RESUMEN
Neutrophil extracellular traps contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of human NETs on barrier function in primary human bronchial epithelial and a human airway epithelial cell line. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance and increasing paracellular flux, partially by NET-induced airway cell apoptosis. NETs selectively impact the expression of tight junction genes claudins 4, 8 and 11. Bronchial epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, decreases bronchial cell apoptosis and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, which are limited by exogenous A1AT.
Asunto(s)
Asma , Trampas Extracelulares , Humanos , Trampas Extracelulares/metabolismo , Asma/metabolismo , Bronquios , Línea Celular , Cadherinas/metabolismoRESUMEN
BACKGROUND: Interleukin (IL)-33 is implicated in the pathophysiology of asthma and allergic diseases. However, our knowledge is limited regarding how IL-33 release is controlled. The transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a key role in antioxidant response regulation. OBJECTIVE: The goal of this project was to investigate the role of cellular oxidative stress in controlling IL-33 release in airway epithelium. METHODS: Complementary approaches were used that included human bronchial epithelial cells and mouse models of airway type-2 immunity that were exposed to fungus Alternaria extract. The clinically available Nrf2 activator 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me) was used to evaluate the role of Nrf2-induced antioxidant molecules. RESULTS: Human bronchial epithelial cells produced reactive oxygen species (ROS) when they were exposed to Alternaria extract. ROS scavengers, such as glutathione (GSH) and N-acetyl cysteine, prevented extracellular secretion of ATP and increases in intracellular calcium concentrations that precede IL-33 release. Administration of CDDO-Me to mice enhanced expression of a number of antioxidant molecules in the lungs and elevated lung levels of endogenous GSH. Importantly, CDDO-Me treatment reduced allergen-induced ATP secretion and IL-33 release by airway epithelial cells in vitro and protected mice from IL-33 release and asthma-like pathological changes in the lungs. CONCLUSIONS: The balance between oxidative stress and antioxidant responses plays a key role in controlling IL-33 release in airway epithelium. The therapeutic potential of Nrf2 activators needs to be considered for asthma and allergic airway diseases.
Asunto(s)
Interleucina-33/metabolismo , Estrés Oxidativo , Adenosina Trifosfato/metabolismo , Alérgenos/inmunología , Animales , Antioxidantes/metabolismo , Calcio/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacología , Especies Reactivas de Oxígeno/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismoRESUMEN
Stimulation of adult rat alveolar epithelial cells with terbutaline was previously shown to activate Cl- channels in the apical membrane. In this study, we show that terbutaline stimulates net transepithelial (apical-to-basolateral) Cl- absorption from 0.19 +/- 0.13 to 1.43 +/- 0.31 mmol x cm-2 x hr-1. Terbutaline also increases net Cl- efflux across the basolateral membrane under conditions where an outward [K+] gradient exists and the membrane voltage is clamped at zero mV. When the [K+] gradient is eliminated, the effect of terbutaline on net Cl- efflux is inhibited to the extent that no significant Cl- efflux can be detected across the basolateral membrane. RT-PCR experiments detected mRNA for three KCl cotransport isoforms (KCC1, KCC3 and KCC4) in monolayer cultures of alveolar epithelial cells. Western blot analysis using antibodies to the four cloned isoforms of KCl cotransporters revealed the presence of KCC1 and KCC4 isoforms in monolayer cultures of these cells. These results provide evidence suggesting a role for KCl cotransport in terbutaline-stimulated transepithelial Cl- absorption.
Asunto(s)
Cloro/farmacocinética , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Simportadores/metabolismo , Terbutalina/farmacología , Animales , Transporte Biológico Activo/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Transporte Iónico/fisiología , Masculino , Isoformas de Proteínas/metabolismo , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Ratas , Ratas Sprague-Dawley , Cotransportadores de K ClRESUMEN
The role of phosphodiesterase (PDE) isoforms in regulation of transepithelial Cl secretion was investigated using cultured monolayers of T84 cells grown on membrane filters. Identification of the major PDE isoforms present in these cells was determined using ion exchange chromatography in combination with biochemical assays for cGMP and cAMP hydrolysis. The most abundant PDE isoform in these cells was PDE4 accounting for 70-80% of the total cAMP hydrolysis within the cytosolic and membrane fractions from these cells. The PDE3 isoform was also identified in both cytosolic and membrane fractions accounting for 20% of the total cAMP hydrolysis in the cytosolic fraction and 15-30% of the total cAMP hydrolysis observed in the membrane fraction. A large portion of the total cGMP hydrolysis detected in cytosolic and membrane fractions of T84 cells was mediated by PDE5 (50-75%). Treatment of confluent monolayers of T84 cells with various PDE inhibitors produced significant increases in short-circuit current (Isc). The PDE3-selective inhibitors terqinsin, milrinone and cilostamide produced increases in Isc with EC50 values of 0.6 nM, 8.0 nM and 0.5 microM respectively. These values were in close agreement with the IC50 values for cAMP hydrolysis. The effects of the PDE1-(8-MM-IBMX) and PDE4-(RP-73401) selective inhibitors on Isc were significantly less potent than PDE3 inhibitors with EC50 values of >7 microM and >50 microM respectively. However, the effects of 8-MM-IBMX and terqinsin on Cl secretion were additive, suggesting that inhibition of PDE1 also increases Cl secretion. The effect of PDE inhibitors on Isc were significantly blocked by apical treatment with glibenclamide (an inhibitor of the CFTR Cl channel) and by basolateral bumetanide, an inhibitor of Na-K-2Cl cotransport activity. These results indicate that inhibition of PDE activity in T84 cells stimulates transepithelial Cl secretion and that PDE1 and PDE3 are involved in regulating the rate of secretion.
Asunto(s)
Cloruros/metabolismo , Colon/metabolismo , AMP Cíclico/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Bumetanida/farmacología , Bovinos , Canales de Cloruro/metabolismo , Cromatografía por Intercambio Iónico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Conductividad Eléctrica , Gliburida/farmacología , Humanos , Técnicas In Vitro , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacologíaRESUMEN
Eosinophils play a primary role in the pathophysiology of asthma. In the lung, the activation state of the infiltrating eosinophils determines the extent of tissue damage. Interleukin-5 (IL-5) and leukotriene B4 (LTB4) are important signaling molecules involved in eosinophil recruitment and activation. However, the physiological processes that regulate these activation events are largely unknown. In this study we have examined the mechanisms of human eosinophil NADPH oxidase regulation by IL-5, LTB4, and phorbol ester (PMA). These stimuli activate a Zn2+-sensitive plasma membrane proton channel, and treatment of eosinophils with Zn2+ blocks superoxide production. We have demonstrated that eosinophil intracellular pH is not altered by IL-5 activation of NADPH oxidase. Additionally, PKCdelta inhibitors block PMA, IL-5 and LTB4 mediated superoxide formation. Interestingly, the PKCdelta-selective inhibitor, rottlerin, does not block proton channel activation by PMA indicating that the oxidase and the proton conductance are regulated at distinct phosphorylation sites. IL-5 and LTB4, but not PMA, stimulated superoxide production is also blocked by inhibitors of PI 3-kinase indicating that activation of this enzyme is an upstream event common to both receptor signaling pathways. Our results indicate that the G-protein-coupled LTB4 receptor and the IL-5 cytokine receptor converge on a common signaling pathway involving PI 3-kinase and PKCdelta to regulate NADPH oxidase activity in human eosinophils.
Asunto(s)
Eosinófilos/enzimología , Isoenzimas/fisiología , NADPH Oxidasas/metabolismo , Proteína Quinasa C/fisiología , Acetofenonas/farmacología , Benzopiranos/farmacología , Células Cultivadas , Cloruros/farmacología , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Humanos , Concentración de Iones de Hidrógeno , Interleucina-5/farmacología , Isoenzimas/antagonistas & inhibidores , Cinética , Leucotrieno B4/farmacología , Técnicas de Placa-Clamp , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-delta , Bombas de Protones/fisiología , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Compuestos de Zinc/farmacologíaRESUMEN
The effect of beta-adrenergic receptor stimulation on Cl- channel activation was investigated in alveolar epithelial cells grown in monolayer culture and in freshly isolated cells. Monolayers cultured under apical air interface conditions exhibited enhanced amiloride-sensitive Na+ transport compared to apical liquid interface monolayers. Amiloride or benzamil inhibited most (66%) of the basal short circuit current (Isc) with half-maximal inhibitory concentration (IC50) values of 0.62 microm and 0.09 microm respectively. Basolateral addition of terbutaline (2 microm) produced a rapid decrease in Isc followed by a slow recovery that exceeded the basal Isc. When Cl- was replaced with methanesulfonate in either intact monolayers or basolateral membrane permeabilized monolayers, the response to terbutaline (2 microm) was completely inhibited. No effect of terbutaline on amiloride-sensitive Na+ current was detected. beta-Adrenergic agonists and 8-chlorothiophenyl cyclic adenosine monophosphate (8-ctp cAMP) directly stimulated a Cl- channel in freshly isolated alveolar epithelial cells. The current was blocked by glibenclamide (100 microm) and had a reversal potential of -22 mV. No increase in amiloride-sensitve current was detected in response to terbutaline or 8-cpt cAMP stimulation. These data support the conclusion that beta-adrenergic agonists produce acute activation of apical Cl- channels and that monolayers maintained under apical air interface conditions exhibit increased Na+ absorption.
Asunto(s)
Adrenérgicos/farmacología , Canales de Cloruro/metabolismo , Células Epiteliales/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Aire , Amilorida/farmacología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Polaridad Celular , Células Cultivadas , Canales de Cloruro/antagonistas & inhibidores , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Gliburida/farmacología , Inmunohistoquímica , Concentración 50 Inhibidora , Masculino , Nitrobenzoatos/farmacología , Técnicas de Placa-Clamp , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Terbutalina/farmacologíaRESUMEN
The objective of this study was to investigate acute and long-term effects of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) on basal ion transport activity of glandular endometrial epithelial cells in primary culture. The effects of EGF on insulin-dependent regulation of Na+ transport across this epithelium was also investigated. Addition of 1.6 nM EGF or 2 nM TGFalpha to the basolateral, but not the apical, solution inhibited both basal and insulin-stimulated Na+ transport with a maximum response within 45-60 min. This effect was mimicked by the calcium ionophore ionomycin. Incubation with EGF for 4 days inhibited insulin-stimulated Na absorption in a concentration-dependent fashion with an IC(50) value of 0.3 nM. Experiments using amphotericin B-permeabilized monolayers demonstrated that EGF inhibited Na transport by decreasing apical membrane Na conductance without affecting insulin-dependent stimulation of the Na+-K+ ATPase. Addition of EGF or TGFalpha for 24 h resulted in increased basal Cl- secretion in addition to inhibition of Na absorption. The EGF-induced increase in Cl- secretion was inhibited in part by indomethacin, suggesting that long-term regulation by EGF involves stimulation of arachidonic acid synthesis and prostaglandin release. The EGF-induced increase in indomethacin-insensitive Cl- secretion was prevented by the protein synthesis inhibitor cyclohexamide, and by the DNA transcription inhibitor actinomycin D indicating that EGF-stimulated anion secretion required DNA transcription and protein synthesis. The results of these studies demonstrated that the basal transport properties of endometrial epithelial cells are differentially regulated by EGF, TGFalpha, and insulin.
Asunto(s)
Endometrio/citología , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/fisiología , Sodio/metabolismo , Factor de Crecimiento Transformador alfa/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Cloruros/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Dactinomicina/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Indometacina/farmacología , Insulina/farmacología , Ionomicina/farmacología , Cinética , Nitrobenzoatos/farmacología , Prostaglandinas/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , PorcinosRESUMEN
We investigated the amino acid specificity of a Na-dependent amino acid cotransport system that contributes to transepithelial Na absorption in the apical membrane of cultured adult rat alveolar epithelial cell monolayers. Short-circuit current was increased by basic, uncharged polar, and nonpolar amino acids but not by L-aspartic acid or L-proline. EC(50) values for L-lysine and L-histidine were 0.16 and 0.058 mM, respectively. The L-lysine-stimulated short-circuit current was Na dependent, with a concentration causing a half-maximal stimulation by Na of 44.24 mM. L-Serine, L-glutamine, and L-cysteine had EC(50) values of 0.095, 0.25, and 0.12 mM, respectively. L-Alanine had the highest affinity, with an EC(50) of 0.027 mM. We conclude that monolayer cultures of adult rat alveolar epithelial cells possess a broad-specificity Na-dependent amino acid cotransport system with properties consistent with system B(0,+). We suggest that this cotransport system plays a critical role in recycling of constituent amino acids that make up glutathione, thus ensuring efficient replenishment of this important antioxidant within the alveolar fluid.
Asunto(s)
Aminoácidos/metabolismo , Aminoácidos/farmacología , Proteínas Portadoras/metabolismo , Alveolos Pulmonares/fisiología , Mucosa Respiratoria/fisiología , Sodio/metabolismo , Amilorida/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Células Cultivadas , Lisina/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Alveolos Pulmonares/citología , Ratas , Ratas Sprague-Dawley , Mucosa Respiratoria/citología , Especificidad por SustratoRESUMEN
In this review, we discuss evidence that supports the hypothesis that adrenergic stimulation of transepithelial Na absorption across the alveolar epithelium occurs indirectly by activation of apical Cl channels, resulting in hyperpolarization and an increased driving force for Na uptake through amiloride-sensitive Na channels. This hypothesis differs from the prevailing idea that adrenergic-receptor activation increases the open probability of Na channels, leading to an increase in apical membrane Na permeability and an increase in Na and fluid uptake from the alveolar space. We review results from cultured alveolar epithelial cell monolayer experiments that show increases in apical membrane Cl conductance in the absence of any change in Na conductance after stimulation by selective beta-adrenergic-receptor agonists. We also discuss possible reasons for differences in Na-channel regulation in cells grown in monolayer culture compared with that in dissociated alveolar epithelial cells. Finally, we describe some preliminary in vivo data that suggest a role for Cl-channel activation in the process of amiloride-sensitive alveolar fluid absorption.
Asunto(s)
Canales de Cloruro/fisiología , Alveolos Pulmonares/metabolismo , Sodio/metabolismo , Animales , Transporte Biológico/fisiología , Células Epiteliales/metabolismo , Humanos , Alveolos Pulmonares/citologíaRESUMEN
The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(+) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(+) transport two- to threefold by increasing Na(+)-K(+) ATPase transport activity and basolateral membrane K(+) conductance without increasing the apical membrane amiloride-sensitive Na(+) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(+) absorption with a further increase in Na(+)-K(+) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(+) conductance. The effect of insulin on the Na(+)-K(+) ATPase was the result of an increase in V(max) for extracellular K(+) and intracellular Na(+), and an increase in affinity of the pump for Na(+). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of alpha-1 and alpha-2 isoforms, but not the alpha-3 isoform of Na(+)-K(+) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(+) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(+) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(+)-K(+) ATPase activation.
Asunto(s)
Endometrio/metabolismo , Activadores de Enzimas/farmacología , Células Epiteliales/metabolismo , Insulina/farmacología , Fosfoproteínas Fosfatasas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Transporte Biológico Activo/efectos de los fármacos , Western Blotting , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Electrofisiología , Endometrio/citología , Endometrio/efectos de los fármacos , Activación Enzimática/fisiología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Femenino , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/fisiología , Ouabaína/metabolismo , Canales de Potasio/metabolismo , Estimulación Química , PorcinosRESUMEN
Alveolar epithelial cells were isolated from adult Sprague-Dawley rats and grown to confluence on membrane filters. Most of the basal short-circuit current (Isc; 60%) was inhibited by amiloride (IC50 0. 96 microM) or benzamil (IC50 0.5 microM). Basolateral addition of terbutaline (2 microM) produced a rapid decrease in Isc, followed by a slow recovery back to its initial amplitude. When Cl- was replaced with methanesulfonic acid, the basal Isc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of -28 mV. Increasing Cl- concentration in the basolateral solution shifted the reversal potential to more depolarized voltages. These results were consistent with the existence of a terbutaline-activated Cl- conductance in the apical membrane. Terbutaline did not increase the amiloride-sensitive Na+ conductance. We conclude that beta-adrenergic stimulation of adult alveolar epithelial cells results in an increase in apical Cl- permeability and that amiloride-sensitive Na+ channels are not directly affected by this stimulation.
Asunto(s)
Canales de Cloruro/fisiología , Alveolos Pulmonares/metabolismo , Receptores Adrenérgicos beta/fisiología , Sodio/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/efectos de los fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Electrofisiología , Células Epiteliales/metabolismo , Masculino , Alveolos Pulmonares/citología , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio , Terbutalina/farmacología , Tionucleótidos/farmacologíaRESUMEN
1. The objective of this study was to investigate the mechanism of PGE2 regulation of Cl- transport across glandular endometrial cells grown in primary culture. 2. Most of the basal short circuit current (Isc) was inhibited by luminal addition of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or glibenclamide, suggesting the presence of a basally active Cl- conductance in the apical membrane. 3. Basolateral addition of 10 microM PGE2 increased Isc by 41 +/- 3 microA. A similar response was observed when cells were treated with 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Pretreatment of monolayers with NPPB and glibenclamide blocked the PGE2 and cAMP-mediated increase in Isc, suggesting that the effects of PGE2 and cAMP were dependent on the activity of an apical NPPB- and glibenclamide-sensitive conductance. 4. Addition of 50 nM antiPGE2 antibody to the basolateral bathing solution decreased basal Isc by 20 % and shifted the threshold response to exogenous PGE2. This result suggests autocrine regulation of electrogenic Cl- transport by PGE2. 5. Experiments with amphotericin B-permeabilized monolayers revealed that the apical PGE2-activated, NPPB- and glibenclamide-sensitive conductance was Cl- dependent and that the current-voltage relationship and anion permeation properties (SCN->Br- > Cl- > I-) were characteristic of the cystic fibrosis transmembrane conductance regulator (CFTR). 6. Cultured porcine endometrial epithelial cells were specifically labelled with an antibody to a peptide sequence within the regulatory domain of CFTR. 7. The effect of PGE2 was blocked by basolateral addition of bumetanide and furosemide at concentrations that are selective for inhibition of Na+-K+-2Cl-cotransport activity. The effect of bumetanide on Isc was Cl- dependent, suggesting a role for the bumetanide-sensitive transport pathway in Cl- secretion. 8. PGE2 and cAMP also activated an outwardly rectifying basolateral K+ channel which presumably sustains the driving force for electrogenic Cl- efflux across the apical membrane. 9. The concentration-conductance and concentration-Isc response relationships for PGE2 showed that basolateral K+ permeability was rate limiting with respect to transepithelial anion secretion and that activation of a basolateral K+ channel by PGE2 was necessary to achieve maximum rates of Cl- secretion.
Asunto(s)
Canales de Cloruro/fisiología , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Dinoprostona/farmacología , Endometrio/fisiología , Secuencia de Aminoácidos , Anfotericina B/farmacología , Animales , Aniones/metabolismo , Anticuerpos/farmacología , Bumetanida/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Canales de Cloruro/efectos de los fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Dinoprostona/inmunología , Endometrio/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Femenino , Furosemida/farmacología , Gliburida/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Biológicos , Nitrobenzoatos/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Simportadores de Cloruro de Sodio-Potasio , Porcinos , Tionucleótidos/farmacología , Factores de TiempoRESUMEN
In the present paper, the mechanisms underlying the neural and hormonal regulation of mucosal ion transport in the pig intestinal tract are reviewed. The active transport of NaCl by isolated sheets of porcine intestinal mucosa is modulated by cholinergic and non-cholinergic neurons of undetermined neurochemical identity that lie in the submucosa. The application of electrical field stimulation to mucosa-submucosa preparations from porcine jejunum, ileum, or colon produces rapid elevations in short-circuit current which are inhibited by tetrodotoxin or omega-conotoxin GVIA, blockers of neuronal Na+ and Ca2+ channels, respectively. In porcine ileum, these elevations in current are mimicked in large part by cholinergic agonists and have been attributed to anion secretion. The majority of classical neurotransmitters and gut peptides that have been examined to date increase active transepithelial anion secretion through interactions with G protein-coupled receptors associated with submucosal neurons or situated on the basolateral membranes of epithelial cells. A small number of neuropeptides interact with neuronal receptors to augment NaCl absorption or decrease anion secretion. Noradrenergic control of intestinal transport differs in the porcine small and large intestines, and displays considerable inter-species variability in its cellular underpinnings. Transport regulation by bombesin-like peptides may be mediated by receptors distributed in both the apical and basolateral membrane domains of epithelial cells in porcine colon. The transport process affected by these peptides may be linked to epithelial growth and differentiation. The pig intestinal tract appears to be a useful biological model for resolving the cellular mechanisms by which gut neurotransmitters and hormones act in regulating transepithelial ion fluxes. Its general relevance to human intestinal function is discussed.
Asunto(s)
Hormonas Gastrointestinales/fisiología , Mucosa Intestinal/metabolismo , Transporte Iónico/fisiología , Neurotransmisores/fisiología , Porcinos/metabolismo , Animales , Bombesina/fisiología , Humanos , Receptores Adrenérgicos/fisiologíaRESUMEN
Previous studies in our laboratory have shown that Na absorption across the porcine endometrium is stimulated by PGF2alpha and cAMP-dependent activation of a barium-sensitive K channel located in the basolateral membrane of surface epithelial cells. In this study, we identify and characterize this basolateral, barium-sensitive K conductance. Porcine uterine tissues were mounted in Ussing chambers and bathed with KMeSO4 Ringer solution. Amphotericin B (70 microM) was added to the luminal solution to permeabilize the apical membrane and determine the current-voltage relationship of the basolateral K conductance after activation by 100 microM CPT-cAMP. An inwardly rectifying current was identified which possessed a reversal potential of -53 mV when standard Ringer solution was used to bathe the serosal surface. The K:Na selectivity ratio was calculated to be 12:1. Administration of 5 mM barium to the serosal solution completely inhibited the current activated by cAMP under these conditions. In addition to these experiments, amphotericin-perforated whole cell patch clamp recordings were obtained from primary cultures of porcine surface endometrial cells. The isolated cells displayed an inwardly rectifying current under basal conditions. This current was significantly stimulated by CPT-cAMP and blocked by barium. These results together with our previous studies demonstrate that cAMP increases Na absorption in porcine endometrial epithelial cells by activating an inwardly rectifying K channel present in the basolateral membrane. Similar patch clamp experiments were conducted using cells from a human endometrial epithelial cell line, RL95-2. An inwardly rectifying current was also identified in these cells which possessed a reversal potential of -56 mV when the cells were bathed in standard Ringer solution. This current was blocked by barium as well as cesium. However, the current from the human cells did not appear to be activated by cAMP, indicating that distinct subtypes of inwardly rectifying K channels are present in endometrial epithelial cells from different species.
Asunto(s)
AMP Cíclico/metabolismo , Endometrio/metabolismo , Canales de Potasio/metabolismo , Sodio/metabolismo , Absorción , Animales , Bario/farmacología , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Endometrio/citología , Endometrio/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Técnicas In Vitro , Transporte Iónico/efectos de los fármacos , Potenciales de la Membrana , Modelos Biológicos , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Especificidad de la Especie , Porcinos , Tionucleótidos/farmacologíaRESUMEN
The purpose of this study was to investigate the regulation of electrolyte transport across the porcine endometrium by gastrin-releasing peptide (GRP) and substance P (SP). Luminal addition of GRP, neuromedin B (NMB), SP, or neurokinin A(NKA) to mucosal tissues mounted in Ussing chambers produced a multiphasic change in short-circuit current (Isc) characterized by an initial rapid increase and subsequent decrease in current. A similar response was obtained after addition of ionomycin or thapsigargin to the tissues. The Isc response to the peptides or Ca ionophore was inhibited by pretreatment of the tissues with luminal amiloride or benzamil. GRP and SP were more potent [50% effective concentration (EC50) of 3 nM] than NMB or NKA (EC50 values of 46 and 26 nM, respectively) in producing the decrease in Isc. Pretreatment with the GRP receptor antagonist 3-Phe-His-Trp-Ala-Val-D-Ala-His-D-Pro-psi Phe-NH2 blocked the Isc response to GRP and NMB but not to SP or NKA, whereas the NMB receptor antagonist D-Nal-[Cys-Try-D-Trp-Orn-Val-Cys]-Nal-NH2 was ineffective in inhibiting the Isc response to any of the peptides. In contrast, pretreatment of the tissue with the nonpeptide SP receptor antagonist CP-99,994 blocked the Isc response to SP and NKA but not to GRP or NMB. Experiments with amphotericin B-permeabilized tissues showed that GRP, SP, ionomycin, and thapsigargin increased current through an outwardly rectifying K conductance located on the apical membrane of the cells. The K-to-Na selectivity ratio of this conductance was calculated to be 2.5:1. These experiments showed that GRP and SP, acting through different receptors, produced an increase in K efflux through a Ca-dependent K conductance present in the apical membrane of surface endometrial epithelial cells. In addition, immunohistochemistry data showed that GRP-like immunoreactivity was localized to surface and glandular epithelial cells, whereas GRP receptor antibody labeling was observed in both epithelial and stromal cells. These results suggest that GRP functions as both an autocrine and paracrine regulatory peptide in the endometrium.
Asunto(s)
Electrólitos/metabolismo , Miometrio/fisiología , Péptidos/farmacología , Sustancia P/farmacología , Amilorida/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Femenino , Péptido Liberador de Gastrina , Hormonas Gastrointestinales/farmacología , Ionomicina/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Membrana Mucosa/citología , Membrana Mucosa/efectos de los fármacos , Membrana Mucosa/fisiología , Miometrio/citología , Miometrio/efectos de los fármacos , Neuroquinina A/farmacología , Neuroquinina B/análogos & derivados , Neuroquinina B/farmacología , Antagonistas del Receptor de Neuroquinina-1 , Piperidinas/farmacología , Potasio/metabolismo , Porcinos , Tapsigargina/farmacologíaRESUMEN
Alveolar fluid is resorbed using active Na+ transport primarily through basolateral sodium-potassium-adenosinetriphosphatase (Na-K-ATPase) and apical Na+ channels that are particularly dense on the alveolar type II (ATII) epithelial cells. During lung injury with pulmonary edema, continued or accelerated Na+ and fluid resorption is critical for a favorable outcome. However, little is known of how ATII cell Na+ transport is affected during injury. These experiments examined the effects of acute lung injury on ATII cell Na-K-ATPase activity and expression using an established model of rats exposed to 100% O(2) for 60 h. Na-K-ATPase activity of ATII cells isolated immediately after exposure was assessed by ouabain-sensitive (86)Rb+ uptake in intact cells and by ouabain-sensitive P(i) production by cell membranes. In the presence of 1 mM ouabain, ouabain-sensitive Rb+ uptake was not different between normoxic and hyperoxic cells, but the apparent Na-K-ATPase maximal velocity (Vmax) of hyperoxic cell membranes was 75 +/- 8% of normoxic membranes (P < 0.05). On Western blots of ATII cell membranes, alpha1-subunit protein significantly decreased with hyperoxia (35 +/- 9% of normoxia; P < 0.05), whereas the amounts of the beta-subunit were unchanged (P > 0.05). On Northern blots of ATII cell total RNA, steady-state levels of both the alpha1- and beta1-subunit mRNA increased after hyperoxia (alpha1 = 2.5 +/- 1.3-fold; beta1 = 4.6 +/- 2.5-fold). Thus despite hyperoxic decreases in Na-K-ATPase Vmax and the amount of alpha1-protein, Rb+ uptake by Na-K-ATPase in intact cells was unchanged. The mRNA levels, protein amounts, and enzyme activity did not respond in parallel to hyperoxic injury, and the activity in intact cells correlated best with the amounts of the beta-subunit, the limiting component in de novo pump assembly in many tissues.
Asunto(s)
Hiperoxia/enzimología , Alveolos Pulmonares/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Transporte Biológico Activo , Northern Blotting , Western Blotting , Bumetanida/farmacología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Cinética , Masculino , Ouabaína/farmacología , Oxidación-Reducción , Fosfatos/metabolismo , Potasio/metabolismo , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Rubidio/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
The functional properties of the G protein-coupled P2Y1 receptor were investigated in Xenopus oocytes. Incubation of oocytes expressing either the human or turkey P2Y1 receptor with adenine nucleotide agonists resulted in an increase in Cl- current and activation of a novel cation current with an inwardly rectifying current-voltage relationship. Activation of either the human P2Y2 (P2U-purinergic) or M1 muscarinic receptor expressed in oocytes resulted in an increase in Cl- current similar to that observed in P2Y1 receptor-expressing oocytes but had no effect on cation current. P2 receptor agonists stimulated both the cation current and Cl- current in P2Y1 receptor-expressing oocytes with EC50 values and an order of potency (2-methylthioadenosine diphosphate > 2-methylthioadenosine triphosphate (2MeSATP) > ATP > UTP) that were similar to those previously observed for activation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human or turkey P2Y1 receptor. The P2Y receptor antagonists suramin and pyridoxal phosphate 6-azophenyl-2'-4'-disulfonic acid both shifted to the right the concentration-response relationship for 2MeSATP for stimulation of oocyte currents. Although injection of oocytes with either GDPbetaS (guanyl-5'-yl thiophosphate) or GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) resulted in loss of adenine nucleotide-promoted Cl- channel activation, neither guanine nucleotide altered the 2MeSATP-stimulated cation current. These data are consistent with the view that activation of the novel cation current by the P2Y1 receptor does not involve a G protein. Tail current analysis of the novel P2Y1 receptor-associated cation conductance revealed that the open channel current-voltage relationship was outwardly rectifying with a reversal potential of -38 mV for the turkey P2Y1 receptor and -36 mV for the human P2Y1 receptor. Replacement of Na+ with K+ ions in the bathing solution produced a shift in reversal potential to near zero mV, but significant outward rectification remained. The cation current was not permeable to either Ca2+ or Ba2+ and exhibited steady-state inactivation at holding potentials below -60 mV. These results indicate that the P2Y1 receptor exhibits both metabotropic properties and a novel G protein-independent ionotropic response when expressed in Xenopus oocytes.
Asunto(s)
Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/análogos & derivados , Receptores Purinérgicos P2/metabolismo , Tionucleótidos/metabolismo , Animales , Clonación Molecular , Femenino , Proteínas de Unión al GTP/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Activación del Canal Iónico , Oocitos/metabolismo , Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Pavos , Xenopus laevisRESUMEN
Of particular interest for voltage-gated K+ channels are the effects of membrane voltage and pharmacologic agents on channel kinetics. We have characterized in detail properties of Kv1.2 channel expressed in oocytes as the basis for investigation of its structure-function relationships. This channel exhibited a voltage-dependent rate of activation with a V1/2 of -21 mV. Voltage-dependent steady-state inactivation overlapped the activation curve with half-maximal inactivation occurring at -22 mV. Dendrotoxin inhibited channel activation with an IC50 of 8.6 nM at + 35 mV. Charybdotoxin also blocked this K+ channel (IC50 = 5.6 nM). While dendrotoxin block was not affected by channel activation, charybdotoxin exhibited additional accumulation of block following activation, which was relieved with a time constant of 0.5 s upon repolarization of the membrane. The deactivation of this channel was accelerated in the presence of charybdotoxin while not significantly affected by dendrotoxin.
Asunto(s)
Caribdotoxina/farmacología , Venenos Elapídicos/farmacología , Neurotoxinas/farmacología , Canales de Potasio/efectos de los fármacos , Animales , Activación del Canal Iónico , Cinética , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Relación Estructura-Actividad , Xenopus laevisRESUMEN
Regulation of electrolyte transport across porcine distal colon epithelium by gastrin-releasing peptide (GRP) was examined using mucosal sheets mounted in Ussing chambers. Serosal GRP produced a biphasic response consisting of a transient increase in short-circuit current (ISC) followed by a long-lasting decrease. Indomethacin and tetrodotoxin inhibited the ISC increase without affecting the secondary decrease. Addition of GRP to the mucosal solution produced a decrease in ISC similar to that observed with serosal treatment, but no transient increase in ISC was observed. GRP and bombesin (50% effective concentrations of 26 and 30 nM, respectively) were more effective than neuromedin B in decreasing the ISC, and the GRP receptor antagonist [D-Phe(6)]bombesin(6-13)-O-methyl produced a sixfold dextral shift in the GRP concentration-response relationship. The GRP-stimulated decrease was reduced in the absence of Cl and by serosal bumetanide. Flux measurements showed that GRP increased Rb and Na secretion while having no effect on transepithelial Cl transport. Phosphoinositide turnover was increased by GRP, suggesting that the ion transport changes may be mediated by intracellular Ca concentration. The results of this study demonstrate that GRP stimulates K and Na secretion across the porcine distal colon epithelium and that these processes are dependent, in part, on a bumetanide-sensitive transport pathway located in the basolateral membrane.
Asunto(s)
Colon/fisiología , Mucosa Intestinal/fisiología , Péptidos/farmacología , Potasio/metabolismo , Sodio/metabolismo , Animales , Electrofisiología , Péptido Liberador de Gastrina , Hormonas Gastrointestinales/farmacología , Técnicas In Vitro , Indometacina/farmacología , Mucosa Intestinal/efectos de los fármacos , Cinética , Potenciales de la Membrana , Fosfatidilinositoles/metabolismo , Porcinos , Tetrodotoxina/farmacologíaRESUMEN
The purpose of this study was to examine the potential modulatory effects of gastrin-releasing peptide (GRP) on prostaglandin (PG) E2-stimulated electrolyte transport across the distal colon epithelium. In an earlier study, PGE2 was shown to reduce net Cl absorption without altering the serosal-to-mucosal unidirectional Cl flux in porcine distal colon (19). In the present study, tissues were pretreated with serosal or mucosal GRP and subsequently stimulated with PGE2. The resulting increase in short-circuit current (ISC) was 152% (serosal GRP) and 49% (mucosal GRP) greater than control PGE2 responses alone. Serosal, but not mucosal, GRP also enhanced the ISC response to vasoactive intestinal peptide. On the basis of flux measurements, the combined effects of serosal GRP and PGE2 resulted in the activation of a transcellular pathway for Cl secretion, which was not activated by either mediator alone. The time course of the PGE2 response was also affected by GRP. Serosal GRP shortened the time to maximum ISC by 35%, whereas mucosal peptide lengthened the time to maximum ISC by 68% These results suggest that GRP acts as a modulator of PG action on electrolyte transport in the distal colon.