RESUMO
Electronic nicotine delivery systems, or e-cigarettes, utilize a liquid solution that normally contains propylene glycol (PG) and vegetable glycerin (VG) to generate vapor and act as a carrier for nicotine and flavorings. Evidence indicated these "carriers" reduced growth and survival of epithelial cells including those of the airway. We hypothesized that 3% PG or PG mixed with VG (3% PG/VG, 55:45) inhibited glucose uptake in human airway epithelial cells as a first step to reducing airway cell survival. Exposure of H441 or human bronchiolar epithelial cells (HBECs) to PG and PG/VG (30-60 min) inhibited glucose uptake and mitochondrial ATP synthesis. PG/VG inhibited glycolysis. PG/VG and mannitol reduced cell volume and height of air-liquid interface cultures. Mannitol, but not PG/VG, increased phosphorylation of p38 MAPK. PG/VG reduced transepithelial electrical resistance, which was associated with increased transepithelial solute permeability. PG/VG decreased fluorescence recovery after photobleaching of green fluorescent protein-linked glucose transporters GLUT1 and GLUT10, indicating that glucose transport function was compromised. Puffing PG/VG vapor onto the apical surface of primary HBECs for 10 min to mimic the effect of e-cigarette smoking also reduced glucose transport. In conclusion, short-term exposure to PG/VG, key components of e-cigarettes, decreased glucose transport and metabolism in airway cells. We propose that this was a result of PG/VG reduced cell volume and membrane fluidity, with further consequences on epithelial barrier function. Taking these results together, we suggest these factors contribute to reduced defensive properties of the epithelium. We propose that repeated/chronic exposure to these agents are likely to contribute to airway damage in e-cigarette users.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Células Epiteliais/efeitos dos fármacos , Glucose/metabolismo , Sistema Respiratório/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Glicerol/farmacologia , Humanos , Propilenoglicol/farmacologiaRESUMO
OBJECTIVE: To compare the risk of fetal death on the day of childbirth, with the risk of death at other ages, and with the risks of some hazardous activities, on a common scale of risk per day. DESIGN: Review of publicly available data. SETTING UK SAMPLE: Data extracted from the Office of National Statistics and other sources. METHODS: Data from the Office of National Statistics and other sources were used to calculate death rates at different ages expressed as rates per day of life. Death rates for different activities were also calculated as risks per day, or risks per activity, as appropriate. All risks were expressed in micromorts, the number of one in a million chances of dying. Figures on life expectancy (LE) were used to compare potential life years lost. MAIN OUTCOME MEASURES: Daily, or unit of activity, risk of dying for different activities compared with the risk of dying on the day of childbirth. RESULTS: The risk of dying on the day of birth (0.43 per 1000, or 430 micromorts) exceeds that of any other average day of life until the 92nd year. It is comparable with other apparently more dangerous activities, such as undergoing major surgery. For comparison, the average risk of non-natural death per day and the increased risk from smoking one cigarette or travelling 200 miles by car are all about 1 micromort. CONCLUSIONS: The lifetime risk of death in childbirth is low, but is concentrated in a short period, making being born a high-risk activity. Parents considering interventions to reduce these risks should be made aware of this.
Assuntos
Mortalidade Infantil , Mortalidade Materna , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Comportamento de Redução do Risco , Natimorto , Adulto , Fatores Etários , Causas de Morte , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Recém-Nascido , Expectativa de Vida , Masculino , Computação Matemática , Gravidez , Medição de Risco , Fatores de Risco , Natimorto/epidemiologia , Reino Unido/epidemiologia , Estados Unidos/epidemiologiaRESUMO
BACKGROUND AND PURPOSE: Pulmonary transepithelial Na(+) transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na(+) channels and basolateral Na(+) K(+) ATPase activity. EXPERIMENTAL APPROACH: H441 human airway epithelial cells were used to examine the effects of hypoxia on Na(+) transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS. KEY RESULTS: AMPK was activated by exposure to 3% or 0.2% O(2) for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm(-2) ) was added to the apical surface of cells grown at the air-liquid interface. Only 0.2% O(2) activated AMPK in cells grown at the air-liquid interface. AMPK activation was associated with elevation of cellular AMP:ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I(sc) (I(ouabain) ) and apical amiloride-sensitive Na(+) conductance (G(Na+) ). Modification of AMPK activity prevented the effect of hypoxia on I(ouabain) (Na(+) K(+) ATPase) but not apical G(Na+) . Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G(Na+) (epithelial Na(+) channels). CONCLUSIONS AND IMPLICATIONS: Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na(+) channels and basolateral Na(+) K(+) ATPase activity to decrease transepithelial Na(+) transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Transporte Biológico/fisiologia , Células Epiteliais/fisiologia , Oxigênio/farmacologia , Mucosa Respiratória/citologia , Sódio/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Vetores Genéticos , Humanos , Lentivirus , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
BACKGROUND AND PURPOSE The transepithelial absorption of Na(+) in the lungs is crucial for the maintenance of the volume and composition of epithelial lining fluid. The regulation of Na(+) transport is essential, because hypo- or hyperabsorption of Na(+) is associated with lung diseases such as pulmonary oedema or cystic fibrosis. This study investigated the effects of the gaseous signalling molecule hydrogen sulphide (H(2) S) on Na(+) absorption across pulmonary epithelial cells. EXPERIMENTAL APPROACH Ion transport processes were electrophysiologically assessed in Ussing chambers on H441 cells grown on permeable supports at air/liquid interface and on native tracheal preparations of pigs and mice. The effects of H(2)S were further investigated on Na(+) channels expressed in Xenopus oocytes and Na(+) /K(+)-ATPase activity in vitro. Membrane abundance of Na(+) /K(+)-ATPase was determined by surface biotinylation and Western blot. Cellular ATP concentrations were measured colorimetrically, and cytosolic Ca(2+) concentrations were measured with Fura-2. KEY RESULTS H(2)S rapidly and reversibly inhibited Na(+) transport in all the models employed. H(2)S had no effect on Na(+) channels, whereas it decreased Na(+) /K(+)-ATPase currents. H(2)S did not affect the membrane abundance of Na(+) /K(+)-ATPase, its metabolic or calcium-dependent regulation, or its direct activity. However, H(2)S inhibited basolateral calcium-dependent K(+) channels, which consequently decreased Na(+) absorption by H441 monolayers. CONCLUSIONS AND IMPLICATIONS H(2) S impairs pulmonary transepithelial Na(+) absorption, mainly by inhibiting basolateral Ca(2+)-dependent K(+) channels. These data suggest that the H(2)S signalling system might represent a novel pharmacological target for modifying pulmonary transepithelial Na(+) transport.
Assuntos
Células Epiteliais/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Sódio/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Células Epiteliais/fisiologia , Canais Epiteliais de Sódio/fisiologia , Humanos , Técnicas In Vitro , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Oócitos , Canais de Potássio Cálcio-Ativados/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Suínos , Xenopus laevisRESUMO
Bacterial lipopolysaccharides (LPS) are potent inducers of proinflammatory signaling pathways via the activation of nuclear factor-kappa B (NF-kappaB) and mitogen-activated protein kinase (MAPK), causing changes in the processes that control lung fluid homeostasis and contributing to the pathogenesis of lung disease. In human H441 airway epithelial cells, incubation of cells with 15 microg ml(-1) LPS caused a significant reduction in amiloride-sensitive I (sc) from 15 +/- 2 to 8 +/- 2 microA cm(-2) (p = 0.01, n = 13) and a shift in IC(50) amiloride of currents from 6.8 x 10(-7) to 6.4 x 10(-6) M. This effect was associated with a decrease in the activity of 5 pS, highly Na(+) selective, amiloride-sensitive <1 microM channels (HSC) and an increase in the activity of approximately 18 pS, nonselective, amiloride-sensitive >10 microM cation channels (NSC) in the apical membrane. LPS decreased alphaENaC mRNA and protein abundance, inferring that LPS inhibited alphaENaC gene expression. This correlated with the decrease in HSC activity, indicating that these channels, but not NSCs, were comprised of at least alphaENaC protein. LPS increased NF-kappaB DNA binding activity and phosphorylation of extracellular signal-related kinase (ERK)1/2, but decreased phosphorylation of ERK5 in H441 cells. Pretreatment of monolayers with PD98059 (20 microM) inhibited ERK1/2 phosphorylation, promoted phosphorylation of ERK5, increased alphaENaC protein abundance, and reversed the effect of LPS on I (sc) and the shift in amiloride sensitivity. Inhibitors of NF-kappaB activation were without effect. Taken together, our data indicate that LPS acts via ERK signaling pathways to decrease alphaENaC transcription, reducing HSC/ENaC channel abundance, activity, and transepithelial Na(+) transport in H441 airway epithelial cells.
Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Lipopolissacarídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Amilorida/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Canais Epiteliais de Sódio/genética , Flavonoides/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , NF-kappa B/metabolismo , Técnicas de Patch-Clamp , Transdução de Sinais/fisiologia , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
H441 cells are a model of absorptive airway epithelia that are characterised by a pronounced apical Na+ flux through amiloride-sensitive Na+ channels. The flux of Na+ is intimately linked to Na+ handling by the cell as well as the membrane potential across the apical membrane. As KCNQ-encoded K+ channels influence chloride secretion in gastrointestinal epithelia, the goal of the present study was to ascertain the expression of KCNQ genes in H441 cells and determine the functional role of the expression products. Message for KCNQ3 and KCNQ5 was detected by RT-polymerase chain reaction and the translated proteins were observed by immunocytochemistry. Ussing experiments showed that the pan-KCNQ channel blocker XE991, but not KCNQ1 selective blockers, reduced the short circuit current and the amiloride-sensitive component. These data show for the first time that potassium channels encoded by KCNQ3 or KCNQ5 are crucial determinants of epithelial Na+ flux.
Assuntos
Células Epiteliais/metabolismo , Canais de Potássio KCNQ/metabolismo , Pulmão/citologia , Isoformas de Proteínas/metabolismo , Sódio/metabolismo , Antracenos/metabolismo , Anticonvulsivantes/metabolismo , Encéfalo/metabolismo , Carbamatos/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Humanos , Canais de Potássio KCNQ/antagonistas & inibidores , Canais de Potássio KCNQ/genética , Miocárdio/metabolismo , Peptídeos/metabolismo , Fenilenodiaminas/metabolismo , Bloqueadores dos Canais de Potássio/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genéticaRESUMO
Transepithelial transport of Na(+) across the lung epithelium via amiloride-sensitive Na(+) channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na(+) conductance (G(Na+)) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to G(Na+): a 5-pS highly Na(+) selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to -38 mV and 0 mV to -35 mV. Amiloride at 1 microM inhibited HSC activity and 56% of short-circuit current (I(sc)), whereas 10 microM amiloride partially reduced NSC activity and inhibited a further 30% of I(sc). Neither conductance was associated with CNG channels as there was no effect of 10 microM pimoside on I(sc), HSC, or NSC activity, and 8-bromo-cGMP (0.3-0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na(+)-permeable channels by a mechanism that likely reduces channel open probability.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Canais Epiteliais de Sódio/metabolismo , Pulmão/citologia , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Amilorida/farmacologia , Aminoimidazol Carboxamida/farmacologia , Fenômenos Biofísicos/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Células Epiteliais/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Metformina/farmacologia , Técnicas de Patch-Clamp , Permeabilidade/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (I(amiloride)) and ouabain-sensitive basolateral (I(ouabain)) short circuit current in H441 lung epithelial cells. EXPERIMENTAL APPROACH: H441 cells were grown on permeable filters at air interface. I(amiloride), I(ouabain) and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. KEY RESULTS: Phenformin, AICAR and metformin increased AMPK (alpha1) activity and decreased I(amiloride). The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I(ouabain) across H441 monolayers and decreased monolayer resistance. The decrease in I(amiloride) was closely related to I(ouabain) with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. CONCLUSIONS AND IMPLICATIONS: Activation of alpha1-AMPK is associated with inhibition of apical amiloride-sensitive Na(+) channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I(ouabain), with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Complexos Multienzimáticos/efeitos dos fármacos , Fenformin/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Sódio/metabolismo , Proteínas Quinases Ativadas por AMP , Nucleotídeos de Adenina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Amilorida , Aminoimidazol Carboxamida/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Células Epiteliais , Canais Epiteliais de Sódio/efeitos dos fármacos , Fluorescência , Humanos , Pulmão , Microscopia Confocal , Complexos Multienzimáticos/metabolismo , Ouabaína , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
BACKGROUND: Hyperglycaemia is associated with poor outcomes from pneumonia, myocardial infarction and stroke, but the effect of blood glucose on outcomes from acute exacerbations of chronic obstructive pulmonary disease (AECOPD) has not been established. Recent UK guidelines do not comment on measurement or control of blood glucose in AECOPD. A study was therefore undertaken to determine the relationship between blood glucose concentrations, length of stay in hospital, and mortality in patients admitted with AECOPD. METHODS: Data were retrieved from electronic records for patients admitted with AECOPD with lower respiratory tract infection in 2001-2. The patients were grouped according to blood glucose quartile (group 1, <6 mmol/l (n = 69); group 2, 6.0-6.9 mmol/l (n = 69); group 3, 7.0-8.9 mmol/l (n = 75); and group 4, >9.0 mmol/l (n = 71)). RESULTS: The relative risk (RR) of death or long inpatient stay was significantly increased in group 3 (RR 1.46, 95% CI 1.05 to 2.02, p = 0.02) and group 4 (RR 1.97, 95% CI 1.33 to 2.92, p < 0.0001) compared with group 1. For each 1 mmol/l increase in blood glucose the absolute risk of adverse outcomes increased by 15% (95% CI 4 to 27), p = 0.006. The risk of adverse outcomes increased with increasing hyperglycaemia independent of age, sex, a previous diagnosis of diabetes, and COPD severity. Isolation of multiple pathogens and Staphylococcus aureus from sputum also increased with increasing blood glucose. CONCLUSION: Increasing blood glucose concentrations are associated with adverse clinical outcomes in patients with AECOPD. Tight control of blood glucose reduces mortality in patients in intensive care or following myocardial infarction. A prospective study is now required to determine whether control of blood glucose can also improve outcomes from AECOPD.
Assuntos
Hiperglicemia/complicações , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Aguda , Idoso , Glicemia/metabolismo , Feminino , Hospitalização , Humanos , Hiperglicemia/mortalidade , Masculino , Prognóstico , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/mortalidade , Fatores de Risco , Espirometria , Escarro/microbiologiaRESUMO
Rhinorrhoea is a clinical sign of cerebrospinal fluid (CSF) leakage in patients with skull fracture, but can also be attributable to respiratory secretions or tears. Laboratory tests confirming the presence of CSF are not sufficiently rapid to support clinical decision making in the emergency department and may not be universally available. Detection of glucose in nasal discharge was traditionally used to diagnose CSF leak at the bedside, but has fallen into disuse as it has poor positive predictive value. We propose an algorithm to improve the diagnostic value of this test taking into consideration factors we have found to affect the glucose concentration of respiratory secretions. In patients at risk of CSF leak, nasal discharge is likely to contain CSF if glucose is present in the absence of visible blood, if blood glucose is <6 mmol x L(-1), and if there are no symptoms of upper respiratory tract infection.
Assuntos
Algoritmos , Rinorreia de Líquido Cefalorraquidiano/diagnóstico , Glucose Oxidase , Glucose/líquido cefalorraquidiano , Glicemia/análise , Rinorreia de Líquido Cefalorraquidiano/líquido cefalorraquidiano , Rinorreia de Líquido Cefalorraquidiano/etiologia , Traumatismos Craniocerebrais/complicações , Humanos , Muco/química , Fitas ReagentesRESUMO
The secretion of uterine luminal fluid initially provides a transport and support medium for spermatozoa and unimplanted embryos, while the absorption of uterine luminal fluid in early pregnancy results in the closure of the lumen and allows blastocysts to establish intimate contact with the uterine epithelium. We have established an in vivo perfusion technique of the lumen to study the hormonal control of the events in the peri-implantation period. Fluorescein-labelled dextran was included in the perfusion medium to monitor fluid movements and the concentrations of Na(+) and CI(-) ions in the effluent were monitored. Using an established regimen of steroid treatment of ovariectomized rats mimicking early pregnancy, oestradiol caused fluid secretion, while progesterone resulted in an amiloride-sensitive fluid absorption. Fluid absorption peaked at about the expected time of implantation. The effect of progesterone could be inhibited by treatment with a high dose of oestradiol, by the anti-progestin RU486, and by the presence of an intra-uterine contraceptive device. Studies of expression of Na(+) and CI(-) channels (ENaC, CFTR) indicated that these channels were subject to tissue-specific regulation within the uterus, but more work is required to determine their role and the factors controlling their abundance and localization in early pregnancy.
Assuntos
Cloro/metabolismo , Estradiol/metabolismo , Prenhez/metabolismo , Progesterona/metabolismo , Sódio/metabolismo , Útero/metabolismo , Animais , Líquidos Corporais/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Canais Epiteliais de Sódio , Feminino , Hormônios/metabolismo , Ovariectomia , Gravidez , Ratos , Ratos Wistar , Canais de Sódio/metabolismoRESUMO
A range of easily identifiable characteristics is often used by researchers and general practitioners to categorise primary care practices. In the United Kingdom, for example, practices can be defined as dispensing, single-handed or training. The availability of routinely collected data has led to a growing research literature that links practice characteristics to their workload, performance and costs. This paper examines the use and limitations of this 'characteristics approach' and argues that this type of research is often undertaken because it is easy to perform rather than because it is the most appropriate way to study primary care. Using this approach may lead to failure to do the following: to account for the environmental factors that determine the effects particular characteristics manifest; to identify the true relationships between the observed characteristics; to control for changes in the effects of characteristics over time; to differentiate between the behaviour of individual members of a group with the same characteristic and that of the group as a whole; to assign the correct causality to relationships between practice characteristics, workloads, performance, and costs. The characteristics approach should be used with great caution by general practice researchers.
Assuntos
Medicina de Família e Comunidade , Pesquisa sobre Serviços de Saúde/métodos , Viés , Pesquisa sobre Serviços de Saúde/normas , Humanos , Medicina Estatal , Reino UnidoRESUMO
Removal of fetal lung fluid at birth is crucial to survival. In vivo, a reversal in the direction of vectorial, amiloride-sensitive Na+) transport can be stimulated by ETYA, a nonmetabolizable analogue of the naturally occurring unsaturated fatty acid, arachidonate. Using the patch-clamp technique, fetal guinea pig alveolar type II pneumocyte single Na+ channel activity was robustly activated by 10 microM arachidonate, ETYA, oleate and stearate; this was unaffected by cyclooxygenase and 5'lipoxygenase inhibitors. The Na+ channel expressed in fetal guinea pig alveolar epithelial type II pneumocytes has biophysical properties compatible with species-specific coexpression of a novel variant of alphaENaC with betaENaC. gammaENaC is either not expressed in this tissue or shares very little homology with the rat and human gamma subunit. Thus, dramatic stimulation of this channel by arachidonate explains the in vivo observation of gestation-dependent reversal of fetal transepithelial driving force and may, therefore, be of physiological significance during the transition to breathing air at birth.
Assuntos
Amilorida/metabolismo , Ácidos Graxos/metabolismo , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Eletrofisiologia , Canais Epiteliais de Sódio , Cobaias , Humanos , Cinética , Pulmão/citologia , Pulmão/embriologia , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Canais de Sódio/genética , Canais de Sódio/fisiologiaRESUMO
Monolayer cultures of rat fetal distal lung epithelial (FDLE) cells generated larger spontaneous short circuit currents (ISC) when maintained (48 h) at neonatal alveolar PO2 (100 mmHg) than at fetal PO2 (23 mmHg). When cells were shifted between these atmospheres in order to impose a rise in PO2 equivalent to that seen at birth, no rise in ISC was seen after 6 h but the response was fully established by 24 h. Studies of basolaterally permeabilised cells revealed a small rise in apical Na+ conductance (GNa) 6 h after PO2 was raised but no further change had occurred by 24 h. A substantial rise was, however, seen after 48 h. Reporter gene assays showed that no activation of the -ENaC (epithelial Na+ channel -subunit) promoter was discernible 24 h after PO2 was raised but increased transcriptional activity was seen at 48 h. Studies of apically permeabilised cells showed that a small rise in Na+ pump capacity was evident 6 h after PO2 was raised and, in common with the rise in ISC, this effect was fully established by 24 h. The rise in ISC thus develops 6-24 h after PO2 is raised and is due, primarily, to increased Na+ pump capacity. The increase in GNa thus coincides with activation of the -ENaC promoter but these effects occur after the rise in ISC is fully established and so cannot underlie this physiological response. The increased transcription may be an adaptation to increased Na+ transport and not its cause.
Assuntos
Células Epiteliais/metabolismo , Pulmão/metabolismo , Oxigênio/metabolismo , Canais de Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Amilorida/farmacologia , Animais , Polaridade Celular , Células Cultivadas , Genes Reporter , Transporte de Íons , Pulmão/citologia , Pulmão/embriologia , Potenciais da Membrana/fisiologia , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Subunidades Proteicas , Ratos , Ratos Sprague-Dawley , Canais de Sódio/genética , ATPase Trocadora de Sódio-Potássio/genética , Fatores de Tempo , Transcrição GênicaRESUMO
1. Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (for 48 h) at PO2 levels between 23 and 142 mmHg. Higher PO2 levels between 23 and 142 mmHg. Higher PO2 was associated with increased short circuit current (ISC) and increased abundance of the Na+ channel protein alpha-ENaC. PO2 had no effect upon ISC remaining after apical application of amiloride (10 microM). 2. Studies of cells maintained (for 48 h) at PO2 levels of 23 mmHg or 100 mmHg, and subsequently nystatin permeabilized (50 microM), showed that high PO2 increased Na+ pump capacity. This response was apparent 24 h after PO2 was raised whilst it took 48 h for the rise in ISC seen in intact cells to become fully established. Both parameters were unaffected by raising PO2 for only 30 min. 3. Basolateral application of isoprenaline (10 microM) did not affect ISC in cells maintained at 23 mmHg but evoked progressively larger responses at higher PO2. The response seen at 142 mmHg was larger than at 100 mmHg, the normal physiological alveolar PO2. 4. Isoprenaline had no effect on Na+ pump capacity at PO2 levels of 23 mmHg or 100 mmHg, but stimulated Na+ extrusion at 142 mmHg. Increasing PO2 above normal physiological levels thus allows the Na+ pump to be controlled by isoprenaline. This may explain the enhanced sensitivity to isoprenaline seen under these slightly hyperoxic conditions. 5. Changes in PO2 mimicking those occurring at birth thus exert profound influence over Na+ transport in FDLE cells and the Na+ pump could be an important locus at which this control is exercised.
Assuntos
Pulmão/metabolismo , Oxigênio/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Amilorida/farmacologia , Animais , Cardiotônicos/farmacologia , Células Cultivadas , Diuréticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Isoproterenol/farmacologia , Pulmão/citologia , Pulmão/embriologia , Ouabaína/farmacologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Ratos , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
We have studied factors that potentially modulate the expression of mRNA coding for subunits of the amiloride-sensitive sodium channel, alphaENaC and betaENaC, in lungs of vaginally and Caesarean (CS)-delivered late gestation fetal guinea-pigs. Expression of alphaENaC and betaENaC mRNAs was developmentally regulated in the late gestation fetus, reaching peak levels at term (68 days post conception, PC) and postnatally, respectively. In animals delivered by CS at 65 days PC and term, alphaENaC mRNA expression was significantly increased by day 1 post partum, reaching levels greater than those normally achieved in vaginally delivered animals at term. In contrast, betaENaC mRNA levels remained significantly lower postnatally in animals delivered by CS at 65 days PC compared with those in vaginally and CS-delivered animals at term. Plasma cortisol and total triiodothyronine (T3) levels increased towards term, were higher 1 day after vaginal delivery but declined towards pre-term levels by day 3. Cortisol levels also increased rapidly in the CS-delivered animals, reaching levels similar to those in vaginally delivered animals at day 1. Plasma T3 levels at days 1 and 3 were significantly lower in animals delivered by CS at 65 days PC. The increase in alphaENaC mRNA paralleled the increase in plasma cortisol after delivery, but not T3, and inhibition of cortisol synthesis with 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) after CS delivery suppressed the increase in alphaENaC mRNA expression. Concomitant with the increase in alphaENaC mRNA expression after CS delivery at 65 days PC was an increase in the amiloride-blockable component of lung fluid clearance by day 3 postnatally. We conclude that in late gestation guinea-pigs delivered by CS there is a significant increase in lung alphaENaC expression postnatally, which is mediated, in part, by the postnatal rise in cortisol at delivery. This in turn leads to an increase in amiloride-sensitive lung fluid clearance, which is unrelated to labour.
Assuntos
Parto Obstétrico/métodos , Hormônios/sangue , Pulmão/metabolismo , Canais de Sódio/metabolismo , Animais , Animais Recém-Nascidos , Líquidos Corporais/metabolismo , Cesárea , Meio Ambiente , Canais Epiteliais de Sódio , Feto , Idade Gestacional , Cobaias , Hidrocortisona/biossíntese , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Metirapona/farmacologia , Oxigênio , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Respiração , Canais de Sódio/genéticaRESUMO
Apical ATP, ATP, UTP and UDP evoked transient increases in short circuit current (I(SC), a direct measure of transepithelial ion transport) in confluent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors. Experiments using cells grown on glass coverslips showed that ATP and UTP consistently increased intracellular free calcium ([Ca(2+)](i)) whilst sensitivity to UDP was variable. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. Messenger RNA transcripts corresponding to the P2Y(2), P2Y(4) and P2Y(6) receptors genes were detected in cells grown on Transwell membranes by the reverse transcriptase - polymerase chain reaction. Identical results were obtained for cells grown on glass. Experiments in which I(SC) and [Ca(2+)](i) were monitored simultaneously in cells on Transwell membranes, confirmed that apical ATP and UTP increased both parameters and showed that the UDP-evoked increase in I(SC) was accompanied by a [Ca(2+)](i)-signal. Ionomycin consistently increased [Ca(2+)](i) in such polarized cells but caused no discernible change in I(SC). However, subsequent application of apical ATP or UTP evoked a small rise in I(SC) but no rise in [Ca(2+)](i). UDP evoked no such response. As well as evoking increases in [Ca(2+)](i), the ATP/UTP-sensitive receptors present in Caco-2 cells thus allow direct control over ion channels in the apical membrane. The UDP-sensitive receptors, however, appear to simply evoke a rise in [Ca(2+)](i).
Assuntos
Membrana Celular/fisiologia , Células Epiteliais/fisiologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/farmacologia , Células CACO-2 , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y2 , Transcrição Gênica , Uridina Trifosfato/farmacologiaRESUMO
1 Rat foetal distal lung epithelial cells were plated onto permeable supports where they became integrated into epithelial sheets that spontaneously generated short circuit current (ISC). 2 Apical ATP (100 microM) evoked a transient fall in ISC that was followed by a rise to a clear peak which, in turn, was succeeded by a slowly developing decline to a value below control. Apical UTP evoked an essentially identical response. 3 UDP and ADP were ineffective whilst ATP had no effect when added to the basolateral solution. These effects thus appear to be mediated by apical P2Y2 receptors. 4 The rising phase of the responses to ATP/UTP was selectively inhibited by anion transport inhibitors but persisted in the presence of amiloride, which abolished the inhibitory effects of both nucleotides. Thus, apical nucleotides appear to evoke a transient stimulation of anion secretion and sustained inhibition of Na+ absorption. 5 Basolateral isoprenaline (10 microM) elicited a rise in ISC but subsequent addition of apical ATP reversed this effect. Conversely, isoprenaline restored ISC to its basal level following stimulation with ATP. Apical P2Y2 receptors and basolateral beta-adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.