RESUMEN
Acute mountain sickness (AMS) can cause capillary hyper-permeability and vasogenic edema. However, its underlying mechanisms remained unclear and there is no previous in vitro study on AMS. We therefore conducted an in vitro study and examined whether continuous hypobaric hypoxia (CHH) could alter expression of junctional protein complex of vascular endothelial cells, causing hyper-permeabilization. EA.hy926 human endothelial cells were exposed to either CHH or normoxia for up to 24 h. Flow cytometry using annexin V/propidium iodide co-staining demonstrated that cell death had no significant difference at 12-h, but was increased by CHH at 24-h. Transendothelial resistance (TER) of endothelial cell monolayer was progressively decreased by CHH from 1-h to 24-h. Western blot analysis and immunofluorescence study demonstrated decreased expression levels of VE-cadherin, PECAM-1 and ZO-1 junctional proteins at both 12-h and 24-h exposure time-points. Interestingly, while the main form of ZO-1 (220 kDa) was decreased, its degraded form (100 kDa) was increased by 24-h CHH that might be linked to the increased cell death. Our data have demonstrated that CHH caused vascular endothelial hyper-permeability and defective junctional protein complex by reducing expression levels of VE-cadherin, PECAM-1, and ZO-1. Taken together, these data may explain pathophysiology underlying vascular hyper-permeability in AMS.
Asunto(s)
Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Regulación hacia Abajo , Hipoxia/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Permeabilidad Capilar , Muerte Celular , Línea Celular , Células Endoteliales/metabolismo , HumanosRESUMEN
Nearly 90% of premature infants experience the stress of intermittent hypoxia (IH) as a consequence of recurrent apneas (periodic cessation of breathing). In neonates, catecholamine secretion from the adrenal medulla is critical for maintaining homeostasis under hypoxic stress. We recently reported that IH treatment enhanced hypoxia-evoked catecholamine secretion and [Ca2+]i responses in neonatal rat adrenal chromaffin cells and involves reactive oxygen species (ROS). The purpose of the present study was to identify the source(s) of ROS generation and examine the mechanisms underlying the enhanced catecholamine secretion by IH. Neonatal rats of either sex (postal day 0-5) were exposed to either IH or normoxia. IH treatment increased NADPH oxidase (NOX) activity, upregulated NOX2 and NOX4 transcription in adrenal medullae, and a NOX inhibitor prevented the effects of IH on hypoxia-evoked chromaffin cell secretion. IH upregulated Cav3.1 and Cav3.2 T-type Ca2+ channel mRNAs via NOX/ROS signaling and augmented T-type Ca2+ current in IH-treated chromaffin cells. Mibefradil, a blocker of T-type Ca2+ channels attenuated the effects of hypoxia on [Ca2+]i and catecholamine secretion in IH-treated cells. In Ca2+-free medium, IH-treated cells exhibited higher basal [Ca2+]i levels and more pronounced [Ca2+]i responses to hypoxia compared with controls, and blockade of ryanodine receptors (RyRs) prevented these effects. RyR2 and RyR3 mRNAs were upregulated, RyR2 was S-glutathionylated in IH-treated adrenal medullae, and NOX/ROS inhibitors prevented these effects. These results demonstrate that neonatal IH treatment leads to NOX/ROS-dependent recruitment of T-type Ca2+ channels and RyRs, resulting in augmented [Ca2+]i mobilization and catecholamine secretion.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Catecolaminas/metabolismo , Células Cromafines/fisiología , Exocitosis/fisiología , Hipoxia/patología , NADPH Oxidasas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Animales Recién Nacidos , Cloruro de Cadmio/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/genética , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Electroquímica/métodos , Inhibidores Enzimáticos/farmacología , Exocitosis/efectos de los fármacos , Femenino , Hipoxia/fisiopatología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Ratas , Canal Liberador de Calcio Receptor de Rianodina/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
We recently reported that adrenomedullary chromaffin cells (AMC) from neonatal rats treated with intermittent hypoxia (IH) exhibit enhanced catecholamine secretion by hypoxia (Souvannakitti D, Kumar GK, Fox A, Prabhakar NR. J Neurophysiol 101: 2837-2846, 2009). In the present study, we examined whether neonatal IH also facilitate AMC responses to nicotine, a potent stimulus to chromaffin cells. Experiments were performed on rats exposed to either IH (15-s hypoxia-5-min normoxia; 8 h/day) or to room air (normoxia; controls) from ages postnatal day 0 (P0) to P5. Quantitative RT-PCR analysis revealed expression of mRNAs alpha(3-), alpha(5-), alpha(7-), and beta(2-) and beta(4-)nicotinic acetylcholine receptor (nAChR) subunits in adrenal medullae from control P5 rats. Nicotine-elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) in AMC and nAChR antagonists prevented this response, suggesting that nAChRs are functional in neonatal AMC. In IH-treated rats, nAChR mRNAs were downregulated in AMC, which resulted in a markedly attenuated nicotine-evoked elevation in [Ca(2+)](i) and subsequent catecholamine secretion. Systemic administration of antioxidant prevented IH-evoked downregulation of nAChR expression and function. P35 rats treated with neonatal IH exhibited reduced nAChR mRNA expression in adrenal medullae, attenuated AMC responses to nicotine, and impaired neurogenic catecholamine secretion. Thus the response to neonatal IH lasts for at least 30 days. These observations demonstrate that neonatal IH downregulates nAChR expression and function in AMC via reactive oxygen species signaling, and the effects of neonatal IH persist at least into juvenile life, leading to impaired neurogenic catecholamine secretion from AMC.
Asunto(s)
Células Cromafines/metabolismo , Hipoxia/metabolismo , Receptores Nicotínicos/biosíntesis , Animales , Animales Recién Nacidos , Antioxidantes/farmacología , Calcio/metabolismo , Células Cultivadas , Células Cromafines/fisiología , Femenino , Regulación de la Expresión Génica , Nicotina/farmacología , Oxígeno/fisiología , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/genética , Receptores Nicotínicos/fisiologíaRESUMEN
Gaseousmessengers, nitric oxide and carbon monoxide, have been implicated in O2 sensing by the carotid body, a sensory organ that monitors arterial blood O2 levels and stimulates breathing in response to hypoxia. We now show that hydrogen sulfide (H2S) is a physiologic gasotransmitter of the carotid body, enhancing its sensory response to hypoxia. Glomus cells, the site of O2 sensing in the carotid body, express cystathionine gamma-lyase (CSE), an H2S-generating enzyme, with hypoxia increasing H2S generation in a stimulus-dependent manner. Mice with genetic deletion of CSE display severely impaired carotid body response and ventilatory stimulation to hypoxia, as well as a loss of hypoxia-evoked H2S generation. Pharmacologic inhibition of CSE elicits a similar phenotype in mice and rats. Hypoxia-evoked H2S generation in the carotid body seems to require interaction of CSE with hemeoxygenase-2, which generates carbon monoxide. CSE is also expressed in neonatal adrenal medullary chromaffin cells of rats and mice whose hypoxia-evoked catecholamine secretion is greatly attenuated by CSE inhibitors and in CSE knockout mice.
Asunto(s)
Cuerpo Carotídeo/fisiología , Sulfuro de Hidrógeno/metabolismo , Oxígeno/fisiología , Animales , Cistationina gamma-Liasa/deficiencia , Cistationina gamma-Liasa/metabolismo , Hipoxia/metabolismo , Masculino , Ratones , Ratones Noqueados , RatasRESUMEN
In the present study we examined the effects of intermittent (IH) and sustained hypoxia (SH) on low PO(2)-evoked catecholamine (CA) secretion from neonatal rat chromaffin cells. Experiments were performed on chromaffin cells isolated from rat pups exposed to either IH (P0-P5; 15 s hypoxia-5 min normoxia;8 h/day) or SH (hypobaric hypoxia; 0.4ATM). CA secretion from chromaffin cells was monitored by amperometry. Control chromaffin cells, from P5 rat pups, exhibited robust CA secretion in response to acute hypoxia. IH facilitated whereas SH attenuated hypoxia-evoked CA secretion. IH increased the epinephrine and norepinephrine content of the adrenal medulla whereas SH had no effect. These results demonstrate that neonatal exposures IH and SH exert diametrically opposed effects on acute hypoxia-evoked CA secretion from chromaffin cells and CA contents.
Asunto(s)
Catecolaminas/metabolismo , Células Cromafines/metabolismo , Hipoxia/metabolismo , Oxígeno/metabolismo , Médula Suprarrenal/metabolismo , Animales , Animales Recién Nacidos , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The objective of the present study was to examine the effects of intermittent hypoxia (IH) and sustained hypoxia (SH) on hypoxia-evoked catecholamine (CA) secretion from chromaffin cells in neonatal rats and assess the underlying mechanism(s). Experiments were performed on rat pups exposed to either IH (15-s hypoxia/5-min normoxia; 8 h/day) or SH (hypobaric hypoxia, 0.4 atm) or normoxia (controls) from P0 to P5. IH treatment facilitated hypoxia-evoked CA secretion and elevations in the intracellular calcium ion concentration ([Ca(2+)](i)) and these responses were attenuated, but not abolished, by treatments designed to eliminate Ca(2+) flux into cells (Ca(2+)-free medium or Cd(2+)), indicating that intracellular Ca(2+) stores were augmented by IH. Norepinephrine (NE) and epinephrine (E) levels of adrenal medullae were elevated in IH-treated pups. IH treatment increased reactive oxygen species (ROS) production in adrenal medullae and antioxidant treatment prevented IH-induced facilitation of CA secretion, elevations in [Ca(2+)](i) by hypoxia, and the up-regulation of NE and E. The effects of neonatal IH treatment on hypoxia-induced CA secretion and elevation in [Ca(2+)](i), CA, and ROS levels persisted in rats reared under normoxia for >30 days. In striking contrast, chromaffin cells from SH-treated animals exhibited attenuated hypoxia-evoked CA secretion. In SH-treated cells hypoxia-evoked elevations in [Ca(2+)](i), NE and E contents, and ROS levels were comparable with controls. These observations demonstrate that: 1) neonatal IH and SH evoke opposite effects on hypoxia-evoked CA secretion from chromaffin cells, 2) ROS signaling mediates the faciltatory effects of IH, and 3) the effects of neonatal IH on chromaffin cells persist into adult life.
Asunto(s)
Médula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Células Cromafines/metabolismo , Hipoxia/patología , Animales , Animales Recién Nacidos , Cadmio/farmacología , Calcio/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Electroquímica/métodos , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Metaloporfirinas/farmacología , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O(2). A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2alpha is an orthologue of HIF-1alpha, we examined the effects of IH on HIF-2alpha, the O(2)-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1alpha, HIF-2alpha was down-regulated by IH. Similar down-regulation of HIF-2alpha was also seen in carotid bodies and adrenal medullae from IH-exposed rats. Inhibitors of calpain proteases (ALLM, ALLN) prevented IH-evoked degradation of HIF-2alpha whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2alpha degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2alpha prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2alpha degradation and restored SOD2 activity, thereby preventing oxidative stress and hypertension. These observations demonstrate that, unlike continuous hypoxia, IH leads to down-regulation of HIF-2alpha via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.
Asunto(s)
Apnea/enzimología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Calpaína/metabolismo , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Animales , Apnea/mortalidad , Sistema Nervioso Autónomo/efectos de los fármacos , Sistema Nervioso Autónomo/patología , Señalización del Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Calpaína/antagonistas & inhibidores , Hipoxia de la Célula/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Unión Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/antagonistas & inhibidoresRESUMEN
OBJECTIVE: Determine the prevalence of overweight and obesity in the Royal Thai Army (RTA) personnel. MATERIAL AND METHOD: A cross-sectional study was performed on 4,276 RTA personnel aged 20-60 years between January and July 2005. They were interviewed for general health status, history of smoking, alcohol consumption, and physical activity. Blood pressure, weight, height, waist, and hip circumference were measured. RESULT: The prevalence of overweight and obesity in RTA personnel were 27.1% and 4.9% respectively. The mean value of age, weight, body mass index, waist, and hip circumference, waist hip ratio, blood pressure in overweight, and obese personnel were significantly higher than those of non-overweight personnel. Logistic regression analysis showed that significant risks of being overweight were positively associated with age, male, and working in a combat unit. Current smoking was inversely associated with overweight and obesity, whereas physical activity > or = 3 times/wk was inversely associated with obesity. Overweight and obesity were also associated with hypertension. CONCLUSION: These baseline data are useful for future trends monitoring and may be used for health promotion planning to prevent obesity in RTA personnel.
Asunto(s)
Medicina Militar , Personal Militar/estadística & datos numéricos , Obesidad/epidemiología , Sobrepeso , Adulto , Índice de Masa Corporal , Estudios Transversales , Femenino , Encuestas Epidemiológicas , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Factores de Riesgo , Encuestas y Cuestionarios , Tailandia/epidemiologíaRESUMEN
Chronic intermittent hypoxia (CIH) augments physiological responses to low partial pressures of O2 in the arterial blood. Adrenal medullae from adult rats, however, are insensitive to direct effects of acute hypoxia. In the present study, we examined whether CIH induces hypoxic sensitivity in the adult rat adrenal medulla and, if so, by what mechanism(s). Experiments were performed on adult male rats exposed to CIH (15 s of 5% O2 followed by 5 min of 21% O2; 9 episodes h(-1); 8 h d(-1); for 3 or 10 days) or to comparable, cumulative durations of continuous hypoxia (CH; 4 h of 7% O2 followed by 20 h of 21% O2 for 1 or 10 days). Noradrenaline (NA) and adrenaline (ADR) effluxes were monitored from ex vivo adrenal medullae. In adrenal medullae of rats exposed to CIH, acute hypoxia evoked robust NA and ADR effluxes, whereas these responses were absent in control rats or in those exposed to CH for 1 or 10 days. Hypercapnia (10% CO2; either acidic, pH 6.8, or isohydric, pH 7.4) was ineffective in eliciting catecholamine (CA) efflux from control, CIH or CH rats. Nicotine (100 microM) evoked NA and ADR effluxes in control rats, and this response was abolished in CIH but not in CH rats. Systemic administration of 2-deoxyglucose depleted ADR content in control rats, and CIH attenuated this response, indicating downregulation of neurally regulated CA secretion. Cytosolic and mitochondrial aconitase enzyme activities decreased in CIH adrenal medullae, suggesting increased generation of superoxide anions. Systemic administration of antioxidants reversed the effect of CIH on the adrenal medulla. Rats exposed to CIH exhibited increased blood pressures and elevated plasma CA, and antioxidants abolished these responses. These observations demonstrate that CIH induces hypoxic sensing in the adult rat adrenal medulla via mechanisms involving increased generation of superoxide anions and suggest that hypoxia-evoked CA efflux from the adrenal medulla contributes, in part, to elevated blood pressure and plasma CA.