RESUMO
Cystic fibrosis (CF), a lethal hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene coding for an epithelial chloride channel, is characterized by an imbalanced homeostasis of ion and water transports in secretory epithelia. As the disease is single-gene based, transcript therapy using therapeutic mRNA is a promising concept of treatment in order to correct many aspects of the fatal pathology on a cellular level. Hence, we developed chitosan nanocapsules surface-loaded with wtCFTR-mRNA to restore CFTR function. Furthermore, we loaded the nanocapsules with capsaicin, aiming to enhance the overall efficiency of transcript therapy by reducing sodium hyperabsorption by the epithelial sodium channel (ENaC). Dynamic light scattering with non-invasive back scattering (DLS-NIBS) revealed nanocapsules with an average hydrodynamic diameter of ~200 nm and a Zeta potential of ~+60 mV. The results of DLS-NIBS measurements were confirmed by asymmetric flow field-flow fractionation (AF4) with multidetection, while transmission electron microscopy (TEM) images confirmed the spherical morphology and size range. After stability measurements showed that the nanocapsules were highly stable in cell culture transfection medium, and cytotoxicity was ruled out, transfection experiments were performed with the CF cell line CFBE41o-. Finally, transepithelial measurements with a new state-of-the-art Ussing chamber confirmed successfully restored CFTR function in transfected cells. This study demonstrates that CS nanocapsules as a natural and non-toxic delivery system for mRNA to target cells could effectively replace risky vectors for gene delivery. The nanocapsules are not only suitable as a transcript therapy for treatment of CF, but open aspiring possibilities for safe gene delivery in general.
RESUMO
Nanoscale drug delivery systems exhibit a broad range of applications and promising treatment possibilities for various medical conditions. Nanomedicine is of great interest, particularly for rare diseases still lacking a curative treatment such as cystic fibrosis (CF). CF is defined by a lack of Cl- secretion through the cystic fibrosis transmembrane conductance regulator (CFTR) and an increased Na+ absorption mediated by the epithelial sodium channel (ENaC). The imbalanced ion and water transport leads to pathological changes in many organs, particularly in the lung. We developed a non-viral delivery system based on the natural aminopolysaccharide chitosan (CS) for the transport of antisense oligonucleotides (ASO) against ENaC to specifically address Na+ hyperabsorption. CS-ASO electrostatic self-assembled nanocomplexes were formed at varying positive/negative (P/N) charge ratios and characterized for their physicochemical properties. Most promising nanocomplexes (P/N 90) displayed an average size of ~150 nm and a zeta potential of ~+30 mV. Successful uptake of the nanocomplexes by the human airway epithelial cell line NCI-H441 was confirmed by fluorescence microscopy. Functional Ussing chamber measurements of transfected NCI-H441 cells showed significantly decreased Na+ currents, indicating successful downregulation of ENaC. The results obtained confirm the promising characteristics of CS as a non-viral and non-toxic delivery system and demonstrate the encouraging possibility to target ENaC with ASOs to treat abnormal ion transport in CF.
Assuntos
Brônquios/citologia , Quitosana/química , Portadores de Fármacos/química , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/genética , Nanoestruturas/química , Oligonucleotídeos Antissenso/química , Linhagem Celular , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismoRESUMO
In vitro disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these in vitro CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial ΔF508 cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of CFTR exons 11 to 27, into exon 11 and re-activated endogenous CFTR expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous CFTR promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in CFTR exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5' end of exon 11.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/patologia , Éxons/genética , Edição de Genes/métodos , Loci Gênicos/genética , Sequência de Bases , Linhagem Celular , Fibrose Cística/genética , DNA Complementar/genética , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Células Epiteliais/metabolismo , Genótipo , Humanos , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Dedos de ZincoRESUMO
Successful gene therapy requires the development of suitable vehicles for the selective and efficient delivery of genes to specific target cells at the expense of minimal toxicity. In this work, we investigated a non-viral gene delivery system based on chitosan (CS) to specifically address cystic fibrosis (CF). Thus, electrostatic self-assembled CS-pEGFP and CS-pEGFP-siRNA complexes were prepared from high-pure fully characterized CS (Mw â¼ 20 kDa and degree of acetylation â¼ 30%). The average diameter of positively-charged complexes (i.e. ζ â¼+25 mV) was â¼ 200 nm. The complexes were found relatively stable over 14h in Opti-MEM. Cell viability study did not show any significant cytotoxic effect of the CS-based complexes in a human bronchial cystic fibrosis cell line (CFBE41o-). We evaluated the transfection efficiency of this cell line with both CS-pEGFP and co-transfected with CS-pEGFP-siRNA complexes at (N/P) charge ratio of 12. We reported an increase in the fluorescence intensity of CS-pEGFP and a reduction in the cells co-transfected with CS-pEGFP-siRNA. This study shows proof-of-principle that co-transfection with chitosan might be an effective delivery system in a human CF cell line. It also offers a potential alternative to further develop therapeutic strategies for inherited disease treatments, such as CF.
Assuntos
Quitosana/administração & dosagem , Proteínas de Fluorescência Verde/genética , RNA Interferente Pequeno/administração & dosagem , Transfecção/métodos , Linhagem Celular , Quitosana/química , Fibrose Cística , Humanos , Plasmídeos , RNA Interferente Pequeno/químicaRESUMO
Cystic fibrosis (CF) is the most common life shortening autosomal inherited disorder, affecting 1 in 2500 newborns in the Caucasian population. In CF the lung pathology is associated with dehydration of the airways epithelial surface which in part results from Na(+) hyperabsorption via the epithelial sodium channel (ENaC). The molecular mechanisms of this Na(+) hyperabsorption and its correlation with the underlying genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) are not fully understood. However, it is obvious that a reduced Cl(-) secretion by CFTR and an enhanced Na+ absorption through ENaC lead to the so far incurable disease. Therefore, it could be indicated to pursue a double-tracked strategy in that way enabling Cl(-) secretion by a reconstitution of the defect CFTR as well as blocking ENaC to prevent Na(+) hyperabsorption. Since the cloning of CFTR great efforts have been done in delivery of CFTR for the correction of the reduced Cl(-) secretion. Positive benefits for the inhibition of the CF related Na(+) hyperabsorption offer technologies using small molecule inhibitors like ASOs or siRNA, which target translation and knockdown of ENaC, respectively. In this review we discuss possible CFTR/ENaC interactions in the context of CF, describe ENaC structure as well as some of the numerous attempts that were performed to prevent the Na(+) hyperabsorption in CF related lung disease. Thus, we give a short summary of e.g. amiloride therapy approaches and focus on inventive blocking efforts using ASOs and siRNA.
Assuntos
Fibrose Cística/tratamento farmacológico , Bloqueadores do Canal de Sódio Epitelial/uso terapêutico , Canais Epiteliais de Sódio/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Amilorida/farmacologia , Amilorida/uso terapêutico , Ensaios Clínicos como Assunto , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Humanos , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Bibliotecas de Moléculas Pequenas/farmacologia , Sódio/metabolismoRESUMO
BACKGROUND: Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP- and ATP-dependent Cl(-) channel and central regulatory protein in epithelia. CFTR influences the fluid composition of the mucus in the respiratory tract. The most common mutation inducing CF, ΔF508, impairs CFTR processing within the cell and thus prevents functional CFTR expression in the apical membrane. The present study aimed to investigate the functional restoration of CFTR in human CF airway epithelia after transfection with optimized wild-type (wt)CFTR-mRNA. METHODS: We used primary cultured human nasal epithelial (HNE) cells and the human bronchial epithelial cell line CFBE41o(-) that stably expresses ΔF508-CFTR and carried out transepithelial Ussing chamber measurements after transfection with optimized wtCFTR-mRNA. We confirmed the data obtained using immunofluorescence and protein biochemical approaches. RESULTS: Transfection of the CFBE41o(-) cells with wtCFTR-mRNA restored cAMP-induced CFTR currents similar to the values seen in control cells (16HBE14o(-)). Using immunofluorescence approaches, we demonstrated that a considerable amount of CFTR is located at the apical surface in the CF cells after transfection. Western blot analyses of wtCFTR-mRNA transfected CFBE41o(-) cells confirmed these findings. Furthermore, we demonstrated physiological relevance by using primary cultured HNE cells and showed an almost two-fold increase in the cAMP-stimulated CFTR current after transfection. CONCLUSIONS: From these data, we conclude that CFTR-mRNA transfection could comprise a novel alternative for gene therapy to restore impaired CFTR function.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Técnicas de Transferência de Genes , RNA Mensageiro , Animais , Linhagem Celular , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Terapia Genética , Humanos , Oócitos/metabolismo , Cultura Primária de Células , Transfecção/métodos , Xenopus laevisRESUMO
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 (EC(50) (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 (EC(50) = 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.Molecular Therapy - Nucleic Acids (2013) 2, e65; doi:10.1038/mtna.2012.57; published online 15 January 2013.
RESUMO
The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na(+) channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na(+) transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na(+) homeostasis. To explore this process, we examined the coordinated effect of this protease on Na(+) and Cl(-) transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current (I(sc)) was used to measure transport of these ions. The I(sc) exhibited an ENaC-dependent Na(+) component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl(-) component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated I(ENaC) but was without effects on I(CFTR). Subtilisin an exogenous S8 protease that mimics endogenous furin-type proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na(+) and Cl(-) transport pathways in renal epithelia by extracellular luminal proteases. They also indicate that baseline urinary proteolytic activity is sufficient to modify Na(+) and Cl(-) transport in these epithelia.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Canais Epiteliais de Sódio/fisiologia , Serina Proteases/metabolismo , Calicreínas Teciduais/metabolismo , Amilorida/farmacologia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Cães , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Células Madin Darby de Rim Canino , Ratos , Serina Proteases/farmacologia , Serina Proteases/urina , Subtilisina/farmacologia , Tripsina/farmacologiaRESUMO
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 (I(m)), conductance (G(m)), and capacitance (C(m)) were determined. Sildenafil increased I(m), G(m), and C(m) of wt-CFTR and functionally restored ΔF508-CFTR in oocytes. These effects were also seen in CFBE41o(-) and 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.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Sulfonas/farmacologia , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Linhagem Celular , Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Imunofluorescência , Humanos , Inibidores de Fosfodiesterase/uso terapêutico , Piperazinas/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Citrato de Sildenafila , Sulfonas/uso terapêutico , Xenopus laevisRESUMO
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.
Assuntos
Canais Epiteliais de Sódio/biossíntese , Regulação da Expressão Gênica/fisiologia , Mucosa Nasal/metabolismo , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Canais Epiteliais de Sódio/genética , HumanosRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Oócitos/metabolismo , Xenopus laevis/metabolismo , Animais , Canais de Cálcio/química , DNA Complementar/metabolismo , Difusão , Eletrofisiologia/métodos , Canais Iônicos/metabolismo , Íons , Canais de Potássio/química , RNA Mensageiro/metabolismo , Canais de Sódio/química , Água/químicaRESUMO
BACKGROUND: 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 alpha-ENaC subunit. METHODS: We heterologously expressed ENaC in oocytes of Xenopus laevis for mass screening of AON. Additionally, primary cultures of human nasal epithelia were transfected with AON and were used for Ussing chamber experiments, as well as biochemical and fluorescence optical analyses. RESULTS: Screening of several AON by co-injection or sequential microinjection of AON and ENaC mRNA in X. laevis oocytes led to a sustained decrease in amiloride-sensitive current and conductance. Using primary cultures of human nasal epithelia, we show that AON effectively suppress amiloride-sensitive Na(+) absorption mediated by ENaC in CF and non-CF tissues. In western blot experiments, it could be shown that the amount of ENaC protein is effectively reduced after AON transfection. CONCLUSIONS: Our data comprise an initial step towards a preclinical test with AON to reduce Na(+) hyperabsorption in CF epithelia.
Assuntos
Fibrose Cística/metabolismo , Bloqueadores do Canal de Sódio Epitelial , Oligonucleotídeos Antissenso/farmacologia , Sódio/metabolismo , Animais , Sequência de Bases , Primers do DNA , Canais Epiteliais de Sódio/genética , Humanos , Microeletrodos , Microscopia de Fluorescência , Mucosa Nasal/metabolismo , RNA Mensageiro/genética , Xenopus laevisRESUMO
The expression of the epithelial Na+ channel (ENaC) is tissue-specific and dependent on a variety of mediators and interacting proteins. Here we examined the role of intracellular Na+ ([Na+](i)) as a modulator of the expression of rat ENaC in Xenopus laevis oocytes. We manipulated [Na+](i) of ENaC-expressing oocytes in the range of 0-20 mM by incubating in extracellular solutions of different [Na+](o). Electrophysiological, protein biochemical and fluorescence optical methods were used to determine the effects of different [Na+]i on ENaC expression and membrane abundance. In voltage-clamp experiments we found that amiloride-sensitive ENaC current (Iami) and conductance (Gami) peak at a [Na+](i) of approximately 10 mM Na+, but were significantly reduced in 5 mM and 20 mM [Na+](i). Fluorescence intensity of EGFP-ENaC-expressing oocytes also followed a bell-shaped curve with a maximum at approximately 10 mM [Na+](i). In Western blot experiments with specific anti-ENaC antibodies the highest protein expression was found in ENaC-expressing oocytes with [Na+](i) of 10-15 mM. Since ENaC is also highly permeable for Li+, we incubated ENaC-expressing oocytes in different Li+ concentrations and found a peak of Iami and Gami with 5 mM Li+. The influence of [Na+](i) on the expression is not ENaC-specific, since expression of a Cl(-) channel (CFTR) and a Na+/glucose cotransporter (SGLT1) showed the same dependence on [Na+](i). We conclude that specific concentrations of Na+ and Li+ influence the expression and abundance of ENaC and other transport proteins in the plasma membrane in Xenopus laevis oocytes. Furthermore, we suggest the existence of a general mechanism dependent on monovalent cations that optimizes the expression of membrane proteins.
Assuntos
Proteínas de Transporte/metabolismo , Canais Epiteliais de Sódio/metabolismo , Oócitos/metabolismo , Sódio/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Proteínas de Transporte/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Eletrofisiologia , Canais Epiteliais de Sódio/genética , Expressão Gênica/genética , Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Ratos , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Cystic fibrosis (CF) is characterised by the absence of CFTR function resulting in a reduced Cl(-) secretion and an increase in Na+ absorption. This Na+ hyperabsorption is mediated by the human amiloride-sensitive epithelial sodium channel (ENaC), but the underlying mechanisms are still unknown. After demonstrating functional differences of the Na+ absorption in CF and non-CF epithelia in Ussing chamber experiments with human primary cultures, we compared ENaC sequences from CF and non-CF human nasal tissue (hnENaC), investigated the mRNA transcription levels via real-time PCR and studied the protein expression in Western blot analyses. We found no differences in the sequences of CF and non-CF hnENaC, but identified some polymorphisms. The real-time experiments revealed an enhanced mRNA amount of all three hnENaC subunits in CF tissue. By comparing the two groups on the protein level, we observed differences in the abundance of the Na+ channel. While the alpha- and beta-hnENaC protein amount was increased in CF tissue the gamma-hnENaC was decreased. We conclude that the Na+ hyperabsorption in CF is not caused by mutations in hnENaC, but by an increase in the transcription of the hnENaC subunits. This could be induced by a disturbed regulation of the channel in CF.
Assuntos
Fibrose Cística/metabolismo , Canais Epiteliais de Sódio/metabolismo , Mucosa Olfatória/metabolismo , Sequência de Bases , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Humanos , Transcrição Gênica/fisiologia , Regulação para Cima/fisiologiaRESUMO
Amiloride-sensitive Na+ absorption is a well-described feature of numerous transporting epithelia in vertebrates. Yet, very little is known about this important physiological process regarding invertebrates. In the present paper, we compare vertebrate Na+ absorption mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) and its invertebrate counterpart. We used the dorsal skin of the annelid Hirudo medicinalis as a model for the Na+ absorption of invertebrate epithelia. In applying electrophysiological, molecular, and biochemical techniques we found striking functional and structural differences between vertebrate and invertebrate amiloride-sensitive Na+ absorption. Using modified Ussing chambers, we analyzed the influence of different known blockers and effectors of vertebrate ENaC on leech epithelial Na+ absorption. We demonstrate that the serine protease trypsin had no effect on the Na+ transport across leech integument, while it strongly activates vertebrate ENaC. While protons, and the divalent cations Ni2+ and Zn2+ stimulate vertebrate ENaC, amiloride-sensitive Na+ currents in leech integument were substantially reduced. For molecular studies, we constructed a cDNA library of Hirudo medicinalis and screened it with specific ENaC antibodies. We performed numerous PCR approaches using a vast number of different degenerated and specific ENaC primers to identify ENaC-like structures. Yet, both strategies did not reveal any ENaC-like sequence in leech integument. From these data we conclude that amiloride-sensitive Na+ absorption in leech skin is not mediated by an ENaC-like Na+ channel but by a still unknown invertebrate member of the ENaC/DEG family that we termed lENaTP (leech epithelial Na+ transporting protein).
Assuntos
Amilorida/administração & dosagem , Canais Epiteliais de Sódio/fisiologia , Hirudo medicinalis/fisiologia , Ativação do Canal Iônico/fisiologia , Absorção Cutânea/fisiologia , Sódio/farmacocinética , Vertebrados/fisiologia , Animais , Canais Epiteliais de Sódio/efeitos dos fármacos , Hirudo medicinalis/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Absorção Cutânea/efeitos dos fármacos , Especificidade da EspécieRESUMO
We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na(+) channel from rat distal colon (rENaC). We combined conventional dual-microelectrode voltage-clamp with continuous capacitance (C(m)) measurements and noise analysis to evaluate the effects of cAMP and Ni(2+) on rENaC. Control oocytes or rENaC-expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC-expressing oocytes amiloride induced a marked plateau-shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker-channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio-cAMP and IBMX (cAMP cocktail) increased amiloride-sensitive current (I(ami)) and conductance (G(ami)). Furthermore, C(m) was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single-channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni(2+) (2.5 mM) inhibited about 60% of the rENaC current and conductance while C(m) remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single-channel current did not change significantly. These observations argue for direct effects of Ni(2+) on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.
Assuntos
AMP Cíclico/farmacologia , Níquel/farmacologia , Oócitos/metabolismo , Canais de Sódio/biossíntese , Canais de Sódio/efeitos dos fármacos , Amilorida/farmacologia , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Capacitância Elétrica , Inibidores Enzimáticos/farmacologia , Canais Epiteliais de Sódio , Exocitose/efeitos dos fármacos , Líquido Intracelular/metabolismo , Microinjeções , Oócitos/citologia , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Processamento de Sinais Assistido por Computador , Sódio/metabolismo , Canais de Sódio/genética , Xenopus laevisRESUMO
We expressed the three subunits of the epithelial amiloride-sensitive Na(+) channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na(+) accumulation we performed all experiments in low-Na(+) solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f(c) (2 pi f(c) plot) we determined the rate constants k(on) and k(off) for the amiloride-ENaC interaction. At a clamp potential (V(m)) of -60 mV k(on) was 80.8 +/- 5.1 microM(-1) s(-1) and k(off) 15.4 +/- 4.2 s(-1). The half-maximal blocker concentration (K(mic,ami)) was 0.19 microM (V(m)=-60 mV). While k(on) was voltage-independent in the range -50 to -100 mV, k(off) and K(mic,ami) decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na(+)] ([Na(+)](o)) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na(+)](o) and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na(+) for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k(on) and k(off) for amiloride decreased with increasing [Na(+)](o), while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na(+)](o), indicating that Na(+) and amiloride do not compete for the same binding site at the channel.