Katja Sobczak

Aldosterone and amiloride alter ENaC abundance in vascular endothelium

The amiloride-sensitive epithelial sodium channel (ENaC) is usually found in the apical membrane of epithelial cells but has also recently been described in vascular endothelium. Because little is known about the regulation and cell surface density of ENaC, we studied the influence of aldosterone, spironolactone, and amiloride on its abundance in the plasma membrane of human endothelial cells. Three different methods were applied, single ENaC molecule detection in the plasma membrane, quantification by Western blotting, and cell surface imaging using atomic force microscopy. We found that aldosterone increases the surface expression of ENaC molecules by 36% and the total cellular amount by 91%. The aldosterone receptor antagonist spironolactone prevents these effects completely. Acute application of amiloride to aldosterone-pretreated cells led to a decline of intracellular ENaC by 84%. We conclude that, in vascular endothelium, aldosterone induces ENaC expression and insertion into the plasma membrane. Upon functional blocking with amiloride, the channel disappears from the cell surface and from intracellular pools, indicating either rapid degradation and/or membrane pinch-off. This opens new perspectives in the regulation of ENaC expressed in the vascular endothelium.

Endogenous transport systems in the Xenopus laevis oocyte plasma membrane

Oocytes of the South African clawed frog Xenopus laevis are widely used as a heterologous expression system for the characterization of transport systems such as passive and active membrane transporters, receptors and a whole plethora of other membrane proteins originally derived from animal or plant tissues. The large size of the oocytes and the high degree of expression of exogenous mRNA or cDNA makes them an optimal tool, when compared with other expression systems such as yeast, Escherichia coli or eukaryotic cell lines, for the expression and functional characterization of membrane proteins. This easy to handle expression system is becoming increasingly attractive for pharmacological research. Commercially available automated systems that microinject mRNA into the oocytes and perform electrophysiological measurements fully automatically allow for a mass screening of new computer designed drugs to target membrane transport proteins. Yet, the oocytes possess a large variety of endogenous membrane transporters and it is absolutely mandatory to distinguish the endogenous transporters from the heterologous, expressed transport systems. Here, we review briefly the endogenous membrane transport systems of the oocytes.

Characterization of the Epithelial Sodium Channel δ-Subunit in Human Nasal Epithelium

The epithelial sodium channel (ENaC) mediates the first step in Na+ reabsorption in epithelial cells such as kidney, colon, and airways and may consist of four homologous subunits (alpha, beta, gamma, delta). Predominantly, the alpha-subunit is expressed in these epithelia, and it usually forms functional channels with the beta- and gamma-subunits. The delta-subunit was first found in human brain and kidney, but the expression was also detected in human cell lines of lung, pancreatic, and colonic origin. When co-expressed with beta and gamma accessory subunits in heterologous systems, the two known isoforms of the delta-ENaC subunit (delta1 and delta2) can build amiloride-sensitive Na+ channels. In the present study we demonstrate the expression and function of the delta-subunit in human nasal epithelium (HNE). We cloned and sequenced the full-length cDNA of the delta-ENaC subunit and were able to show that in nasal tissue at least isoform 1 is expressed. Furthermore, we performed Western blot analyses and compared the cell surface expression of the delta-subunit with the classically expressed alpha-subunit by using immunofluorescence experiments. Thereby, we could show that the quantity of both subunits is almost similar. In addition, we show the functional expression of the delta-ENaC subunit with measurements in modified Ussing chambers, and demonstrate that in HNE a large portion of the Na+ transport is mediated by the delta-ENaC subunit. Therefore, we suppose that the delta-subunit may possess an important regulatory function and might interact with other ENaC subunits or members of the DEG/ENaC family in the human respiratory epithelium.

Pharmacological Characterization of a Novel ENaCα siRNA (GSK2225745) With Potential for the Treatment of Cystic Fibrosis

Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC50 (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5′ RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC50 = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.

Sildenafil Acts as Potentiator and Corrector of CFTR but Might be not Suitable for the Treatment of CF Lung Disease

The phosphodiesterase-5 inhibitor sildenafil is an established and approved drug to treat symptoms of a variety of human diseases. In the context of cystic fibrosis (CF), a genetic disease caused by a defective CFTR gene (e.g. ΔF508-CFTR), it was assumed that sildenafil could be a promising substance to correct impaired protein expression. This study focuses on the molecular mechanisms of sildenafil on CFTR recovery. We used ΔF508-CFTR/wt-CFTR expressing Xenopus laevis oocytes and human bronchial epithelial cell lines (CFBE41o-/16HBE14o-) to investigate the pathways of sildenafil action. Cells were treated with sildenafil and cAMP-mediated current (Im), conductance (Gm), and capacitance (Cm) were determined. Sildenafil increased Im, Gm, and Cm of wt-CFTR and functionally restored ΔF508-CFTR in oocytes. These effects were also seen in CFBE41oand 16HBE14o-cells. Transepithelial measurements revealed that sildenafil mediated increase (wt-CFTR) and restoration (ΔF508-CFTR) of channel activity. cGMP pathway blocker inhibited the activity increase but not CFTR/ΔF508-CFTR exocytosis. From these data we conclude that sildenafil mediates potentiation of CFTR activity by a cGMP-dependent and initiates cGMP-independent functional insertion of CFTR/ ΔF508-CFTR molecules into the apical membranes. Thus, sildenafil is a corrector and potentiator of CFTR/ΔF508-CFTR. Yet, the necessary high doses of the drug for CFTR recovery demonstrate that sildenafil might not be suited as a therapeutic drug for CF lung disease.

Specific inhibition of epithelial Na + channels by antisense oligonucleotides for the treatment of Na + hyperabsorption in cystic fibrosis

Cystic fibrosis (CF) respiratory epithelia are characterized by a defect Cl− secretion and an increased Na+ absorption through epithelial Na+ channels (ENaC). The present study aimed to find an effective inhibitor of human ENaC with respect to replacing amiloride therapy for CF patients. Therefore, we developed specific antisense oligonucleotides (AON) that efficiently suppress Na+ hyperabsorption by inhibiting the expression of the α‐ENaC subunit.

Antisense oligonucleotides for therapy of cystic fibrosis. Inhibition of sodium absorption mediated by ENaC in nasal epithelial cells

BACKGROUND The genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract. MATERIAL AND METHODS In this project it was investigated whether Na(+) hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out. RESULTS AON transfection significantly inhibits Na(+) absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells. CONCLUSION The inhibition of ENaC associated Na(+) absorption by specific AON could offer a new perspective for the regulation of the Na(+) hyperabsorption in CF patients.ZusammenfassungHintergrundDer Erbkrankheit Mukoviszidose („cystic fibrosis“, CF) liegt neben einer reduzierten Chloridsekretion über den „cystic fibrosis transmembrane conductance regulator“ (CFTR) auch eine Na+-Hyperabsorption über den amiloridsensitiven epithelialen Na+-Kanal (ENaC) zugrunde. Mutationen des CFTR sind ursächlich für die Bildung eines zähflüssigen Mukus und eine gestörte mukoziliäre Reinigung im respiratorischen Trakt.Material und MethodenInnerhalb dieses Projekts sollte die Inhibierung der Na+-Hyperabsorption durch Antisense-Oligonukleotide (AON) erreicht werden. Für funktionale Untersuchungen wurden Monolayer humaner Nicht-CF- und CF-Nasenepithelzellen mittels Ussing-Kammer-Messungen analysiert. Zur Charakterisierung der AON-Wirkung auf Proteinebene wurden Western-Blot-Analysen durchgeführt.ErgebnisseDie AON-Transfektion verringerte die Na+-Absorption über den ENaC in Nicht-CF- und in CF-Zellen deutlich. Außerdem zeigten Western-Blot-Analysen eine Reduzierung des ENaC-Proteins in den mit AON-transfizierten Nicht-CF-Zellen.SchlussfolgerungDie Reduktion der ENaC-vermittelten Na+-Absorption mit spezifischen AON eröffnet eine neue Perspektive, die krankhafte Na+-Hyperabsorption bei CF-Patienten gezielt zu regulieren.AbstractBackgroundThe genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+ hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract.Material and MethodsIn this project it was investigated whether Na+ hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out.ResultsAON transfection significantly inhibits Na+ absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells.ConclusionThe inhibition of ENaC associated Na+ absorption by specific AON could offer a new perspective for the regulation of the Na+ hyperabsorption in CF patients.

Antisense-Oligonukleotide zur Therapie der Mukoviszidose

BACKGROUND The genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract. MATERIAL AND METHODS In this project it was investigated whether Na(+) hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out. RESULTS AON transfection significantly inhibits Na(+) absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells. CONCLUSION The inhibition of ENaC associated Na(+) absorption by specific AON could offer a new perspective for the regulation of the Na(+) hyperabsorption in CF patients.ZusammenfassungHintergrundDer Erbkrankheit Mukoviszidose („cystic fibrosis“, CF) liegt neben einer reduzierten Chloridsekretion über den „cystic fibrosis transmembrane conductance regulator“ (CFTR) auch eine Na+-Hyperabsorption über den amiloridsensitiven epithelialen Na+-Kanal (ENaC) zugrunde. Mutationen des CFTR sind ursächlich für die Bildung eines zähflüssigen Mukus und eine gestörte mukoziliäre Reinigung im respiratorischen Trakt.Material und MethodenInnerhalb dieses Projekts sollte die Inhibierung der Na+-Hyperabsorption durch Antisense-Oligonukleotide (AON) erreicht werden. Für funktionale Untersuchungen wurden Monolayer humaner Nicht-CF- und CF-Nasenepithelzellen mittels Ussing-Kammer-Messungen analysiert. Zur Charakterisierung der AON-Wirkung auf Proteinebene wurden Western-Blot-Analysen durchgeführt.ErgebnisseDie AON-Transfektion verringerte die Na+-Absorption über den ENaC in Nicht-CF- und in CF-Zellen deutlich. Außerdem zeigten Western-Blot-Analysen eine Reduzierung des ENaC-Proteins in den mit AON-transfizierten Nicht-CF-Zellen.SchlussfolgerungDie Reduktion der ENaC-vermittelten Na+-Absorption mit spezifischen AON eröffnet eine neue Perspektive, die krankhafte Na+-Hyperabsorption bei CF-Patienten gezielt zu regulieren.AbstractBackgroundThe genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+ hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract.Material and MethodsIn this project it was investigated whether Na+ hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out.ResultsAON transfection significantly inhibits Na+ absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells.ConclusionThe inhibition of ENaC associated Na+ absorption by specific AON could offer a new perspective for the regulation of the Na+ hyperabsorption in CF patients.

Antisense-Oligonukleotide zur Therapie der Mukoviszidose@@@Antisense oligonucleotides for therapy of cystic fibrosis: Inhibition der ENaC-vermittelten Natriumabsorption in humanen Nasenepithelzellen@@@Inhibition of sodium absorption mediated by ENaC in nasal epithelial cells

BACKGROUND The genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract. MATERIAL AND METHODS In this project it was investigated whether Na(+) hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out. RESULTS AON transfection significantly inhibits Na(+) absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells. CONCLUSION The inhibition of ENaC associated Na(+) absorption by specific AON could offer a new perspective for the regulation of the Na(+) hyperabsorption in CF patients.ZusammenfassungHintergrundDer Erbkrankheit Mukoviszidose („cystic fibrosis“, CF) liegt neben einer reduzierten Chloridsekretion über den „cystic fibrosis transmembrane conductance regulator“ (CFTR) auch eine Na+-Hyperabsorption über den amiloridsensitiven epithelialen Na+-Kanal (ENaC) zugrunde. Mutationen des CFTR sind ursächlich für die Bildung eines zähflüssigen Mukus und eine gestörte mukoziliäre Reinigung im respiratorischen Trakt.Material und MethodenInnerhalb dieses Projekts sollte die Inhibierung der Na+-Hyperabsorption durch Antisense-Oligonukleotide (AON) erreicht werden. Für funktionale Untersuchungen wurden Monolayer humaner Nicht-CF- und CF-Nasenepithelzellen mittels Ussing-Kammer-Messungen analysiert. Zur Charakterisierung der AON-Wirkung auf Proteinebene wurden Western-Blot-Analysen durchgeführt.ErgebnisseDie AON-Transfektion verringerte die Na+-Absorption über den ENaC in Nicht-CF- und in CF-Zellen deutlich. Außerdem zeigten Western-Blot-Analysen eine Reduzierung des ENaC-Proteins in den mit AON-transfizierten Nicht-CF-Zellen.SchlussfolgerungDie Reduktion der ENaC-vermittelten Na+-Absorption mit spezifischen AON eröffnet eine neue Perspektive, die krankhafte Na+-Hyperabsorption bei CF-Patienten gezielt zu regulieren.AbstractBackgroundThe genetic disease cystic fibrosis (CF) is characterised by reduced chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+ hyperabsorption through amiloride-sensitive epithelial sodium channels (ENaC). Mutations in CFTR cause the accumulation of thick mucus and dysfunction of mucociliary clearance in the respiratory tract.Material and MethodsIn this project it was investigated whether Na+ hyperabsorption is inhibited by the use of antisense oligonucleotides (AON). For functional analyses monolayers of human non-CF and CF nasal epithelial cells were measured in modified Ussing chambers. To analyse the AON effects on the protein level Western blotting analyses were carried out.ResultsAON transfection significantly inhibits Na+ absorption via ENaC in non-CF and CF cells. Furthermore, Western blot analyses demonstrate a suppression of the ENaC protein in AON transfected human non-CF cells.ConclusionThe inhibition of ENaC associated Na+ absorption by specific AON could offer a new perspective for the regulation of the Na+ hyperabsorption in CF patients.