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1.
J Biol Chem ; 298(3): 101659, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35101441

RESUMEN

Ion channels use charged amino-acid residues to attract oppositely charged permeant ions into the channel pore. In the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, a number of arginine and lysine residues have been shown to be important for Cl- permeation. Among these, two in close proximity in the pore-Lys95 and Arg134-are indispensable for anion binding and high Cl- conductance, suggesting that high positive charge density is required for pore function. Here we used mutagenesis and functional characterization to show that a nearby pore-lining negatively charged residue (Glu92) plays a functionally additive role with these two positive charges. While neutralization of this negative charge had little effect on anion binding or Cl- conductance, such neutralization was able to reverse the detrimental effects of removing the positive charge at either Lys95 or Arg134, as well as the similar effects of introducing a negative charge at a neighboring residue (Ser1141). Furthermore, neutralization of Glu92 greatly increased the susceptibility of the channel to blockage by divalent S2O32- anions, mimicking the effect of introducing additional positive charge in this region; this effect was reversed by concurrent neutralization of either Lys95 or Arg134. Across a panel of mutant channels that introduced or removed fixed charges at these four positions, we found that many pore properties are dependent on the overall charge or charge density. We propose that the CFTR pore uses a combination of positively and negatively charged residues to optimize the anion binding and Cl- conductance properties of the channel.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Aniones/química , Aniones/metabolismo , Arginina/química , Arginina/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fenómenos Electrofisiológicos , Transporte Iónico , Lisina/química , Lisina/metabolismo , Electricidad Estática
2.
Cell Mol Life Sci ; 78(12): 5213-5223, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-34023918

RESUMEN

Positively charged amino acid side-chains play important roles in anion binding and permeation through the CFTR chloride channel. One pore-lining lysine residue in particular (K95) has been shown to be indispensable for anion binding, conductance, and selectivity. Here, we use functional investigation of CFTR to show that a nearby arginine (R134) plays a functionally analogous role. Removal of this positive charge (in the R134Q mutant) drastically reduces single-channel conductance, weakens binding of both permeant and blocking anions, and abolishes the normal anion conductance selectivity pattern. Each of these functional effects was reversed by a second-site mutation (S1141K) that introduces an ectopic positive charge to a nearby pore-lining residue. Substituted cysteine accessibility experiments confirm that R134-but not nearby residues in the same transmembrane helix-is accessible within the pore lumen. These results suggest that K95 and R134, which are very close together within the inner vestibule of the pore, play analogous, important roles, and that both are required for the normal anion binding and anion conductance properties of the pore. Nevertheless, that fact that both positive charges can be "transplanted" to other sites in the inner vestibule with little effect on channel permeation properties indicates that it is the overall number of charges-rather than their exact locations-that controls pore function.


Asunto(s)
Aniones/metabolismo , Arginina/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Lisina/metabolismo , Mutación , Animales , Arginina/química , Arginina/genética , Células Cultivadas , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Lisina/química , Lisina/genética , Técnicas de Placa-Clamp , Conformación Proteica
3.
Cell Mol Life Sci ; 76(12): 2411-2423, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30758641

RESUMEN

Our molecular understanding of the cystic fibrosis transmembrane conductance regulator (CFTR)-the chloride channel that is mutated in cystic fibrosis-has been greatly enhanced by a number of recent atomic-level structures of the protein in different conformations. One surprising aspect of these structures was the finding that the eighth of CFTR's 12 membrane-spanning segments (TM8) appeared close to the channel pore. Although functional evidence supports a role for other TMs in forming the pore, such a role for TM8 has not previously been reported. Here, we use patch-clamp recording to investigate the functional role of TM8. Using substituted cysteine accessibility mutagenesis, we find that three amino acid side-chains in TM8 (Y913, Y914, and Y917) are exposed to the extracellular, but not the intracellular, solution. Cysteine cross-linking experiments suggest that Y914 and Y917 are in close proximity to L102 (TM1) and F337 (TM6), respectively, suggesting that TM8 contributes to the narrow selectivity filter region of the pore. Different amino acid substitutions suggest that Y914, and to a lesser extent Y917, play important roles in controlling anion flux through the open channel. Furthermore, substitutions that reduce side-chain volume at Y917 severely affect channel gating, resulting in a channel with an extremely unstable open state. Our results suggest that pore-lining TM8 is among the most important TMs controlling the permeation phenotype of the CFTR channel, and also that movement of TM8 may be critically involved in channel gating.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Animales , Células CHO , Línea Celular , Cricetulus , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Modelos Moleculares , Mutación Puntual , Conformación Proteica
4.
J Biol Chem ; 293(15): 5649-5658, 2018 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-29475947

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that apparently has evolved from an ancestral active transporter. Key to the CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals." Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electrodiffusional movement of Cl- ions. However, these portals remain the least well-characterized part of the Cl- transport pathway; even the number of functional portals is uncertain, and if multiple portals do exist, their relative functional contributions are unknown. Here, we used patch-clamp recording to identify the contributions of positively charged amino acid side chains located in CFTR's cytoplasmic transmembrane extensions to portal function. Mutagenesis-mediated neutralization of several charged side chains reduced single-channel Cl- conductance. However, these same mutations differentially affected channel blockade by cytoplasmic suramin and Pt(NO2)42- anions. We considered and tested several models by which the contribution of these positively charged side chains to one or more independent or non-independent portals to the pore could affect Cl- conductance and interactions with blockers. Overall, our results suggest the existence of a single portal that is lined by several positively charged side chains that interact electrostatically with both Cl- and blocking anions. We further propose that mutations at other sites indirectly alter the function of this single portal. Comparison of our functional results with recent structural information on CFTR completes our picture of the overall molecular architecture of the Cl- permeation pathway.


Asunto(s)
Membrana Celular/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mutación , Animales , Línea Celular , Membrana Celular/química , Membrana Celular/genética , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Transporte Iónico/fisiología , Dominios Proteicos
5.
Cell Mol Life Sci ; 75(16): 3027-3038, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29441426

RESUMEN

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd2+ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.


Asunto(s)
Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Activación del Canal Iónico , Conformación Proteica , Animales , Células CHO , Cricetinae , Cricetulus , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Humanos , Metales/química , Metales/metabolismo , Modelos Moleculares , Mutación
6.
Biochim Biophys Acta Biomembr ; 1859(5): 1049-1058, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28235470

RESUMEN

The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the "selectivity filter" of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region. However, little is known about the three-dimensional organization of this region, or how multiple TMs combine to determine its functional properties. In the present study we have used patch clamp recording to identify changes in channel function associated with the formation of disulfide cross-links between cysteine residues introduced into different TMs within the selectivity filter. Cysteine introduced at position L102 in TM1 was able to form disulfide bonds with F337C and T338C in TM6, two positions that are known to play key roles in determining anion permeation properties. Consistent with this proximal arrangement of L102, F337 and T338, different mutations at L102 altered anion selectivity and conductance properties in a way that suggests that this residue plays an important role in determining selectivity filter function, albeit a much lesser role than that of F337. These results suggest an asymmetric three-dimensional arrangement of the key selectivity filter region of the pore, as well as having important implications regarding the molecular mechanism of anion permeation.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Animales , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Disulfuros/química , Ditiotreitol/farmacología , Leucina/química , Técnicas de Placa-Clamp
7.
Am J Physiol Lung Cell Mol Physiol ; 304(1): L4-16, 2013 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-23125251

RESUMEN

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.


Asunto(s)
Músculo Liso/citología , Sistema Respiratorio/citología , Técnicas de Cultivo de Tejidos/métodos , Animales , Asma/fisiopatología , Técnicas de Cocultivo , Expresión Génica , Humanos , Ratones , Modelos Biológicos , Contracción Muscular/fisiología , Células 3T3 NIH , Estrés Mecánico , Ingeniería de Tejidos/métodos
8.
J Pharmacol Exp Ther ; 341(2): 552-63, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22362924

RESUMEN

Oxidative stress is implicated in the pathogenesis of many inflammatory pulmonary diseases, including cystic fibrosis (CF). Delineating how oxidative stress stimulates CF transmembrane conductance regulator (CFTR) in airway epithelial cells is useful, both to increase the understanding of airways host defense and suggest therapeutic approaches to reduce the oxidant stress burden in the CF lung. Using the airway epithelial cell line Calu-3, we investigated the hypothesis that hydrogen peroxide (H2O2), which stimulates anion efflux through CFTR, does so via the production of prostaglandin E2 (PGE2). Using iodide efflux as a biochemical marker of CFTR activity and short circuit current (I(sc)) recordings, we found that the H2O2-stimulated efflux was abolished by cyclooxygenase-1 inhibition and potentially also involves microsomal prostaglandin E synthase-1 activity, implicating a role for PGE2 production. Furthermore, H2O2 application resulted in a rapid release of PGE2 from Calu-3 cells. We additionally hypothesized that the PGE2 subtype 4 (EP(4)) receptor was involved in mediating this response. In the presence of (4Z)-7-[(rel-1S,2S,5R)-5-((1,1'-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848) (which blocks the EP4 receptor), the H2O2-stimulated response was abolished. To investigate this finding in a polarized system, we measured the increase in I(sc) induced by H2O2 addition in the presence and absence of AH23848. H2O2 induced a robust increase in I(sc), which was significantly attenuated in the presence of AH23848, suggesting some role for the EP4 receptor. In conclusion, with H2O2 as a model oxidant stress, stimulation of CFTR seems to involve PGE2 production and likely EP4 receptor activation in Calu-3 airway epithelial cells. This mechanism would be compromised in the CF airways.


Asunto(s)
Células Epiteliales/fisiología , Estrés Oxidativo/fisiología , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Sistema Respiratorio/fisiopatología , Aniones/metabolismo , Proteínas Sanguíneas/farmacología , Línea Celular , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Oxidorreductasas Intramoleculares/metabolismo , Yoduros/metabolismo , Microsomas/metabolismo , Prostaglandina-E Sintasas , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/fisiopatología , Sistema Respiratorio/metabolismo
9.
Pulm Pharmacol Ther ; 24(1): 42-8, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20970516

RESUMEN

Prostaglandin (PG)E2 mediates its effects via activation of four distinct PGE2 receptors, termed EP1₋4, all of which are present on the model human airway epithelial cell line, Calu-3. We previously reported that acute activation of the EP4 subtype of the PGE2 receptor is associated with increased anion efflux from these cells, via the CFTR chloride channel. In the present study we examine the effects of longer term activation of the EP4 receptor in Calu-3 cells in an attempt to determine whether this would prove beneficial or detrimental to the airway epithelial cell environment. Using PGE1-OH, an EP4 receptor selective agonist, we determined that EP4 receptor activation was associated with increased phosphorylation of extracellular signal-related kinases (ERKs) and induction of the transcription factor early growth response factor-1 (Egr-1). Additionally, using specific enzyme-linked immunosorbent assays and quantitative PCR, we detected increased production of PGE2, IL-6, IL-8 and the chemokine monocyte chemotactic protein-1 (MCP-1) at both the protein and gene level in response to EP4 receptor activation. Intriguingly, the enhanced production of PGE2 in response to EP4 receptor activation raises the possibility of a positive feedback situation. Generally, within the airways, PGE2 is considered to have pro-inflammatory effects, whilst the enhanced production of IL-6, IL-8 and MCP-1 would be associated with the recruitment and activation of inflammatory cells to the airways. Thus, we conclude that chronic activation of the EP4 receptor is associated with increased production of mediators likely to increase the pro-inflammatory milieu of airway epithelial cells.


Asunto(s)
Citocinas/biosíntesis , Dinoprostona/biosíntesis , Células Epiteliales/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/fisiología , Tráquea/metabolismo , Células Cultivadas , Quimiocina CCL2/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz/análisis , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis
10.
Cell Biochem Biophys ; 78(1): 15-22, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31893350

RESUMEN

Anions enter from the cytoplasm into the channel pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel not via a central pathway but via a single lateral portal or fenestration. High Cl- conductance is dependent on electrostatic attraction of cytoplasmic Cl- ions by four positively charged amino acid side-chains located within this portal. Here we use a mutagenic approach to investigate the functional effects of transplanting or supplementing these positive charges at nearby portal-lining sites. Using patch clamp recording, we find that the functionally important positive charges at K190 and R303 can be transplanted to four nearby sites (N186, L197, W356, and A367) with little loss of Cl- conductance. Introduction of additional positive charge at these sites had almost no effect on Cl- conductance, but did increase the sensitivity to channel block by intracellular suramin and Pt(NO2)42- anions. We suggest that it is the number of positive charges within the portal, rather than their exact location, that is the most important factor influencing Cl- conductance. The portal appears well optimized in terms of charge distribution to maximize Cl- conductance.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citoplasma/metabolismo , Animales , Aniones/química , Aniones/metabolismo , Línea Celular , Complejos de Coordinación/química , Complejos de Coordinación/metabolismo , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Platino (Metal)/química , Electricidad Estática , Suramina/química , Suramina/metabolismo
11.
Am J Respir Cell Mol Biol ; 38(2): 143-52, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17690331

RESUMEN

Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the peroxidation of membrane polyunsaturated fatty acids. Previous work has demonstrated that the isoprostane 8-iso-prostaglandin E(2) (PGE(2)) stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial anion secretion across the human airway epithelial cell line, Calu-3. Since isoprostanes predominantly achieve their effects via binding to prostanoid receptors, we hypothesized that this 8-iso-PGE(2) stimulation of CFTR activity was the result of the isoprostane binding to a prostanoid receptor. Using RT-PCR, immunoblotting, and immunofluorescence, we here demonstrate that Calu-3 cells express the EP(1-4) and FP receptors, and localize these proteins in polarized cell monolayers. Using iodide efflux as a marker for CFTR-mediated Cl(-) efflux, we investigate whether prostanoid receptor agonists elicit a functional response from Calu-3 cells. Application of the agonists PGE(2), misoprostol (EP(2), EP(3), and EP(4)) and PGE(1)-OH (EP(3) and EP(4)) stimulate iodide efflux; however, iloprost, butaprost, sulprostone, and fluoprostenol (agonists of the EP(1), EP(2), EP(3), and FP receptors, respectively) have no effect. The iodide efflux seen with 8-iso-PGE(2) is abolished by the EP(4) receptor antagonist AH23848, the CFTR inhibitor 172, and inhibition of PKA and the PI3K pathway. In conclusion, we demonstrate that although Calu-3 cells possess numerous prostanoid receptors, only the EP(4) subtype appears capable of eliciting a functional iodide efflux response, which is mediated via the EP(4) receptor. We propose that 8-iso-PGE(2), acting via EP(4) receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.


Asunto(s)
Dinoprostona/análogos & derivados , Yoduros/metabolismo , Isoprostanos/farmacología , Receptores de Prostaglandina E/fisiología , Tráquea/efectos de los fármacos , Aniones , Western Blotting , Línea Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Dinoprostona/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Transporte Iónico , Subtipo EP4 de Receptores de Prostaglandina E , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tráquea/metabolismo
12.
Oncol Rep ; 19(6): 1511-6, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18497958

RESUMEN

Pharmacological inhibitors of the human ether-a-go-go (hEAG) potassium channel, astemizole and imipramine, have been used to demonstrate that hEAG plays a role in cancer cell proliferation. Astemizole and imipramine are, however, relatively non-specific ion channel blockers, as astemizole can also block the related potassium channel, human ether-a-go-go-related (hERG). Therefore, we aimed to determine the molecular target of astemizole, in the human mammary carcinoma cell line MCF-7. We initially confirmed the expression of KCNH1 and KCNH2 mRNA and hEAG and hERG channel protein in MCF-7 cells. Using a [3H]-thymidine incorporation assay we determined that astemizole inhibited MCF-7 cell proliferation, whereas the hERG-specific channel blocker E-4031 had no effect. We then determined that E-4031 inhibited the regulatory volume decrease (RVD) observed in these cells following exposure to hypotonic solutions, confirming that functional hERG channels are present and may be important for cell volume regulation in MCF-7 cells. Our results suggest, for the first time, that hERG is involved in cell volume regulation. In addition, the function of hEAG and hERG in MCF-7 cell proliferation can be separated pharmacologically by utilizing the channel inhibitors astemizole and E-4031. The hEAG channel function in MCF-7 cells appears to be involved in the regulation of cell proliferation, whereas hERG is involved in cell volume regulation.


Asunto(s)
Neoplasias de la Mama/metabolismo , Canales de Potasio Éter-A-Go-Go/fisiología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antialérgicos/farmacología , Antiarrítmicos/farmacología , Astemizol/farmacología , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Humanos , Imipramina/farmacología , Síndrome de QT Prolongado , Piperidinas/farmacología , Piridinas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas
13.
Pediatr Pulmonol ; 37(2): 149-57, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14730660

RESUMEN

The quantity and composition of airway surface liquid (ASL) are essential to host defense. To date, attempts to harvest ASL and measure its composition have yielded conflicting results. We investigated the physical principles underlying two techniques that were proposed for harvesting ASL: filter paper pledgets and polyethylene catheters. We compared the force and pressure generation and the kinematics of capillarity-induced fluid uptake with both techniques. Both have significant limitations for harvesting ASL, generating physiologically significant pressures (filter paper, 60.4 Pa; polyethylene, 14.3 Pa) that could potentially compromise epithelial integrity. Furthermore, filter paper generates a force 85-fold higher than the polyethylene catheter, which is associated with a very high rate of uptake of liquid and a large total amount of liquid relative to ASL thickness. While the PE catheter harvests liquid more gently, it is only effective when ASL surface tension is below 31 mN/m. These limitations likely account for some of the variability in reported ASL composition, and highlight the need for improved methods for harvesting ASL.


Asunto(s)
Líquidos Corporales , Electroforesis Capilar/instrumentación , Mucosa Respiratoria/metabolismo , Cateterismo , Electroforesis Capilar/métodos , Epitelio/metabolismo , Filtración/instrumentación , Humanos , Polietileno , Presión , Mucosa Respiratoria/citología , Propiedades de Superficie , Tensión Superficial
14.
Respir Physiol Neurobiol ; 183(3): 177-85, 2012 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-22772316

RESUMEN

Psoriasin is a low molecular weight Ca(2+)-binding protein with known antimicrobial activity. Since human airway epithelial cells produce a number of powerful antimicrobial agents as part of their host defence, we investigated whether psoriasin was expressed in human bronchial epithelial cell lines. Expression was investigated in 16HBE14o- cells, derived from a normal individual, and compared to CFBE41o- cells, derived from a cystic fibrosis patient. We also examined psoriasin expression following treatment with factors pertinent to the CF lung-oxidant stress and exposure to pro-inflammatory cytokines. CFBE41o- cells demonstrated much reduced psoriasin levels compared to the 16HBE14o- cells. Increased psoriasin expression was seen following treatment with IL-22 and a cytomix of the pro-inflammatory cytokines IL-1ß, TNF-α and IFN-γ; however, the oxidant stressor tert-butyl hydroperoxide had no apparent effect. Over-expression of human psoriasin into both cell lines resulted in increased internalization of Pseudomonas aeruginosa. In conclusion, expression of psoriasin - which has known anti-microbial activity in other systems - appears to be reduced in CFBE410- compared to 16HBE14o- cells, and its expression modified by exposure to pro-inflammatory cytokines.


Asunto(s)
Fibrosis Quística/metabolismo , Regulación de la Expresión Génica , Mucosa Respiratoria/metabolismo , Proteínas S100/antagonistas & inhibidores , Supervivencia Celular/fisiología , Células Cultivadas , Fibrosis Quística/patología , Citocinas/fisiología , Humanos , Mediadores de Inflamación/fisiología , Estrés Oxidativo/fisiología , Mucosa Respiratoria/patología , Proteína A7 de Unión a Calcio de la Familia S100 , Proteínas S100/biosíntesis
15.
Br J Pharmacol ; 167(5): 1062-75, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22612315

RESUMEN

BACKGROUND AND PURPOSE: There is great interest in the development of potentiator drugs to increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis. We tested the ability of several anions to potentiate CFTR activity by a novel mechanism. EXPERIMENTAL APPROACH: Patch clamp recordings were used to investigate the ability of extracellular pseudohalide anions (Co(CN)(6) (3-) , Co(NO(2) )(6) (3-) , Fe(CN)(6) (3-) , IrCl(6) (3-) , Fe(CN)(6) (4-) ) to increase the macroscopic conductance of mutant CFTR in intact cells via interactions with cytoplasmic blocking anions. Mutagenesis of CFTR was used to identify a possible molecular mechanism of action. Transepithelial short-circuit current recordings from human airway epithelial cells were used to determine effects on net anion secretion. KEY RESULTS: Extracellular pseudohalide anions were able to increase CFTR conductance in intact cells, as well as increase anion secretion in airway epithelial cells. This effect appears to reflect the interaction of these substances with a site on the extracellular face of the CFTR protein. CONCLUSIONS AND IMPLICATIONS: Our results identify pseudohalide anions as increasing CFTR function by a previously undescribed molecular mechanism that involves an interaction with an extracellular site on the CFTR protein. Future drugs could utilize this mechanism to increase CFTR activity in cystic fibrosis, possibly in conjunction with known intracellularly-active potentiators.


Asunto(s)
Cobalto/farmacología , Cianuros/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Iridio/farmacología , Compuestos de Platino/farmacología , Animales , Línea Celular , Cricetinae , Humanos , Mutación
16.
Can J Physiol Pharmacol ; 87(5): 337-46, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19448731

RESUMEN

Cells regulate their volume in response to changes in the osmolarity of both their extracellular and their intracellular environments. We investigated the ability of the human airway epithelial cell line Calu-3 to respond to changes in extracellular osmolarity. Although switching Calu-3 cells from an isosmotic to a hyperosmotic environment resulted in cell shrinkage, there was no compensatory mechanism for the cells to return to their original volume. In contrast, switching to a hyposmotic environment resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). Pharmacologic studies demonstrate that the voltage-activated K+ channels Kv4.1 and (or) Kv4.3 play a crucial role in mediating this RVD response, and we demonstrated expression of these channel types at the mRNA and protein levels. Furthermore, inhibition of the large- and intermediate-conductance Ca2+-activated K+ channels KCa1.1 (maxi-K) and KCa3.1 (hIK) also implicated these channels as playing a role in volume recovery in Calu-3 cells. This report describes the nature of volume regulation in the widely used model cell line Calu-3.


Asunto(s)
Bronquios/citología , Tamaño de la Célula , Células Epiteliales/citología , Tráquea/citología , Bronquios/fisiología , Línea Celular , Canales de Cloruro/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Humanos , Canal de Potasio KCNQ1/fisiología , Presión Osmótica , Canales de Potasio/fisiología , Tráquea/fisiología
17.
J Membr Biol ; 221(3): 153-63, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18264812

RESUMEN

Potassium channels play an important role in providing a driving force for anion secretion from secretory epithelia. To investigate the role of KCNQ K+ channels in mediating rates of basal anion secretion across the human airway submucosal gland serous cell model, the Calu-3 cell, we examined the expression, localization and function of these channels. In addition to our previous knowledge that Calu-3 cells express KCNQ1, using reverse transcriptase polymerase chain reaction we determined expression of KCNQ3, KCNQ4 and KCNQ5 mRNA transcripts. Immunoblotting detected KCNQ1, KCNQ3 and KCNQ5 proteins, while KCNQ4 protein was not found. Immunolocalization using polarized Calu-3 cell monolayers revealed that KCNQ1 and KCNQ3 were located in or toward the apical membrane of the cells, while KCNQ5 was detected in the apical and lateral membranes. Transepithelial transport studies revealed a small chromanol 293B-sensitive current at the apical membrane, likely KCNQ1. Application of XE991, an inhibitor of all members of the KCNQ channel family, inhibited the basal short-circuit current when applied to both sides of the cells to a greater extent than 293B, with the largest inhibition seen upon apical application. This result was confirmed using linopiridine, a less potent analogue of XE991, and suggests that functional KCNQ3 and KCNQ5, in addition to KCNQ1, are present at the apical aspect of these cells. These results demonstrate the role of a number of KCNQ channel members in controlling basal anion secretion across Calu-3 cells, while also demonstrating the importance of apically located K+ channels in mediating anion secretion in the airway epithelium.


Asunto(s)
Membrana Celular/metabolismo , Canales de Potasio KCNQ/metabolismo , Mucosa Respiratoria/metabolismo , Aniones/metabolismo , Línea Celular , Polaridad Celular , Cromanos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Canales de Potasio KCNQ/antagonistas & inhibidores , Modelos Biológicos , Bloqueadores de los Canales de Potasio/farmacología , Mucosa Respiratoria/citología , Membrana Serosa/citología , Membrana Serosa/metabolismo , Sulfonamidas/farmacología
18.
Am J Physiol Cell Physiol ; 293(3): C1010-9, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17596298

RESUMEN

Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K(+) channel inhibitors Ba(2+), quinine, and tetraethylammonium chloride, implicating K(+) channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K(+) channel, and also a dominant-negative NH(2)-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K(+) current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K(+) secretion.


Asunto(s)
Células Epiteliales/fisiología , Canal de Potasio KCNQ1/metabolismo , Glándulas Mamarias Humanas/citología , Equilibrio Hidroelectrolítico/fisiología , Adenocarcinoma , Neoplasias de la Mama , Línea Celular Tumoral , Polaridad Celular/fisiología , Células Epiteliales/citología , Líquido Extracelular/metabolismo , Humanos , Soluciones Hipotónicas/farmacología , Soluciones Isotónicas/farmacología , Canal de Potasio KCNQ1/genética , Potenciales de la Membrana/fisiología , Mutagénesis Sitio-Dirigida , Presión Osmótica , Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Mensajero/metabolismo
19.
Pflugers Arch ; 451(5): 631-41, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16311719

RESUMEN

Potassium channels are required for the absorption and secretion of fluids and electrolytes in epithelia. Calu-3 cells possess a secretory phenotype, and are a model human airway submucosal gland serous cell. Short-circuit current (I(sc)) recordings from Calu-3 cells indicated that basal anion secretion was reduced by apical application of the K+ channel inhibitors bupivicaine, lidocaine, clofilium, and quinidine. Application of riluzole resulted in a large increase in I(sc), inhibited by apical application of either bupivicane or the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel blocker DPC. These results suggested that one or more members of the two-pore-domain K+ (K(2P)) channel family could influence anion secretion. Using RT-PCR, we found that Calu-3 cells express mRNA transcripts for TASK-2 (KCNK5), TWIK-1 (KCNK1), TWIK-2 (KCNK6) and TREK-1 (KCNK2). TASK-2, TWIK-2 and TREK-1 protein were detected by Western blotting, while immunolocalization of polarized cells confirmed protein expression of TREK-1 and TWIK-2 at the plasma cell membrane. TASK-2 protein staining was localized to intracellular vesicles, located beneath the apical membrane. While the pro-secretory role of basolateral K+ channels is well established, we suggest that apically located K2P channels, not previously described in airway epithelial cells, also play an important role in controlling the rate of transepithelial anion secretion.


Asunto(s)
Aniones/metabolismo , Células Epiteliales/metabolismo , Canales de Potasio de Dominio Poro en Tándem/fisiología , Mucosa Respiratoria/metabolismo , Western Blotting , Línea Celular , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
20.
Pflugers Arch ; 453(2): 167-76, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17047984

RESUMEN

Cystic fibrosis (CF) is caused by genetic mutations that lead to dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The most common mutation, DeltaF508, causes inefficient trafficking of mutant CFTR protein from the endoplasmic reticulum to the cell membrane. Therapeutic efforts have been aimed at increasing the level of DeltaF508-CFTR protein in the membrane using agents such as sodium butyrate. In this study, we investigated the effects of culturing a human airway epithelial cell line, Calu-3, in the presence of 5 mM sodium butyrate. Within 24 h, butyrate exposure caused a significant decrease in the basal, as well as Ca(2+)-activated, anion secretion by Calu-3 cell monolayers, determined by the change in transepithelial short-circuit current in response to the Ca(2+)-elevating agent thapsigargin. The secretory response to 1-ethyl-2-benzimidazolinone, an activator of the basolateral Ca(2+)-activated K(+) channel KCNN4, was similarly reduced by butyrate treatment. Quantitative PCR revealed that these functional effects were associated with dramatic decreases in mRNA for both KCNN4 and CFTR. Furthermore, the KCNQ1 K(+) channel was upregulated after butyrate treatment. We suggest that prolonged exposure to sodium butyrate downregulates the expression of both KCNN4 and CFTR, leading to a functional loss of Ca(2+)-activated anion secretion. Thus, butyrate may inhibit, rather than stimulate, the anion secretory capacity of human epithelial cells that express wild-type CFTR, particularly in tissues that normally exhibit robust Ca(2+)-activated secretion.


Asunto(s)
Ácido Butírico/farmacología , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/efectos de los fármacos , Bencimidazoles/farmacología , Calcio/metabolismo , Agonistas de los Canales de Calcio/farmacología , Línea Celular , Fibrosis Quística/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Pulmón/metabolismo , Tapsigargina/farmacología
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