Epithelial sodium channel silencing as a strategy to correct the airway surface fluid deficit in cystic fibrosis.

In the respiratory system, Na(+) absorption and Cl(-) secretion are balanced to maintain an appropriate airway surface fluid (ASF) volume and ensure efficient mucociliary clearance. In cystic fibrosis (CF), this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the absence of functional CFTR-dependent Cl(-) secretion. The consequences of defective Cl(-) transport are worsened by the persistence of Na(+) absorption, which contributes to airway surface dehydration. We asked whether normal ASF can be restored to an equal extent by recovering Cl(-) secretion from mutated CFTR or by reducing Na(+) absorption. This is highly relevant in the selection of the best strategy for the treatment of patients with CF. We analyzed the ASF thickness of primary cultured bronchial CF and non-CF epithelia after silencing the epithelial Na(+) channel (ENaC) with specific short, interfering RNAs (siRNAs) and after the pharmacological stimulation of CFTR. Our results indicate that (1) single siRNAs complementary to ENaC subunits are sufficient to reduce ENaC transcripts, Na(+) channel activity, and fluid transport, but only silencing both the α and ß ENaC subunits at the same time leads to an increase of ASF (from nearly 7 µm to more than 9 µm); (2) the ASF thickness obtained in this way is about half that measured after maximal CFTR stimulation in non-CF epithelia (10-14 µm); and (3) the pharmacological rescue of mutant CFTR increases the ASF to the same extent as ENaC silencing. Our results indicate that CFTR rescue and ENaC silencing both produce a significant and long-lasting increase of airway hydration in vitro.

Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH.

Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pH(i)) would alter the binding affinity. Here, we analyzed the effect of pH(i) on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH(i) does modify CFTR maximum current (I(m)) and half-activation concentration (K(d)): I(m) = 127.7, 185.5, and 231.8 µA/cm(2) and K(d) = 32.7, 56.6 and 71.9 µm at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (K(a)) increased at alkaline pH(i), near cysteine pK (K(a) = 1.83, 1.81 and 4.99 µm at pH(i) 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pH(i). Effects of pH(i) on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH(i) on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.

Analysis of ion transport in the airway epithelium using RNA interference.

Knowledge of the physiology of airway epithelium had been limited by the lack of potent and selective inhibitors of the ion channels, transporters and regulators involved in transepithelial electrolyte and fluid transport. The use of siRNA technology to downregulate the expression of a given gene represents a useful method for studying the function of proteins in both native cells and tissues, enabling a more precise assessment of the contribution of single proteins to the general transport process in the airway epithelium and an evaluation of the contribution of these proteins in various respiratory conditions. This review focuses on recent research involving siRNA-based silencing of proteins implicated in ion transport through the airway epithelium, illustrating how this technology has increased our understanding of the function and regulation of the transport pathway, and has helped to identify new targets for drugs and to uncover the function of newly identified proteins.

Epithelial sodium channel inhibition in primary human bronchial epithelia by transfected siRNA.

Na(+) absorption and Cl(-) secretion are in equilibrium to maintain an appropriate airway surface fluid volume and ensure appropriate mucociliary clearance. In cystic fibrosis, this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in the absence of functional CFTR protein, which in turn results in deficient cAMP-dependent Cl(-) secretion and predominant Na(+) absorption. It has been suggested that down-regulation of the epithelial sodium channel, ENaC, might help to restore airway hydration and reverse the airway phenotype in patients with cystic fibrosis. We used an siRNA approach to analyze the possibility of down-regulating ENaC function in bronchial epithelia and examine the resulting effects on fluid transport. siRNA sequences complementary to each of the three ENaC subunits have been used to establish whether single subunit down-regulation is enough to reduce Na(+) absorption. Transfection was performed by exposure to siRNA for 24 hours at the time of cell seeding on permeable support. By using primary human bronchial epithelial cells we demonstrate that (1) siRNA sequences complementary to ENaC subunits are able to reduce ENaC transcripts and Na(+) channel activity by 50 to 70%, (2) transepithelial fluid absorption decreases, and (3) these functional effects last at least 8 days. A decrease in ENaC mRNA results in a significant reduction of ENaC protein function and fluid absorption through the bronchial epithelium, indicating that an RNA interference approach may improve the airway hydration status in patients with cystic fibrosis.

Block of CFTR-dependent chloride currents by inhibitors of multidrug resistance-associated proteins.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that belongs to the same family as multidrug resistance-associated proteins whose main function is to expel xenobiotics and physiological organic anions from the cell interior. Despite the overall structural similarity with these membrane proteins, CFTR is not an active transporter but is instead a Cl- channel. We have tested the ability of known inhibitors of multidrug resistance-associated proteins to affect CFTR Cl- currents. We have found that sulfinpyrazone, probenecid, and benzbromarone are also inhibitors of CFTR activity, with a mechanism involving blockage of the channel pore.

Growth arrest-inducing genes are activated in Dbl-transformed mouse fibroblasts.

The Dbl oncogene is a guanine nucleotide exchange factor for Rho GTPases and its activity has been linked to the regulation of gene transcription. Dbl oncogene expression in NIH3T3 cells leads to changes in morphological and proliferative properties of these cells, inducing a highly transformed phenotype. To gain insights into Dbl oncogene-induced transformation we compared gene expression profiles between Dbl oncogene-transformed and parental NIH3T3 cells by cDNA microarray. We found that Dbl oncogene expression is associated with gene expression modulation involving upregulation of 51 genes and downregulation of 49 genes. Five of the overexpressed genes identified are known to exert antiproliferative functions. Our observations suggest that the expression of Dbl oncogene in NIH3T3 may lead to the induction of genes associated with cell cycle arrest, possibly through the activation of stress-induced kinases.

Detection of electrophysiological indicators of neurotoxicity in human and rat brain slices by a three-dimensional microelectrode array.

Electrophysiological techniques for the assessment of in vitro neurotoxicology have several advantages over other currently-used methods (for example, morphological techniques), including the ability to detect damage at a very early stage. Novel recording techniques based on microelectrode arrays are available, and could improve recording power. In this study, we investigated how a three-dimensional microelectrode array detects the electrophysiological endpoints of neurotoxicity. We conclude that electrophysiology sensitively reveals neurotoxic actions, and that three-dimensional microelectrode arrays could be proposed for use in neurotoxicology as recording tools that allow easy and sensitive multisite recording, from both rodent and human brain tissue.

Changes in extracellular action potential detect kainic acid and trimethyltin toxicity in hippocampal slice preparations earlier than do MAP2 density measurements.

In vitro electrophysiological techniques for the assessment of neurotoxicity could have several advantages over other methods in current use, including the ability to detect damage at a very early stage, and could further assist in replacing animal experimentation in vivo. We investigated how an electrophysiological parameter, the extracellularly-recorded compound action potential ("population spike", PS) could be used as a marker of in vitro neurotoxicity in the case of two well-known toxic compounds, kainic acid (KA) and trimethyltin (TMT). We compared the use of this electrophysiological endpoint with changes in immunoreactivity for microtubule-associated protein 2 (MAP2), a standard histological test for neurotoxicity. We found that both toxic compounds reliably caused disappearance of the PS, and that such disappearance occurred after only 1 hour of exposure to the drug. By contrast, densitometric measurements of MAP2 immunoreactivity were unaffected by both KA and TMT after such a short exposure time. We conclude that, in the case of KA and TMT, the extracellular PS was abolished at a very early time-point, when MAP2 immunoreactivity levels were still comparable to those of the untreated controls. Electrophysiology could be a reliable and early indicator of neurotoxicity, which could improve our ability to test for neurotoxicity in vitro, thus further replacing the need for in vivo experimentation.