactonas/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Óxido Nítrico/metabolismo , Ovalbúmina/farmacología , Purinas/farmacología , Ratas Wistar , Sesquiterpenos/farmacología , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Tráquea/metabolismo , Tráquea/fisiopatologíaRESUMEN
A series of studies have shown that the heavy burdens of diarrheal diseases in the first 2 formative years of life in children living in urban shanty towns have negative effects on physical and cognitive development lasting into later childhood. We have shown that APOE4 is relatively common in shanty town children living in Brazil (13.4 percent) and suggest that APOE4 has a protective role in cognitive development as well as weight-for-height in children with heavy burdens of diarrhea in early childhood (64/123; 52 percent), despite being a marker for cognitive decline with Alzheimers and cardiovascular diseases later in life. APOE2 frequency was higher among children with heaviest diarrhea burdens during the first 2 years of life, as detected by PCR using the restriction fragment length polymorphism method, raising the possibility that ApoE-cholesterol balance might be critical for growth and cognitive development under the stress of heavy diarrhea burdens and when an enriched fat diet is insufficient. These findings provide a potential explanation for the survival advantage in evolution of genes, which might raise cholesterol levels during heavy stress of diarrhea burdens and malnutrition early in life.
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Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Apolipoproteínas E/genética , Diarrea Infantil/genética , Polimorfismo Genético/genética , Apolipoproteínas E/metabolismo , Brasil , Desarrollo Infantil , Cognición , Estudios de Cohortes , Diarrea Infantil/complicaciones , Diarrea Infantil/metabolismo , Frecuencia de los Genes , Genotipo , Mucosa Bucal/citología , Reacción en Cadena de la Polimerasa , Factores SocioeconómicosRESUMEN
Because thalidomide and pentoxifylline inhibit the synthesis and release of tumor necrosis factor-alpha (TNF-alpha), we determined the effect of these drugs on the renal damage induced by supernatants of macrophages activated with Crotalus durissus cascavella venom in order to identify the role of TNF-alpha in the process. Rat peritoneal macrophages were collected with RPMI medium and stimulated in vitro with C.d. cascavella venom (10 µg/ml) in the absence and presence of thalidomide (15 µM) or pentoxifylline (500 µM) for 1 h and washed and kept in culture for 2 h. Supernatant (1 ml) was tested on an isolated perfused rat kidney (N = 6 for each group). The first 30 min of each experiment were used as control. The supernatant was added to the perfusion system. All experiments lasted 120 min. The toxic effect of the preparation of venom-stimulated macrophages on renal parameters was determined. At 120 min, thalidomide (Thalid) and pentoxifylline (Ptx) inhibited (P < 0.05) the increase in perfusion pressure caused by the venom (control = 114.0 ± 1.3; venom = 137.1 ± 1.5; Thalid = 121.0 ± 2.5; Ptx = 121.4 ± 4.0 mmHg), renal vascular resistance (control = 4.5 ± 0.2; venom = 7.3 ± 0.6; Thalid = 4.5 ± 0.9; Ptx = 4.8 ± 0.6 mmHg/ml g-1 min-1), urinary flow (control = 0.23 ± 0.001; venom = 0.44 ± 0.01; Thalid = 0.22 ± 0.007; Ptx = 0.21 ± 0.009 ml g-1 min-1), glomerular filtration rate (control = 0.72 ± 0.06; venom = 1.91 ± 0.11; Thalid = 0.75 ± 0.04; Ptx = 0.77 ± 0.05 ml g-1 min-1) and the decrease in percent tubular sodium transport (control = 77.0 ± 0.9; venom = 73.9 ± 0.66; Thalid = 76.6 ± 1.1; Ptx = 81.8 ± 2.0 percent), percent tubular chloride transport (control = 77.1 ± 1.2; venom = 71.4 ± 1.1; Thalid = 77.6 ± 1.7; Ptx = 76.8 ± 1.2 percent), and percent tubular potassium transport (control = 72.7 ± 1.1; venom = 63.0 ± 1.1; Thalid = 72.6 ± 1.0; Ptx = 74.8 ± 1.0 percent), 30 min before and during the stimulation of macrophages with C.d. cascavella venom. These data suggest the participation of TNF-alpha in the renal effects induced by supernatant of macrophages activated with C.d. cascavella venom.
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Animales , Masculino , Femenino , Ratas , Venenos de Crotálidos , Inmunosupresores , Pentoxifilina , Talidomida , Factor de Necrosis Tumoral alfa , Riñón , Activación de Macrófagos , Macrófagos Peritoneales , Ratas WistarRESUMEN
The excretion ratio of lactulose/mannitol in urine has been used to assess the extension of malabsorption and impairment of intestinal permeability. The recovery of lactulose and mannitol in urine was employed to evaluate intestinal permeability in children with and without diarrhea. Lactulose and mannitol probes were measured using high-performance liquid chromatography with pulsed amperometric detection (HPLC-PAD). Two groups of solutions containing 60 µM sugars were prepared. Group I consisted of glucosamine, mannitol, melibiose and lactulose, and group II of inositol, sorbitol, glucose and lactose. In the study of intra-experiment variation, a sample of 50 µl from each group was submitted to 4 successive determinations. The recovered amounts and retention times of each sugar showed a variation <2 and 1 per cent, respectively. The estimated recovery was >97 per cent. In the study of inter-experiment variation, we prepared 4 independent samples from groups I and II at the following concentrations: 1.0, 0.3, 0.1, 0.03 and 0.01 mM. The amounts of the sugars recovered varied by <10 per cent, whereas the retention times showed an average variation <1 per cent. The linear correlation coefficients were >99 per cent. Retention (k'), selectivity (a) and efficiency (N) were used to assess the chromatographic conditions. All three parameters were in the normal range. Children with diarrhea presented a greater lactulose/mannitol ratio compared to children without diarrhea.
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Humanos , Preescolar , Diarrea/metabolismo , Diuréticos Osmóticos/orina , Fármacos Gastrointestinales/orina , Intestinos/metabolismo , Lactulosa/orina , Manitol/orina , Cromatografía Líquida de Alta Presión , PermeabilidadRESUMEN
In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 ñ 0.19 and Dex = 0.35 ñ 0.13 vs saline (S) = 2.85 ñ 0.59; fMLP: Ptx = 0.43 ñ 0.09 and Dex 0.01 ñ 0.01 vs S = 1.08 ñ 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 ñ 1.4 and Dex = 3.06 ñ 0.76 vs S = 15.94 ñ 3.97; fMLP: Ptx = 3.85 ñ 0.56 and Dex = 0.40 ñ 0.16 vs S = 7.15 ñ 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 µm), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 ñ 0.38 vs S = 4.20 ñ 1.01; fMLP: Dex = 0.25 ñ 0.11 vs S = 2.20 ñ 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 ñ 2.25 vs S = 4.20 ñ 1.01; fMLP: Ptx = 4.66 ñ 1.24 vs S = 2.20 ñ 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.
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Animales , Masculino , Ratas , Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Dexametasona/farmacología , Mesenterio/patología , Neutrófilos/efectos de los fármacos , Toxina del Pertussis/farmacología , Escherichia coli , Inflamación/inducido químicamente , Recuento de Leucocitos , Lipopolisacáridos/efectos adversos , Venas Mesentéricas , N-Formilmetionina Leucil-Fenilalanina/efectos adversos , Ratas WistarRESUMEN
Guanylin is an endogenous peptide synthesized by several mammalian species that mimics the effects of a thermostable enterotoxin of Escherichia coli (STa: NTFYCCELCCNPACAGCY) in the gut. We have cloned a lysine-1 derivative of rat guanylin (Lys-1-NTCEICAYAACTGC) and tested its effects on ileal tissue membranes in Ussing chambers and in the isolated perfused rat kidney. Rabbit ileal mucosa membranes were mounted into a Ussing chamber and the effects of Lys-1 guanylin (Lys-1 G) and STa enterotoxin peptide on chloride secretion were determined by changes in short-circuit current (Isc). Lys-1 G (10 to 100 nM) showed a dose-dependent effect on chloride secretion with a maximal response estimated to be 52 microA/cm2. Lys-1 G mimics the effect of STa peptide, but the enterotoxin elicited a greater maximal effect of 120 microA/cm2 (p<0.01). Lys-1 G (2.5 microg/ml) promoted an increase in both urine flow (from 0.13 +/- 0.07 to 0.40 +/- 0.01 ml g(-1) min(-1), N = 4; P<0.05) and glomerular filtration rate (from 0.68 +/- 0.02 to 0.85 0.00 ml g(-1) min(-1), N = 4; P<0.01) in the isolated perfused kidney and a reduction of the fractional reabsorption of sodium (from 76.0 +/- 0.03 to 59.5 +/- 0.85 percent, N = 4; P<0.01). These maximal effects were accompanied by intense natriuretic effect observed 30 and 60 min after drug administration. The Lys-1 G analog similar to STa enterotoxin elicited intestinal chloride secretion and a natriuretic effect. These data demonstrate that the cloned peptide analog retains the biological activity of the native hormone and presents activity similar to STa. The properties of Lys-1 G resemble those of a factor formed during perfusion of the hypoxic rabbit kidney and named by us factor natriureticus similis (FNS).
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Animales , Masculino , Femenino , Ratas , Conejos , Riñón/efectos de los fármacos , Lisina/análogos & derivados , Natriuresis/efectos de los fármacos , Secreciones Intestinales , Riñón/fisiología , Sodio/metabolismoRESUMEN
Pertussis toxin (Ptx) is a hexameric protein with classical AB architecture produced by Bordetella pertussis. The aim of this study was to investigate the effect og Ptx on migration of polymorphonuclear leukocytes to site of inflamation and on cell- dependent edema. Ptx was purified from the supernatant of the culture medium of B. pertussis using hydroxylapatite chromatography and fetuin affinity chromatography. Ptx induced a maximal clusterin of Chinese hamster ovary cells at concentration as low as 0.1 ng/ ml. Intravenous inection of Ptx (400 ng) significantly blocked the neutrophil migration induced by 200 ng of lipopolysaccharide (LPS from E. coli O111:B4; 2.27 ñ 0.13 vs 0.61 ñ 0.16 per 10**6 neutrophils/ml; P < 0.001; N = 5) and by 200 ng of formylmethionyl-leucyl-phenylalanine(fMLP; 2.53 ñ 0.45 vs 0.75 ñ 0.14 per 10**6 neutrophils/ml; P < 0.01; N=6) into the peritoneal cavities of male Wistar rats (eighing 150-180). In addition, Ptx (400ng) pretreatment also blocked the edema induced by intraplantar injection of 100 µg carrageenin ( increase in volume: 0.667 ñ 0.087 vs 0.313 ñ 0.058 ml; P < 0.01; N = 5) but not the edema induced by 100 µg dextran ( increase in volume: 0.537 ñ 0.06 vs 0.385 ñ 0.076 ml; P > 0.05; N = 5). These data demonstrate that Ptx blocked neutrophil migration induced by a direct f MLP stimulus of a site of inflammation. In addition, this toxin blocks the indirect stimulus of LPS on neutrophil migration. Furthermore, Ptx also inhibits the neutrophil-dependent edema induced by carrageenin, but not the edema induced by dextran that is in part dependent on basophil cells. These results warrant further studies on the mechanisms of Ptx inhibition of neutrophil-dependent edema and cell migration
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Animales , Masculino , Ratas , Inhibición de Migración Celular , Inflamación/fisiopatología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/fisiología , Toxina del Pertussis/farmacología , Carragenina/farmacología , Dextranos/farmacología , Lipopolisacáridos/farmacología , Toxina del Pertussis/aislamiento & purificaciónRESUMEN
Cholera toxin peptide stimulates adenylyl cyclase activity in several tissues and causes severe intestinal water and electrolyte secretion. To evaluate the regulatory function of sodium transport in renal tubules, we studied the effect of cholera toxin peptide on rat kidneys. Isolated kidneys from adult male hooded rats weighting 240-335 g were perfused with Krebs-Henseleit solution containing 60 mg/ml dialyzed bovine serum albumin (BSA). The effects of Vibrio cholerae peptide (CT; molecular weight, approximately 82,000 Dalton) on glomerular filtration rate (GFR), proximal sodium reabsorption ( per cent pTNa+) and urinary flow rate (UF) were studied. All experiments were preceded by a 30-min control period and in another group of kidneys the time course of the variables was followed without toxin infusion, for a paired control. Control kidneys perfused with Krebs-Henseleit solution plus 60 mg/ml BSA presented stable GFR (paired internal control GFR30 min = 0.596 +/- 0.248 ml g-1 min-1 vs GFR120 min = 0.694 +/- 0.362, N = 32; P > 0.05) and per cent pTNa+ ( per cent pTNa+ 30 min = 75 +/- 8.3 vs 84 +/- 1.6 for the internal control, N = 32; P > 0.05). CT caused a dose (0.03, 0.75 and 1.0 microgram/ml)-dependent decrease in GFR starting at 30 min and with a maximal peak of effect at 90 min after toxin infusion (GFRCT = 0.130 +/- 0.086 ml g-1 min-1, N = 12, vs paired internal control GFRControl/30 min = 0.660 +/- 0.132, N = 12; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
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Animales , Masculino , Ratas , Enterotoxinas/farmacología , Sodio/metabolismo , Túbulos Renales Proximales , Vibrio cholerae , Tasa de Filtración Glomerular , Factores de Tiempo , Transporte Biológico ActivoRESUMEN
To investigate the effect of substrates during oral rehydration therapy, we studied intestinal cation cotransport (ICC) with glutamine (Gln) alanine (Ala) and glucose (Glu). The specific aims were to determine the biological effects of these three different cotransport systems on intestinal function. Isolated rabbit ileal mucosa preparations mounted in Ussing chambers were studied. ICC was determined by measuring short-circuit current (Isc) and potential difference (PD) while monitoring toissue resistance (TR). The data are reported as the mean ñ SEM of 4-6 experiments for each amino acid concentration. Increasing concentrations of Gln (10-5 to 10-2 M), Ala (10-5 to 10-1 M) and Glu (10-5 to 10-2 M) caused a significant (P<0.05) increase in ICC. Glin (30 mM) and Ala (0.1 M) had a maximal effects (Em (Glin)=100% anmd Em (Ala)=66%, P<0.05) which was higher than that obtained with 30 mM Glu (Em (Glu)=35%). When sodium was replaced with choline on the mucosal side. Ringer solution completeley abolished the response with Gln, Ala and Glu. The presence of all three substrates (10-2 M gln, 10-1 M Ala and 10-2 M Glu) in Ringer solution on the mucosal side caused a significant increase in ICC ( increase of short circuit current = III ñ 43 uA, P<0.05). These results demonstrate that Glin, Ala and Glu each increased sodium-dependent cation cotransport, and that sodium-dependent intestinal cation cotransport was higher with Gln than with Ala or Glu
Asunto(s)
Conejos , Alanina , Aminoácidos , Fluidoterapia , Glucosa , Glutamina , Íleon , Intestinos/fisiología , Membrana Mucosa , Sodio , CationesRESUMEN
Hyperbolic and allosteric mathematical equations are used extensively to describe cumulative dose-response curves in pharmacology. In 1954, using an axiom described by Clark, Ariëns suggested the following equation for cumulative dose-response curves: Em/E-1 = Kd/[D], where Em = maximum effect, E = effect, Kd = dissociation constant of the drug-receptor complex and [D] = drug concentration. To evaluate the validity of the modified Ariëns equation on the dose-response curves with a Hill coefficient statistically different from one, we used sodium intestinal cotransport (SIC) with glutamine (Gln) and glucose (Glu). Increasing doses (10 microM to 0.1 M) of Gln and Glu were added to the mucosal side of the isolated rabbit ileum mucosal membrane mounted on Ussing chambers. Sodium cotransport was recorded by measuring short-circuit current (Isc), potential difference (PD) and tissue resistance (TR) at 20-min intervals for 2 h. The Em and ED50 for SIC with glucose were 25% and 150 microM, respectively, using the method proposed by Lineweaver-Burk. The same method could not be used to estimate the Em and ED50 in SIC with glutamine, suggesting a specific limitation of the Gln cotransport kinetic curve in the equation. Therefore, we modified the Ariëns equation to Em/E-1 = (Kd/[D])p. With p = Log (S), Log (Kd) = -pD2 and Log[D] = -pDx, we obtain E = Em/(1 + S(pD x -pD2)), where S is a hyperbolic constant.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Animales , Glucosa/metabolismo , Glutamina/metabolismo , Técnicas In Vitro , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Modelos Biológicos , Sodio/metabolismo , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucosa/administración & dosificación , Glutamina/administración & dosificación , Íleon/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Matemática , ConejosRESUMEN
Malnutrition and dehydration are the immediated consequences of diarrheal diseases. To investigate the biological significance ofglutamine, alanine and glucose in the intestinal mucosa, we have used Ussing chambers to determine electrolyte transport by measuring short-circuit current (Isc), potential difference (PD) and tissue resistance (TR) in rabbit intestinal mucosa. Increasing doses (10-5 M to 10-1 M)of glutamine,alanine and glucose cause a significant increase in intestinal cation cotransport. Although glucose had a slightly earlier effect, 30mM glutamine and 0.1 M alanine had a maximal effect which was more than two times that caused by 30 nM glucose. The pD2 values for glucose, glutamine and alanine were 3.0, 2.5, and 2.0, respectively. The dose-response curves of these substrates suggest that the intestinal cotransport kinetics for glutamine is differentfrom that of glucose and alanine. Our results demonstrated that all three substrates cause a significant increase in Isc or PD, suggesting an increase in the intestinal mucosa cation cotransport. Glutamine has a larger effect on cation cotransport than alanine and glucose. These combinations should be studied further for the development of an oral rehydrating solution for diarrhea treatment which could prevent the resulting malnutrition, especially in those cases of prolonged diarrheal diseases