RESUMO
Syntaxins are SNARE proteins and may play a role in epithelial sodium channel (ENaC) trafficking. The aim of the present study was to investigate the effects of syntaxin 2 (STX2), syntaxin 3 (STX3), and syntaxin 4 (STX4) on rat (rENaC) and human ENaC (hENaC). Co-expression of rENaC and STX3 or STX4 in Xenopus laevis oocytes increased amiloride-sensitive whole-cell currents (ΔIami) on average by 50% and 135%, respectively, compared to oocytes expressing rENaC alone. In contrast, STX2 had no significant effect on rENaC. Similar to its effect on rENaC, STX3 stimulated hENaC by 48%. In contrast, STX2 and STX4 inhibited hENaC by 51% and 44%, respectively. Using rENaC carrying a FLAG tag in the extracellular loop of the ß-subunit, we demonstrated that the stimulatory effects of STX3 and STX4 on ΔIami were associated with an increased expression of the channel at the cell surface. Co-expression of STX3 or STX4 did not significantly alter the degree of proteolytic channel activation by chymotrypsin. STX3 had no effect on the inhibition of rENaC by brefeldin A, and the stimulatory effect of STX3 was preserved in the presence of dominant negative Rab11. This indicates that the stimulatory effect of STX3 is not mediated by inhibiting channel retrieval or by stimulating fusion of recycling endosomes. Our results suggest that the effects of syntaxins on ENaC are isoform and species dependent. Furthermore, our results demonstrate that STX3 increases ENaC expression at the cell surface, probably by enhancing insertion of vesicles carrying newly synthesized channels.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Proteínas Qa-SNARE/metabolismo , Sódio/metabolismo , Sintaxina 1/metabolismo , Amilorida/farmacologia , Animais , Membrana Celular/metabolismo , Humanos , Transporte de Íons/fisiologia , Oócitos/metabolismo , Ratos , Xenopus laevis/metabolismoRESUMO
The epithelial Na+ channel (ENaC) is a heteromeric channel composed of three subunits (α, ß, γ). At the C-terminus of each subunit, a PY-motif allows binding of the ubiquitin ligase Nedd4-2 which plays a key role in promoting ENaC retrieval from the plasma membrane. Phosphorylation of Nedd4-2 by the serum and glucocorticoid-inducible kinase 1 (Sgk1) reduces Nedd4-2 binding to the PY-motifs. In ß and γENaC, threonine residues (ßT613, γT623) belong to an extracellular signal-regulated kinase (ERK) motif and directly precede the PY-motifs. Thus, phosphorylation of these residues may modulate the interaction of their adjacent PY-motifs with Nedd4-2. In this study, a phosphospecific antibody was used to demonstrate phosphorylation of ßT613 in Xenopus laevis oocytes heterologously expressing rat αßγENaC. Treating the oocytes with progesterone to stimulate ERK increased phosphorylation of ßT613. Inactivation of the putative phosphorylation sites by mutating both threonine residues to alanine (ßT613A/γT623A) increased ENaC-mediated amiloride-sensitive whole-cell currents (ΔIami) and expression of ßENaC at the cell surface. Co-expression of Nedd4-2 largely reduced ΔIami in oocytes expressing αßγENaC or channels with mutated PY-motifs in α and γENaC or in α and ßENaC. Importantly, the inhibitory effect of co-expressed Nedd4-2 was largely reduced in channels with mutated PY-motifs in α and γENaC when combined with the ßT613A mutation but conserved in channels with mutated PY-motifs in α and ßENaC combined with the γT623A mutation. These results suggest that phosphorylation and dephosphorylation of ßT613 play a prominent role in regulating Nedd4-2-mediated ENaC retrieval from the plasma membrane.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Fosforilação/fisiologia , Subunidades Proteicas/metabolismo , Sódio/metabolismo , Alanina/genética , Animais , Membrana Celular/metabolismo , Canais Epiteliais de Sódio/genética , Feminino , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Mutação/genética , Oócitos/metabolismo , Fosforilação/genética , Subunidades Proteicas/genética , Ratos , Treonina/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
The epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel critically regulate airway surface liquid by driving fluid absorption and secretion, respectively. Their functional interplay is complex and incompletely understood. ENaC is a heteromeric channel with three well-characterized subunits (α, ß, and γ). In humans, an additional δ-ENaC subunit exists in lung and several other tissues, where it may replace the α-subunit to form δßγ-ENaC. Little is known about the physiological role of δßγ-ENaC and its possible interaction with CFTR. The aim of the present study was to investigate the effect of human CFTR on human δßγ-ENaC heterologously expressed in Xenopus laevis oocytes. In oocytes coexpressing δßγ-ENaC and CFTR the ENaC-mediated amiloride-sensitive whole cell current (ΔIami) was reduced by ~50% compared with that measured in oocytes expressing δßγ-ENaC alone. Moreover, basal level of proteolytic ENaC activation was reduced in the presence of CFTR. The inhibitory effect of CFTR on δßγ-ENaC was due to a combination of decreased average open probability (Po) and reduced channel expression at the cell surface. Interestingly, in oocytes expressing δßγ-ENaC, increasing intracellular [cAMP] by IBMX and forskolin increased ΔIami by ~50%. This stimulatory effect was not observed for human and rat αßγ-ENaC and was independent of CFTR coexpression and coactivation. Experiments with a mutant channel (δßS520Cγ-ENaC) which can be converted to a channel with a Po of nearly 1 suggested that cAMP activates δßγ-ENaC by increasing Po In conclusion, our results demonstrate that δßγ-ENaC is inhibited by CFTR but activated by cAMP.
Assuntos
AMP Cíclico/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Canais Epiteliais de Sódio/metabolismo , Oócitos/metabolismo , Subunidades Proteicas/metabolismo , Xenopus laevis/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimotripsina/metabolismo , Colforsina/farmacologia , Feminino , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Probabilidade , Proteólise/efeitos dos fármacos , RatosRESUMO
In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the ß-subunit of ENaC (ßV348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) αßγENaC or mutant αßV348MγENaC in Xenopus laevis oocytes. The ßV348M mutation stimulated amiloride-sensitive whole-cell current (ΔI(ami)) by â¼40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability (P(o)). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (â¼3.0-fold vs. â¼4.2-fold), which is consistent with the increased baseline P(o) of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by â¼2.6-fold) than wt ENaC currents (by â¼3.5-fold). The gain-of-function effect of the ßV348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different ßV348 mutants in oocytes suggests that the ßV348M mutation increases channel P(o) by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the ßV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.
Assuntos
Canais Epiteliais de Sódio/genética , Substituição de Aminoácidos , Animais , Fibrose Cística/genética , Canais Epiteliais de Sódio/metabolismo , Feminino , Células HEK293 , Humanos , Indóis/farmacologia , Mutação , Técnicas de Patch-Clamp , Xenopus laevisRESUMO
Proteolytic activation of the heteromeric epithelial sodium channel (ENaC) is thought to involve the release of inhibitory peptides from the extracellular domains of its α- and γ-subunit. Recently, we demonstrated that an α-13-mer peptide, corresponding to a putative inhibitory region within the extracellular domain of human αENaC, inhibits human αßγENaC. The aim of the present study was to investigate the structural basis of the inhibitory effect of this α-13-mer peptide. Analysis of the peptide by replica exchange molecular dynamics method, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations suggested that a helical turn at the carboxy-terminus is the preferred conformational state of the α-13-mer peptide. From this we predicted that a specific mutation (leucine 188 to alanine) should have a strong effect on the conformational preferences of the peptide. To functionally test this, we compared the effect of the wild-type α-13-mer with that of a mutant α-L188A-13-mer on ENaC currents in Xenopus laevis oocytes heterologously expressing human αßγENaC. We demonstrated that replacing the leucine 188 by alanine abolished the inhibitory effect of the α-13-mer peptide on ENaC. These findings suggest that a helical conformation in its carboxyterminal part is functionally important to mediate ENaC inhibition by the α-13-mer peptide. However, high resolution structural information on the complex of the inhibitory αENaC peptide and the channel are needed to confirm this conclusion.
Assuntos
Canais Epiteliais de Sódio/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Xenopus laevisRESUMO
Increased activity of the epithelial sodium channel (ENaC) in the respiratory airways contributes to the pathophysiology of cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some patients suffering from atypical CF a mutation can be identified in only one CFTR allele. We recently identified in this group of CF patients a heterozygous mutation (W493R) in the alpha-subunit of ENaC. Here, we investigate the functional effects of this mutation by expressing wild-type alpha beta gamma ENaC or mutant alpha(W493R)beta gamma ENaC in Xenopus oocytes. The alpha W493R mutation stimulated amiloride-sensitive whole-cell currents (Delta I(ami)) by approximately 4-fold without altering the single-channel conductance or surface expression of ENaC. As these data suggest that the open probability (P(o)) of the mutant channel is increased, we investigated the proteolytic activation of ENaC by chymotrypsin. Single-channel recordings revealed that chymotrypsin activated near-silent channels in outside-out membrane patches from oocytes expressing wild-type ENaC, but not in membrane patches from oocytes expressing the mutant channel. In addition, the alpha W493R mutation abolished Na(+) self inhibition of ENaC, which might also contribute to its gain-of-function effects. We conclude that the alpha W493R mutation promotes constitutive activation of ENaC by reducing the inhibitory effect of extracellular Na(+) and decreasing the pool of near-silent channels. The resulting gain-of-function phenotype of the mutant channel might contribute to the pathophysiology of CF in patients carrying this mutation.
Assuntos
Fibrose Cística/fisiopatologia , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/fisiologia , Mutação/genética , Sódio/metabolismo , Animais , Células Cultivadas , Quimotripsina/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Retroalimentação Fisiológica/fisiologia , Feminino , Humanos , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Plasmídeos , Xenopus laevisRESUMO
The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (alphabetagamma). In humans an additional delta-subunit (delta-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of alphabetagamma- and deltabetagamma-hENaC and investigated whether deltabetagamma-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (DeltaI(ami)) was about 11-fold larger in oocytes expressing deltabetagamma-hENaC than in oocytes expressing alphabetagamma-hENaC. The 2-fold larger single-channel Na(+) conductance of deltabetagamma-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of deltabetagamma-hENaC must be higher than that of alphabetagamma-hENaC. Experiments exploiting the properties of the known betaS520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on deltabetagamma-hENaC whole-cell currents compared with its effect on alphabetagamma-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for deltabetagamma-hENaC than for alphabetagamma-hENaC. Proteolytic activation of deltabetagamma-hENaC was associated with the appearance of a delta-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human alpha-ENaC inhibited DeltaI(ami) in oocytes expressing alphabetagamma-hENaC but not in those expressing deltabetagamma-hENaC. We conclude that the delta-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica , Oócitos/metabolismo , Animais , Membrana Celular/metabolismo , Quimotripsina/farmacologia , Humanos , Luminescência , Modelos Biológicos , Técnicas de Patch-Clamp , Peptídeos/química , Estrutura Terciária de Proteína , RNA Complementar/metabolismo , Sódio/química , Xenopus laevisRESUMO
Kinases contribute to the regulation of the epithelial sodium channel (ENaC) in a complex manner. For example, SGK1 (serum- and glucocorticoid-inducible kinase type 1) enhances ENaC surface expression by phosphorylating Nedd4-2, thereby preventing ENaC retrieval and degradation. An additional mechanism of ENaC activation by SGK1 involves an SGK consensus motif ((616)RSRYWS(621)) in the C-terminus of the channel's α-subunit. This consensus motif may also be a target for ENaC regulation by protein kinase B α (PKBα) known to be activated by insulin and growth factors. Therefore, we investigated a possible role of PKBα in the regulation of rat ENaC heterologously expressed in Xenopus laevis oocytes. We found that recombinant PKBα included in the pipette solution increased ENaC currents in outside-out patches by about 4-fold within 15-20 min. Replacing the serine residue S621 of the SGK consensus motif by an alanine (S621A) abolished this stimulatory effect. In co-expression experiments active PKBα but not catalytically inactive PKBα significantly increased ENaC whole-cell currents and surface expression by more than 50 % within 24 hours of co-expression. Interestingly, this stimulatory effect was preserved in oocytes expressing ENaC with the S621A mutation. We conclude that the acute stimulatory effect of PKBα involves a specific kinase consensus motif in the C-terminus of the channel's α-subunit. In contrast, the increase in channel surface expression caused by co-expression of PKBα does not depend on this site in the channel and is probably mediated by an effect on channel trafficking.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Substituição de Aminoácidos , Animais , Canais Epiteliais de Sódio/fisiologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/farmacologia , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Xenopus laevis/embriologia , Xenopus laevis/metabolismoRESUMO
Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the alpha-subunit of ENaC (alphaF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type alpha beta gamma-ENaC or mutant alpha(F61L) beta gamma-ENaC in Xenopus laevis oocytes. The alphaF61L mutation reduced the ENaC mediated whole-cell currents by approximately 90%. In contrast, the mutation decreased channel surface expression only by approximately 40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (P(o)). This was confirmed by experiments using the betaS520C mutant ENaC which can be converted to a channel with a P(o) of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average P(o) of ENaC by approximately 75%. Na(+) self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel P(o). The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the alphaF61L mutation. We conclude that the alphaF61L mutation may contribute to respiratory symptoms in atypical CF patients.
Assuntos
Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Mutação , Animais , Quimotripsina/metabolismo , Fibrose Cística/metabolismo , Canais Epiteliais de Sódio/análise , Feminino , Expressão Gênica , Humanos , Oócitos/metabolismo , Sódio/metabolismo , Xenopus laevisRESUMO
We investigated whether mutations in the genes that code for the different subunits of the amiloride-sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)-like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three-fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.-55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R-SCNN1A polymorphism was even found to result in a four-fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R-SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0-10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases.
Assuntos
Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , Mutação , Estudos de Casos e Controles , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Heterozigoto , Humanos , Polimorfismo GenéticoRESUMO
The lipid environment of the epithelial sodium channel (ENaC) and its possible association with so-called lipid rafts may be relevant to its function. The aim of our study was to confirm the association of ENaC with lipid rafts and to analyze the effect of cholesterol depletion of the plasma membrane by methyl-beta-cyclodextrin (MbetaCD) on channel function and regulation. Using sucrose density gradient centrifugation we demonstrated that a significant portion of ENaC protein distributes to low density fractions thought to be typical lipid raft fractions. Importantly, cholesterol depletion of cell lysate by MbetaCD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with MbetaCD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM MbetaCD for three hours. MbetaCD treatment had no consistent effect on baseline whole-cell ENaC currents. In addition to the typical single channel conductance of about 5 pS, subconductance states of ENaC were occasionally observed in patches from MbetaCD treated but not from control oocytes. Importantly, in outside-out patch clamp recordings the stimulatory effect of recombinant SGK1 in the pipette solution was essentially abolished in oocytes pretreated with MbetaCD. These results indicate that ENaC activation by cytosolic SGK1 is compromised by removing cholesterol from the plasma membrane. Thus, ENaC activation by SGK1 may require the presence of an intact lipid environment and/or of lipid rafts as signalling platform.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Canais Epiteliais de Sódio/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Fenômenos Eletrofisiológicos , Microdomínios da Membrana , Oócitos/metabolismo , Xenopus laevis , beta-Ciclodextrinas/farmacologiaRESUMO
Changes in cell function occur by specific patterns of intracellular Ca2+, activating Ca2+-sensitive proteins. The anoctamin (TMEM16) protein family has Ca2+-dependent ion channel activity, which provides transmembrane ion transport, and/or Ca2+-dependent phosphatidyl-scramblase activity. Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano4 function as Ca2+-dependent, non-selective monovalent cation channel; heterologous Ano4 expression in HEK293 cells elicits Ca2+ activated conductance with weak selectivity of K+ > Na+ > Li+. Endogenously expressed Ca2+-dependent cation channels in the retinal pigment epithelium were identified as Ano4 by KO mouse-derived primary RPE cells and siRNA against Ano4. Exchanging a negatively charged amino acid in the putative pore region (AA702-855) into a positive one (E775K) turns Ano4-elicited currents into Cl- currents evidencing its importance for ion selectivity. The molecular identification of Ano4 as a Ca2+-activated cation channel advances the understanding of its role in Ca2+ signaling.
Assuntos
Anoctaminas/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Cátions/metabolismo , Animais , Anoctaminas/genética , Canais de Cálcio/genética , Células HEK293 , Humanos , Camundongos , Camundongos KnockoutRESUMO
During the last years, heart rate variability (HRV) has become a promising risk factor for cardiovascular events. However, the effect of caffeine on HRV in habitual caffeine consumers has barely been investigated. Therefore, we treated 30 male habitual caffeine users in a randomized double-blinded crossover study design with either placebo, 100 or 200 mg caffeine orally and determined parameters of HRV under resting conditions and metronomic breathing. As result, we could not detect significant differences in HRV parameters up to 90 min after drug ingestion. We conclude that modest amounts of caffeine do not reveal negative nor positive effects on HRV within the first 90 min after drug ingestion in young and healthy habitual caffeine consumers. However, further research is necessary to determine the effects of caffeine on HRV in habitual caffeine users, healthy as well as suffering from diabetes, hypertension and postmyocardial infarction.
Assuntos
Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Adulto , Cafeína/administração & dosagem , Estimulantes do Sistema Nervoso Central/administração & dosagem , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Eletrocardiografia , Humanos , Masculino , Estatísticas não ParamétricasRESUMO
Laser Doppler fluxmetry (LDF) or photoplethysmography (PPG) are frequently used as non-invasive tools to assess skin blood flow. They are both thought to be suitable and interchangeable methods for the detection of sympathetically activated vasoconstrictive episodes that can be provoked by a voluntary deep inspiration (DI), the so-called inspiratory gasp response (IGR). However, a rigorous comparison of the two methods has not been reported in the literature. The aim of this study was to compare the detection of IGR with LDF and PPG. We investigated 14 healthy volunteers (five female, nine male, 25.4 +/- 2.7 years). A PPG probe was applied to the tip of the thumb of the dominant hand, a LDF sensor to the tip of the adjacent index finger. After adaptation and baseline measurements the subjects were asked to perform a DI with time intervals of 90, 60, 30 and 15 s. We found that both methods are useful to detect individual IGR. Overall correlation of IGR amplitude detected with LDF and PPG was significant (r=0.433). Surprisingly, there was a continuous increase of the correlation coefficient from the first (r=0.105) or second (r=-0.184) IGR to the fifth (r=0.727) IGR. This implies that experimental data obtained with PPG and LDF are not equivalent and that one has to be cautious regarding the comparison and interpretation of results obtained with these two different methods.
Assuntos
Inalação/fisiologia , Fluxometria por Laser-Doppler/métodos , Microcirculação/fisiologia , Fotopletismografia/métodos , Pele/irrigação sanguínea , Vasoconstrição/fisiologia , Adulto , Velocidade do Fluxo Sanguíneo , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αßγENaC and αßγ(K189A)ENaC in Xenopus laevis oocytes. The γ(K189A) mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γ(RKRK178AAAA)) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γ(RKRK178AAAA;K189A)) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.
Assuntos
Quimotripsina/metabolismo , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/metabolismo , Fibrinolisina/metabolismo , Ativação do Canal Iônico , Motivos de Aminoácidos , Animais , Canais Epiteliais de Sódio/genética , Humanos , Mutação de Sentido Incorreto , Subunidades Proteicas/metabolismo , Proteólise , Serina Endopeptidases/farmacologia , XenopusRESUMO
The effects of sildenafil on heart rate variability were investigated in 20 healthy male subjects aged 24 (21 to 32) years (median; range). Subjects orally received single 100-mg doses of sildenafil and placebo under randomized double-blind crossover conditions on 2 separate study days. Time domain measures of heart rate variability were assessed under conditions of relaxed rest, metronomic breathing (6 cycles per minute), and bicycle ergometry before administration of sildenafil and placebo as well as 60 minutes afterwards. Sildenafil did not alter heart rate nor heart rate variability to a significant extent (P > 0.05).
Assuntos
Frequência Cardíaca/efeitos dos fármacos , Piperazinas/farmacologia , Sulfonas/farmacologia , Adulto , Estudos Cross-Over , Método Duplo-Cego , Eletrocardiografia , Teste de Esforço , Frequência Cardíaca/fisiologia , Humanos , Masculino , Purinas/farmacologia , Respiração , Descanso/fisiologia , Citrato de Sildenafila , Decúbito Dorsal/fisiologia , Vasodilatadores/farmacologiaRESUMO
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR serves as a cAMP-stimulated chloride channel in a wide range of epithelial tissues and its dysfunction is a hallmark of CF. Over 1400 mutations in the CFTR gene are known, but functional data exist only for a minority of the mutant channels. The aim of the present study was to functionally characterize a novel CFTR mutation identified in a patient with atypical CF. Full length sequencing of the patient's CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution. Mutant and wild-type CFTR were heterologously expressed in Xenopus laevis oocytes. CFTR whole-cell currents were studied using the two-electrode voltage-clamp technique. Channel surface expression was assessed by a chemiluminescence assay. Expression of P67S-CFTR resulted in functional CFTR chloride channels. However, the CFTR chloride conductance observed in oocytes expressing the mutant channel averaged only 24% of that in oocytes expressing wild-type CFTR. Similarly, surface expression of the mutant channel was reduced. In contrast, the mutation did not alter the anion selectivity of the channel, and Western blot analysis indicated a similar protein expression level of mutant and wild-type CFTR. Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression. The mild disease phenotype of the patient indicates that the residual function of the mutant channel is sufficient to prevent the development of severe CF symptoms.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/enzimologia , Fibrose Cística/genética , Prolina/genética , Serina/genética , Potenciais de Ação , Adolescente , Substituição de Aminoácidos/genética , Sequência de Bases , Regulador de Condutância Transmembrana em Fibrose Cística/química , Feminino , Humanos , Dados de Sequência Molecular , Prolina/metabolismo , Serina/metabolismoRESUMO
In this placebo controlled double blind cross over study multiple daily dosing with 500 mg acetylic salicylic acid did not influence heart rate variability in 16 healthy male volunteers aged 25 (22-28) years (median; range) to a relevant extent (p > 0.05).
Assuntos
Aspirina/farmacologia , Saúde , Frequência Cardíaca/efeitos dos fármacos , Adulto , Humanos , Masculino , Aptidão Física , Respiração/efeitos dos fármacos , DescansoRESUMO
Regulation of the epithelial sodium channel (ENaC) is highly complex and may involve several aldosterone-induced regulatory proteins. The N-Myc downstream-regulated gene 2 (NDRG2) has been identified as an early aldosterone-induced gene. Therefore, we hypothesized that NDRG2 may affect ENaC function. To test this hypothesis we measured the amiloride-sensitive (2 microm) whole cell current (DeltaI(ami)) in Xenopus laevis oocytes expressing ENaC alone or co-expressing ENaC and NDRG2. Co-expression of NDRG2 significantly increased DeltaI(ami) in some, but not, all batches of oocytes tested. An inhibitory effect of NDRG2 was never observed. Using a chemiluminescence assay we demonstrated that the NDRG2-induced increase in ENaC currents was accompanied by a similar increase in channel surface expression. The stimulatory effect of NDRG2 was preserved in oocytes maintained in a low sodium bath solution to prevent sodium feedback inhibition. These findings suggest that the stimulatory effect of NDRG2 is independent of sodium feedback regulation. Furthermore, the stimulatory effect of NDRG2 on ENaC was at least in part additive to that of Sgk1. A short isoform of NDRG2 also stimulated DeltaI(ami). Overexpression of NDRG2 and ENaC in Fisher rat thyroid cells confirmed the stimulatory effect of NDRG2 on ENaC-mediated short-circuit current (I(SC-ami)). In addition, small interference RNA against NDRG2 largely reduced I(SC-ami) in Fisher rat thyroid cells. Our results indicate that NDRG2 is a likely candidate to contribute to aldosterone-mediated ENaC regulation.
Assuntos
Amilorida/farmacologia , Proteínas do Tecido Nervoso/fisiologia , Sódio/química , Glândula Tireoide/metabolismo , Aldosterona/metabolismo , Animais , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica , Feminino , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Endogâmicos F344 , Bloqueadores dos Canais de Sódio/farmacologia , Xenopus laevisRESUMO
The epithelial sodium channel (ENaC) is the major mediator of sodium transport across the apical membranes of the distal nephron, the distal colon, the respiratory tract and the ducts of exocrine glands. It is subject to feedback inhibition by increased intracellular Na+, a regulatory system wherein the ubiquitin protein ligases, Nedd4 and Nedd4-2, bind to conserved PY motifs in the C-termini of ENaC and inactivate the channel. It has been proposed recently that the kinase Sgk activates the channel as a consequence of phosphorylating Nedd4-2, thus preventing it from inhibiting the channels. This proposal predicts that Sgk should interfere with Na+ feedback regulation of ENaC. We have tested this prediction in Xenopus laevis oocytes and in mouse salivary duct cells and found that in neither system did increased activity of Sgk interrupt Na+ feedback inhibition of ENaC. We found, however, that Sgk stimulation was largely abolished in oocytes expressing ENaC channels with C-terminal truncations or mutated PY motifs. We were also unable to confirm that Sgk directly interacts with Nedd4-2 in vitro. We conclude that the stimulatory effect of Sgk on ENaC requires the presence of the channel's PY motifs, but it is not due to the interruption of Na+ feedback regulation.