RESUMEN
Three water channel proteins, aquaporins, have been shown to be expressed in the placentae of humans and sheep-AQP1, 3, 8; AQP1 is in the vasculature, whereas AQP3, 8 are in the trophoblast cells. In this study we used the sensitive and reproducible technique of real-time PCR to compare the level of expression of the mRNAs for AQP1, 3 and 8 in the ovine placenta at five stages of gestation (27, 45, 66, 100 and 140 days-where term approximately 150 days). AQP3 was quantitatively the most highly expressed AQP at 66, 100, and 140 days). At 27 days before significant trophoblast development had occurred, the only AQP present was AQP1, in the vasculature. The expression of these aquaporins underlie the high water and urea permeability of the ovine placenta in the last half of pregnancy.
Asunto(s)
Acuaporinas/genética , Expresión Génica , Canales Iónicos/genética , Placenta/química , Ovinos , Animales , Acuaporina 1 , Acuaporina 3 , Femenino , Edad Gestacional , Reacción en Cadena de la Polimerasa , Embarazo , ARN Mensajero/análisis , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The concept that 'life before birth' or the 'first environment' is important in determining subsequent risk for the development of cardiovascular/metabolic disease is now gaining acceptance. There are substantial data from animal experiments that complement and enhance the epidemiological data from human studies. We argue that any factor which disrupts nephrogenesis, and lowers nephron number, during the period of active nephrogenesis, will induce malapadaptive changes in the future functioning of that kidney and predispose to the onset of adult hypertension. Such factors include exposure of the mother, to a particular low-protein diet, excess synthetic or natural glucocorticoid at certain critical periods, mild vitamin A deficiency, elevated blood glucose, unilateral nephrectomy during the period of nephrogenesis, as well as the deletion of one allele of a gene (GDNF) involved in normal metanephric development. All of these stresses are associated with a reduction (20-40 per cent) in total nephron number in the adult, and the development of hypertension. In some hypertensive models, (rats) there is evidence of alterations in the components of the hippocampal/hypothalamic/pituitary/adrenal axis, whereas in others (sheep) there are alterations in the expression of angiotensinogen (hypothalamus) and angiotensin II receptor type I (AT(1)) in the medulla oblongata. The surprising finding is that the period when the kidney and brain are most vulnerable is very early in development, when both organs are in an extremely primitive state of development.
Asunto(s)
Encéfalo/embriología , Sistema Cardiovascular/embriología , Hipertensión/embriología , Hipertensión/etiología , Riñón/embriología , Angiotensinas/fisiología , Animales , Encéfalo/efectos de los fármacos , Sistema Cardiovascular/efectos de los fármacos , Modelos Animales de Enfermedad , Desarrollo Embrionario y Fetal/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucocorticoides/administración & dosificación , Glucocorticoides/fisiología , Humanos , Riñón/efectos de los fármacos , Masculino , Embarazo , OvinosRESUMEN
Fetal lung development is dependent upon secretion of liquid into the future airways which must be cleared at birth to establish air-breathing. Aquaporins (AQP) 1, 3, 4 and 5 are membranous water channel proteins that are present in the lung after birth in rodents, with little expression before birth. Our aim was to describe the changes in AQP1, 3, 4 and 5 expression and protein levels in the fetal lung of a long-gestation species (sheep) and in response to physiological factors known to alter fetal lung liquid dynamics. Both mRNA and high protein levels were detected for AQP1, 3, 4 and 5 by day 100 (term is ~150 days in ovine fetuses). A cortisol infusion (120-131 days) significantly (P < 0.05) increased AQP1 (0.9 +/- 0.2 (n = 4) vs.1.8 +/- 0.3 (n = 5)) and AQP5 (8.8 +/- 0.6 vs. 14.1 +/- 1.2) mRNA levels in fetal lung (measured by real-time PCR). Ten days of tracheal obstruction significantly (P < 0.05) decreased AQP5 mRNA levels (6.1 +/- 0.9 (n = 5) vs. 2.7 +/- 0.3 (n = 5)). Immunohistochemistry was used to show that protein levels changed in parallel with the mRNA changes. These findings suggest that AQPs could be involved in lung liquid production and reabsorption during fetal development in long-gestation species.
Asunto(s)
Acuaporinas/biosíntesis , Acuaporinas/genética , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Pulmón/metabolismo , Envejecimiento/genética , Envejecimiento/fisiología , Algoritmos , Secuencia de Aminoácidos , Animales , Antiinflamatorios/farmacología , Western Blotting , Clonación Molecular , ADN Complementario/biosíntesis , Femenino , Hidrocortisona/farmacología , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/genética , Embarazo , ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ovinos , Tráquea/fisiologíaRESUMEN
IL-6 expression in skeletal muscle is stimulated by contractions. We sought to examine whether hyperinsulinaemia increases IL-6 mRNA in skeletal muscle and whether any increase is modified in insulin resistant muscle. We hypothesized that intramuscular IL-6 mRNA would be increased in response to insulin, but such an affect would be unaffected by insulin resistance because the primary insulin sensitive signalling protein responsible for activating IL-6 functions normally in insulin resistant muscle. Transgenic rats over-expressing the gluconeogenic regulatory enzyme phosphoenolpyruvate carboxykinase (PEPCK) were studied. White gastrocnemius muscle samples were obtained under hyperinsulinaemic, euglycaemic clamp (4 mU kg(-1)min(-1) insulin, plasma glucose concentration 4-6 mmol L(-1)) and basal conditions in both PEPCK (basal n=4; insulin n=5) and wild-type (CON) (basal n=5; insulin n=4) rats, which were previously injected with a bolus of 2-[1-14C]deoxyglucose (2-DG) into the carotid artery. Muscle samples were assayed for 2-DG uptake and IL-6 mRNA. No differences in 2-DG uptake or IL-6 mRNA were observed when comparing groups under basal conditions. Under clamp conditions, 2-DG uptake was lower (P<0.05) in PEPCK compared with CON. Insulin stimulation in CON did not change IL-6 mRNA compared with basal levels. In contrast, there was an approximately 8-fold increase (P<0.05) in IL-6 mRNA in insulin-stimulated PEPCK compared with CON basal levels. Insulin stimulation increases IL-6 gene expression in insulin resistant, but not healthy, skeletal muscle, suggesting that IL-6 expression in skeletal muscle is sensitive to changes in insulin in circumstances of insulin resistance. It is likely that the differences observed when comparing healthy with insulin resistant muscle are due to the differential activation of insulin sensitive signalling proteins responsible for activating IL-6.
Asunto(s)
Resistencia a la Insulina/fisiología , Insulina/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Músculo Esquelético/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Glucemia/metabolismo , Desoxiglucosa/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , RatasRESUMEN
1. When pregnant ewes and their fetuses are exposed to the synthetic glucocorticoid dexamethasone for 2 days early in pregnancy (days 26-28; term 145-150 days), female offspring have increased blood pressure relative to a control group. In one series, this was shown to be due to increased cardiac output, concomitant with a reset mean arterial pressure/heart rate reflex. The first group of such animals had, by the age of 7 years, left ventricular hypertrophy and reduced cardiac functional capacity. 2. The elevation in blood pressure is not maintained by any change in the peripheral renin-angiotensin system (RAS). 3. There is, however, preliminary evidence that some aspects of local RAS (particularly in the kidney and brain) could have participated in the 'programming' event. The levels of mRNA for angiotensin II receptors (AT1, AT2) and angiotensinogen are increased in the kidney of such dexamethasone-treated fetuses in late gestation (130 days), some 100 days after steroid treatment. Similar increases in AT1 mRNA in the medulla oblongata of the fetal brain and large increases of mRNA for angiotensinogen occur in the hypothalamus. 4. These findings, together with evidence from the literature, suggest that both the kidney and parts of the brain are affected by events that also 'program' high blood pressure in the offspring of animals in which the intra-uterine environment has been perturbed at some stage.
Asunto(s)
Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda/fisiopatología , Riñón/anomalías , Efectos Tardíos de la Exposición Prenatal , Sistema Renina-Angiotensina/fisiología , Animales , Presión Sanguínea , Femenino , Hipertensión/inducido químicamente , Riñón/crecimiento & desarrollo , Modelos Animales , Embarazo , Sistema Renina-Angiotensina/efectos de los fármacos , Factores Sexuales , Ovinos , Esteroides/farmacologíaRESUMEN
1. The present study was undertaken to examine the effects of exercise and carbohydrate (CHO) ingestion on interleukin-6 (IL-6) gene expression in skeletal muscle and plasma IL-6 concentration. 2. Seven moderately trained men completed 60 min of exercise at a workload corresponding to each individual's lactate threshold on four randomised occasions. Two trials were conducted on a bicycle ergometer (Cyc) and two on a running treadmill (Run) either with (CHO) or without (Con) the ingestion of a CHO beverage throughout the exercise. Muscle biopsies were obtained from the vastus lateralis before and immediately after exercise and IL-6 gene expression in these samples was determined using real-time PCR. In addition, venous blood samples were collected at rest, and after 30 min during and at the cessation of exercise. These samples were analysed for plasma IL-6. 3. Irrespective of exercise mode or CHO ingestion, exercise resulted in a 21 +/- 4-fold increase (P < 0.01; main exercise effect) in IL-6 mRNA expression. In contrast, while the mode of exercise did not affect the exercise-induced increase in plasma IL-6, CHO ingestion blunted (P < 0.01) this response. 4. These data demonstrate that CHO ingestion attenuates the plasma IL-6 concentration during both cycling and running exercise. However, because IL-6 mRNA expression was unaffected by CHO ingestion, it is likely that the ingestion of CHO during exercise attenuates IL-6 production by tissues other than skeletal muscle.
Asunto(s)
Carbohidratos de la Dieta/administración & dosificación , Ejercicio Físico/fisiología , Interleucina-6/sangre , Interleucina-6/genética , Músculo Esquelético/fisiología , Adulto , Biopsia , Glucemia , Prueba de Esfuerzo , Expresión Génica/fisiología , Glucógeno/análisis , Humanos , Masculino , Músculo Esquelético/química , Músculo Esquelético/citología , ARN Mensajero/análisisRESUMEN
BACKGROUND: At 27 days of gestation in the ovine fetus (term = 145 to 150 days), the only kidney is the mesonephros, and allantoic fluid represents fetal urine. The hypothesis tested in this study was that functional glucocorticoid receptors (GRs) are present in this early mesonephric kidney. METHODS: Pregnant ewes, between 26 and 30 days, were infused with saline, dexamethasone (0.48 mg/hour), cortisol (5 mg/hour), or aldosterone (10 microg/hour) for 48 hours and were then killed for collection of fetuses and fetal fluids. GR mRNA was measured by real-time polymerase chain reaction in whole fetuses, and the location of gene expression was determined by hybridization histochemistry. RESULTS: Significant changes in allantoic fluid composition were produced by the exposure of the fetus to maternally infused synthetic (dexamethasone) and natural (cortisol) glucocorticoids, over a period of two days, compared with fetuses of ewes infused with vehicle (isotonic saline; N = 8) or aldosterone (N = 8). Volume of fluid was unchanged by any treatment, but both dexamethasone (N = 10) and cortisol (N = 8) caused significant (P < 0.05) decreases in sodium and chloride concentrations and increases in concentrations of potassium, urea, glucose, and fructose. GR mRNA was detected in equivalent concentrations in the whole fetuses of saline, dexamethasone, and cortisol treatments. The GR mRNA levels were significantly decreased in the aldosterone group. By hybridization histochemistry, GR mRNA was detected in most of the tubular cells of the mesonephros. CONCLUSION: These results suggest that functional GRs are present in the early ovine mesonephros.
Asunto(s)
Feto/metabolismo , Mesonefro/metabolismo , Receptores de Glucocorticoides/metabolismo , Ovinos/embriología , Alantoides/metabolismo , Líquido Amniótico/metabolismo , Animales , Líquidos Corporales/efectos de los fármacos , Líquidos Corporales/metabolismo , Sistemas de Computación , Dexametasona/farmacología , Femenino , Glucocorticoides/farmacología , Histocitoquímica , Hidrocortisona/farmacología , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Embarazo , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/genética , Ovinos/sangreRESUMEN
Recent evidence suggests that heat shock proteins (Hsps) may have an important systemic role as a signal to activate the immune system. Since acute exercise is known to induce Hsp72 (the inducible form of the 70-kDa family of Hsp) in a variety of tissues including contracting skeletal muscle, we hypothesized that such exercise would result in the release of Hsp72 from stressed cells into the blood. Six humans (5 males, 1 female) ran on a treadmill for 60 minutes at a workload corresponding to 70% of their peak oxygen consumption. Blood was sampled from a forearm vein at rest (R), 30 minutes during exercise, immediately postexercise (60 minutes), and 2, 8, and 24 hours after exercise. These samples were analyzed for serum Hsp72 protein. In addition, plasma creatine kinase (CK) was measured at these time points as a crude marker of muscle damage. With the exception of the sample collected at 30 minutes, muscle biopsies (n = 5 males) were also obtained from the vastus lateralis at the time of blood sampling and analyzed for Hsp72 gene and protein expression. Serum Hsp72 protein increased from rest, both during and after exercise (0.13 0.10 vs 0.87+/-0.24 and 1.02+/-0.41 ng/mL at rest, 30 and 60 minutes, respectively, P < 0.05, mean SE). In addition, plasma CK was elevated (P < 0.05) 8 hours postexercise. Skeletal muscle Hsp72 mRNA expression increased 6.5-fold (P < 0.05) from rest 2 hours postexercise, and although there was a tendency for Hsp72 protein expression to be elevated 2 and 8 hours following exercise compared with rest, results were not statistically significant. The increase in serum Hsp72 preceded any increase in Hsp72 gene or protein expression in contracting muscle, suggesting that Hsp72 was released from other tissues or organs. This study is the first to demonstrate that acute exercise can increase Hsp72 in the peripheral circulation, suggesting that during stress these proteins may indeed have a systemic role.
Asunto(s)
Ejercicio Físico , Proteínas HSP70 de Choque Térmico/sangre , Adulto , Biopsia , Creatina Quinasa/sangre , Prueba de Esfuerzo , Femenino , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Humanos , Masculino , Contracción Muscular , Músculo Esquelético/metabolismo , Consumo de Oxígeno , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Renal and cardiovascular responses to an intravenous infusion of ANG II (1 microg/h) or saline for 3 days were examined in ovine fetuses at midgestation (75-85 days of gestation, term 150 days). ANG II caused an increase in fetal blood pressure (36 +/- 2 to 44 +/- 3 mmHg) and urine flow rate (8 +/- 2 to a maximum of 18 +/- 6 ml/h). Plasma renin concentrations decreased in ANG II-infused fetuses. Fetal fluids (amniotic and allantoic) did not differ in volume or composition between the groups when measured at postmortem. There was no difference in the expression levels of the mRNA for the angiotensin (AT(1) or AT(2)) receptors between the two groups when measured by an RNase protection assay. However, there was a significant decline in renin and AT(1) receptor gene expression when measured by a real-time polymerase chain reaction method. These results indicate that ANG II is diuretic and pressor when infused at midgestation. ANG II can feedback to decrease renin secretion by the fetal kidney, and this may occur by decreased renin gene expression.
Asunto(s)
Angiotensina II/farmacología , Desarrollo Embrionario y Fetal , Feto/fisiología , Tasa de Filtración Glomerular/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/embriología , Angiotensina II/administración & dosificación , Animales , Presión Sanguínea/efectos de los fármacos , Diuresis/efectos de los fármacos , Diuresis/fisiología , Femenino , Feto/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Edad Gestacional , Infusiones Intravenosas , Reacción en Cadena de la Polimerasa , Embarazo , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/genética , Renina/genética , OvinosRESUMEN
To examine the effect of exercise on heat shock protein (HSP) 72 mRNA expression in skeletal muscle, five healthy humans (20 +/- 1 yr; 64 +/- 3 kg; peak O(2) uptake of 2.55 +/- 0.2 l/min) cycled until exhaustion at a workload corresponding to 63% peak O(2) uptake. Muscle was sampled from the vastus lateralis, and muscle temperature was measured at rest (R), 10 min of exercise (Min10), approximately 40 min before fatigue (F-40 = 144 +/- 7 min), and fatigue (F = 186 +/- 15 min). Muscle samples were analyzed for HSP72 mRNA expression, as well as glycogen and lactate concentration. Muscle temperature increased (P < 0.05) during the first 10 min of exercise but then remained constant for the duration of the exercise. Similarly, lactate concentration increased (P < 0.05) when Min10 was compared with R but decreased (P < 0.05) thereafter, such that concentrations at F-40 and F were not different from those at R. In contrast, muscle glycogen concentration fell progressively throughout exercise (486 +/- 74 vs. 25 +/- 7 mmol/kg dry weight for R and F, respectively; P < 0.05). HSP72 mRNA was detected at R but did not increase by Min10. However, HSP72 mRNA increased (P < 0.05) 2.2 +/- 0.5- and 2.6 +/- 0.9-fold, respectively, when F-40 and F were compared with R. These data demonstrate that HSP72 mRNA increases progressively during acute cycling, suggesting that processes that take place throughout concentric exercise are capable of initiating a stress response.
Asunto(s)
Ejercicio Físico/fisiología , Expresión Génica/fisiología , Proteínas de Choque Térmico/genética , Músculo Esquelético/fisiología , Resistencia Física , Adulto , Ciclismo , Glucógeno/metabolismo , Proteínas del Choque Térmico HSP72 , Homeostasis , Humanos , Ácido Láctico/metabolismo , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
A sensitive and highly reproducible method has been used to show that Aquaporin 3 (AQP(3)) mRNA is present in the ovine placenta and chorion from at least 60 days of gestation (term=145-150d) with levels increasing substantially (>16 fold) at 100 days, and remaining constant thereafter. By immuno- and hybridization histochemistry, the epithelial cells expressing AQP(3)were found to be the trophoblast cells. Some AQP(3)was expressed in fibroblasts of the amnion and allantois but none was expressed in the epithelia of these membranes. AQP(1)was expressed in endothelial cells of fetal and maternal blood vessels but not in any epithelial cell of the ovine placenta and fetal membranes. The level of AQP(3)expression is consistent with known ovine placental permeabilities to water, glycerol and urea.
Asunto(s)
Acuaporinas/metabolismo , Membranas Extraembrionarias/metabolismo , Placenta/metabolismo , Secuencia de Aminoácidos , Animales , Acuaporina 1 , Acuaporina 3 , Acuaporinas/genética , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/genética , Membranas Extraembrionarias/crecimiento & desarrollo , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Hibridación in Situ , Datos de Secuencia Molecular , Permeabilidad , Placentación , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , OvinosRESUMEN
Arginine vasopressin interacts with the vasopressin type 1a receptor (V1aR) to initiate physiological effects such as vasoconstriction of blood vessels and glycogenolysis. AVP is also involved in central nervous effects such as body homeostasis and blood pressure control. The complete genomic organization of the sheep V1aR gene has been determined, including the presence of one major and two minor transcriptional start sites at -321, -206 and -91bp respectively, relative to the ATG codon. Another more distal minor transcriptional start site was also localized between nucleotides -997 and -892 relative to the ATG codon. One intron exists in the sheep V1aR gene and potential cis- and trans- acting sites were identified in the sheep V1aR promoter. The promoter was also compared to the rat V1aR promoter. The sheep V1aR promoter displays features typical of housekeeping genes, although tissue-specific expression does not support this. V1aR mRNA is absent in the adult sheep liver but not the kidney. One copy of the V1aR gene exists in the sheep genome, which was localized to chromosome 3q23-24, and to the homoeologous position, 5q23-24 in cattle.