Intracellular cholesterol stimulates ENaC by interacting with phosphatidylinositol­4,5­bisphosphate and mediates cyclosporine A-induced hypertension.

We have previously shown that blockade of ATP-binding cassette transporter A1 (ABCA1) with cyclosporine A (CsA) stimulates the epithelial sodium channel (ENaC) in cultured distal nephron cells. Here we show that CsA elevated systolic blood pressure in both wild-type and apolipoprotein E (ApoE) knockout (KO) mice to a similar level. The elevated systolic blood pressure was completely reversed by inhibition of cholesterol (Cho) synthesis with lovastatin. Inside-out patch-clamp data show that intracellular Cho stimulated ENaC in cultured distal nephron cells by interacting with phosphatidylinositol­4,5­bisphosphate (PIP2), an ENaC activator. Confocal microscopy data show that both α­ENaC and PIP2 were localized in microvilli via a Cho-dependent mechanism. Deletion of membrane Cho reduced the levels of γ­ENaC in the apical membrane. Reduced ABCA1 expression and elevated intracellular Cho were observed in old mice, compared to young mice. In parallel, cell-attached patch-clamp data from the split-open cortical collecting ducts (CCD) show that ENaC activity was significantly increased in old mice. These data suggest that elevation of intracellular Cho due to blockade of ABCA1 stimulates ENaC, which may contribute to CsA-induced hypertension. This study also implies that reduced ABCA1 expression may mediate age-related hypertension by increasing ENaC activity via elevation of intracellular Cho.

Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli.

BACKGROUND/AIMS: The epithelial sodium channel (ENaC) in cortical collecting duct (CCD) principal cells plays a critical role in regulating systemic blood pressure. We have previously shown that cholesterol (Cho) in the apical cell membrane regulates ENaC; however, the underlying mechanism remains unclear. METHODS: Patch-clamp technique and confocal microscopy were used to evaluate ENaC activity and density. RESULTS: Here we show that extraction of membrane Cho with methyl-ß-cyclodextrin (MßCD) significantly reduced amiloride-sensitive current and ENaC single-channel activity. The effects were reproduced by inhibition of Cho synthesis in the cells with lovastatin. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP2), an ENaC activator, is predominantly located in the microvilli, a specialized apical membrane domain. Here, our confocal microscopy data show that α-ENaC was co-localized with PIP2 in the microvilli and that Cho was also co-localized with PIP2 in the microvilli. Either extraction of Cho with MßCD or inhibition of Cho synthesis with lovastatin consistently reduced the levels of Cho, PIP2, and ENaC in the microvilli. CONCLUSIONS: Since PIP2 can directly stimulate ENaC and also affect ENaC trafficking, these data suggest that depletion of Cho reduces ENaC apical density and activity at least in part by decreasing PIP2 in the microvilli.

Hydrogen peroxide suppresses TRPM4 trafficking to the apical membrane in mouse cortical collecting duct principal cells.

A Ca2+-activated nonselective cation channel (NSCCa) is found in principal cells of the mouse cortical collecting duct (CCD). However, the molecular identity of this channel remains unclear. We used mpkCCDc14 cells, a mouse CCD principal cell line, to determine whether NSCCa represents the transient receptor potential (TRP) channel, the melastatin subfamily 4 (TRPM4). A Ca2+-sensitive single-channel current was observed in inside-out patches excised from the apical membrane of mpkCCDc14 cells. Like TRPM4 channels found in other cell types, this channel has an equal permeability for Na+ and K+ and has a linear current-voltage relationship with a slope conductance of ~23 pS. The channel was inhibited by a specific TRPM4 inhibitor, 9-phenanthrol. Moreover, the frequency of observing this channel was dramatically decreased in TRPM4 knockdown mpkCCDc14 cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14 cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14 cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ ionophore), but not by ATP (a purinergic P2 receptor agonist). These data suggest that the NSCCa current previously described in CCD principal cells is actually carried through TRPM4 channels. However, the physiological role of this channel in the CCD remains to be further determined.

Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.

Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-ß,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

Percutaneous coronary intervention delays pacemaker implantation in coronary artery disease patients with established bradyarrhythmias.

BACKGROUND: Pacemakers have long been used to assist the heart under pathological conditions, and they are the first choice in the treatment of systematic bradyarrhythmias. However, the effect of percutaneous coronary intervention (PCI) in patients with coronary artery disease as well as bradyarrhythmias remains unknown. METHODS: In the present study, 42 patients with chest pain and/or abnormal stress test results were surveyed. Before coronary angiography, patients underwent complete examination, including a 24 h dynamic electrocardiogram, which was used to diagnose bradyarrhythmias that were not suitable for pacemaker implantation due to a lack of arrhythmia-related symptoms. All patients underwent PCI but did not undergo pacemaker implantation. Forty-one patients with chest pain and/or abnormal stress test results, as well as symptom-free bradyarrhythmias, were selected as the control group. All of the patients in the control group were committed to treatments without PCI. RESULTS: During a mean (±SD) of 3.3±0.5 years of follow-up (range 2.5 to 4.5 years), 24 of 42 patients who received PCI underwent pacemaker implantation for arrhythmia-related symptoms, eight were shown by Holter monitoring to have worsened but still exhibited no symptoms, and the remainder did not show any changes according to the examinations performed. In the control group, 31 patients underwent pacemaker implantation for arrhythmia-related symptoms, eight were shown by Holter monitoring to have worsened but still exhibited no symptoms, and two did not show any changes according to the examinations performed. Nevertheless, the rates of pacemaker implantation each year (from the first to the third year) between the two groups were 7.1% versus 39.0% (P=0.001); 33.3% versus 63.4% (P=0.006); and 57.1% versus 75.6%, (P=0.075), respectively. CONCLUSIONS: The present study found that PCI delayed the demand for pacemaker implantation among coronary artery disease patients.

Cystatin C associates with the prediction of in-stent restenosis among patients receiving stent implantation: results of the 1-year follow-up.

BACKGROUND: For a long time, serum cystatin C (Cys C) was only considered an indicator of renal function besides creatinine. Recently, it has been found that it is also a good biomarker showing the oxidative status of the human body. We hypothesized that serum Cys C levels are associated with in-stent restenosis (ISR) among patients receiving stent implantation. METHODS: Here, we enrolled 145 (men, 110; mean age, 59.6±11.1) consecutive patients admitted to the Fourth Affiliated Hospital of Harbin Medical University for stent implantation. At 11.2±3.2 months after stent implantation, all of the patients were administered coronary angiography to evaluate ISR. The serum levels of total bilirubin (TB), high-sensitive C-reactive protein (hs-CRP), and Cys C were obtained to determine the relationship of ISR with these oxidative-related biomarkers. RESULTS: A total of 163 stents were studied; patients experiencing ISR had considerably higher levels of serum hs-CRP and Cys C and lower levels of TB compared with patients without ISR (TB, 13.7±3.5 vs. 11.8±4.0 µmol/l, P=0.007; hs-CRP, 11.8±6.0 vs. 5.9±6.4 µmol/l, P=0.001; Cys C, 1.68±0.85 vs. 0.94±0.24 mg/l, P<0.001). Furthermore, the Cox proportional hazard model showed that serum TB [odds ratio (OR)=2.42, 95% confidence interval (CI), 2.21-2.68, P=0.028], hs-CRP (OR=2.89, 95% CI, 2.76-3.02, P=0.003), and Cys C (OR=80.46, 95% CI, 11.10-2977.60, P<0.001) are all independent predictors of ISR. CONCLUSION: Cys C, together with TB and hs-CRP, is an independent risk factor associated with ISR.

Lovastatin attenuates effects of cyclosporine A on tight junctions and apoptosis in cultured cortical collecting duct principal cells.

We used mouse cortical collecting duct principal cells (mpkCCDc14 cell line) as a model to determine whether statins reduce the harmful effects of cyclosporine A (CsA) on the distal nephron. The data showed that treatment of cells with CsA increased transepithelial resistance and that the effect of CsA was abolished by lovastatin. Scanning ion conductance microscopy showed that CsA significantly increased the height of cellular protrusions near tight junctions. In contrast, lovastatin eliminated the protrusions and even caused a modest depression between cells. Western blot analysis and confocal microscopy showed that lovastatin also abolished CsA-induced elevation of both zonula occludens-1 and cholesterol in tight junctions. In contrast, a high concentration of CsA induced apoptosis, which was also attenuated by lovastatin, elevated intracellular ROS via activation of NADPH oxidase, and increased the expression of p47phox. Sustained treatment of cells with lovastatin also induced significant apoptosis, which was attenuated by CsA, but did not elevate intracellular ROS. These results indicate that both CsA and lovastatin are harmful to principal cells of the distal tubule, but via ROS-dependent and ROS-independent apoptotic pathways, respectively, and that they counteract probably via mobilization of cellular cholesterol levels.

Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations.

Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPγS), adenosine 5'-(ß,γ-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPγS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPγS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPγS removal (τdeac ∼ 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPγS activated even the strongest GOF mutant as strongly as ATP. ATPγS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPγS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.

The level of serum bilirubin associated with coronary lesion types in patients with coronary artery disease.

BACKGROUND: Serum bilirubin has been proven to be associated with coronary artery disease (CAD). However, how serum bilirubin is related to the complexity of coronary artery lesions is still unknown. METHODS AND RESULTS: One thousand two hundred and sixty patients (men 775, 61.5%, mean age, 59.3  ±â€Š 8.2 years) diagnosed with unstable angina were enrolled in the study. Patients were categorized into three major groups and group III was further divided into four subgroups according to the guidelines of AHA/ACC 1993 described in the Methods section. The total serum bilirubin levels showed significant differences among the three major groups (group I vs. group II, 14.8 ± 5.8 vs. 13.7 ± 4.7 µmol/l, P=0.017; group I vs. group III, 14.8 ± 5.8 vs. 12.6 ±  4.4 µmol/l, P<0.001; group II vs. group III, 13.7 ± 4.7 vs. 12.6 ± 4.4 µmol/l, P=0.009). The difference was further seen among the subgroups. Logistic regression analysis demonstrated that age, male sex, histories of hypertension and diabetes, and total serum bilirubin were independent risk factors for CAD. However, in the subgroups, only age, male sex, history of hypertension and total serum bilirubin were associated with CAD. Total serum bilirubin showed the strongest relationship (odds ratio=0.95, 95% confidence interval 0.91-0.98, P=0.001). CONCLUSION: Total serum bilirubin level is an independent risk factor for CAD. It has a strong relationship with coronary artery lesion types.

Cystatin C levels are associated with the prognosis of systolic heart failure patients.

BACKGROUND: Cystatin C, which has long been regarded as a biomarker that indicates kidney functions, has recently been recognized as an inflammatory marker in the human body. AIM: To elucidate how cystatin C is related to the prognosis of systolic heart failure patients. METHODS: Patients with systolic heart failure who were admitted to the fourth affiliated hospital of Harbin Medical University between January and April 2008 were enrolled in this study. Serum homocysteine, high-sensitivity C-reactive protein (hs-CRP) and cystatin C levels were determined and all the patients received an average of 2 years of follow-up for occurrence of death, heart transplantation or readmission with worsening heart failure. RESULTS: Of 138 patients enrolled, those who experienced adverse outcomes (e.g. cardiac death, heart transplantation or progressive heart failure) (n = 21) had considerably higher mean levels of serum homocysteine (28.6 ± 13.4 vs 14.4 ± 6.3mg/L; P < 0.01), hs-CRP (17.5 ± 14.1 vs 6.4 ± 7.7 µmol/L; p < 0.01) and cystatin C (1.63 ± 0.81 vs 0.91 ± 0.27 mg/L; P < 0.01) than those without adverse outcomes (n = 117). Furthermore, the Cox proportional hazards model demonstrated that serum homocysteine, hs-CRP and cystatin C are all independent predictors of adverse outcomes. CONCLUSIONS: Cystatin C, together with hs-CRP and homocysteine, is an independent risk factor that is important in the prognosis of patients with systolic heart failure.

The status of coronary artery lesions in patients with conduction disturbance.

BACKGROUND: Conduction disturbances are related to coronary artery lesions. However, the types of coronary artery lesions prevalent among patients having conduction disturbances are still uncertain. OBJECTIVE: To investigate the status of coronary artery lesions in 107 consecutive symptomatic patients admitted to the fourth affiliated hospital of Harbin Medical University between 2005 and 2009 to receive pacemaker implantation. METHODS: All of the patients previously underwent coronary angiography for detection of symptoms related to coronary artery disease. The coronary angiographic study included stenosis severity and classification of pathological anatomy. Eighty patients had obvious coronary artery lesions; therefore, 80 age-matched individuals with more than 50% coronary artery lesions but without conduction disturbance composed the control group. The differences in the status of coronary artery lesions were studied between the groups. RESULTS: Third-degree atrioventricular block (AVB) was diagnosed in 51 patients (47.7%); 49 (45.8%) had sick sinus syndrome and 7 (6.5%) had Mobitz II, second-degree AVB. Individuals with type IV lesions accounted for 51.3% of the patients with coronary artery lesions, whereas those with type II lesions had the second highest prevalence (26.3%). Patients with type III and I lesions accounted for 15 and 7.5% of the cases, respectively. This distribution was significantly different from those in the control group in which the most prevalent types were type III and IV (46.8 and 31.6%, P < 0.05, respectively). CONCLUSION: The prevalence of coronary lesion types was different for patients with conduction disturbance from those without it; both left anterior descending artery and right coronary artery appeared to be involved in patients with conduction disturbance.

Postexposure administration of a {beta}2-agonist decreases chlorine-induced airway hyperreactivity in mice.

Exposure to chlorine (Cl(2)) damages airway and alveolar epithelia, resulting in acute lung injury and reactive airway dysfunction syndrome. We evaluated the efficacy and mechanisms by which arformoterol, a long-term ß(2)-agonist, administered after exposure, mitigated the extent of this injury. Exposure of C57BL/6 mice to 400 ppm Cl(2) for 30 minutes increased respiratory system resistance and airway responsiveness to aerosolized methacholine (assessed by FlexiVent) up to 6 days after exposure, and decreased Na(+)-dependent alveolar fluid clearance (AFC). Inducible Nitric Oxide Synthase (iNOS) knockout mice developed similar degrees of airway hyperreactivity as wild-type controls after Cl(2) exposure, indicating that reactive intermediates from iNOS do not contribute to Cl(2)-induced airway dysfunction in our model. Intranasal administration of arformoterol mitigated the Cl(2) effects on airway reactivity and AFC, presumably by increasing lung cyclic AMP level. Arformoterol did not modify the inflammatory responses, as evidenced by the number of inflammatory cells and concentrations of IL-6 and TNF-α in the bronchoalveolar lavage. NF-κB activity (assessed by p65 Western blots and electrophoretic mobility shift assay) remained at control levels up to 24 hours after Cl(2) exposure. Our results provide mechanistic insight into the effectiveness of long-term ß(2)-agonists in reversing Cl(2)-induced reactive airway dysfunction syndrome and injury to distal lung epithelial cells.

Inhibition of epithelial sodium channels by respiratory syncytial virus in vitro and in vivo.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and children worldwide. Infection of mice with RSV decreased sodium (Na(+)) dependent alveolar fluid clearance (AFC), resulting in increased lung water content and hypoxemia. RSV infection resulted in higher levels of pyrimidines and purines in the alveolar space. Intratracheal administration of UTP or UDP also decreased AFC. The effects of RSV on AFC and oxygen saturation of Balb/c mice were reversed by intraalveolar administration of antagonists of P2Y nucleotide receptors, enzymes that enhance the breakdown of pyrimidines and systemic or intranasal administration of inhibitors of the de novo pathway of pyrimidine synthesis. RSV infection of H441 or mouse tracheal epithelial cells decreased the amiloride-sensitive Na(+) currents and pretreatment of H441 cells with A77 prevented this effect. These findings indicate that the harmful effects of RSV on lung epithelia are mediated at least in part via the production of UTP and its paracrine action on ENaC.

Inhibition of lung fluid clearance and epithelial Na+ channels by chlorine, hypochlorous acid, and chloramines.

We investigated the mechanisms by which chlorine (Cl(2)) and its reactive byproducts inhibit Na(+)-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na(+) channels (ENaC) by measuring AFC in mice exposed to Cl(2) (0-500 ppm for 30 min) and Na(+) and amiloride-sensitive currents (I(Na) and I(amil), respectively) across Xenopus oocytes expressing human alpha-, beta-, and gamma-ENaC incubated with HOCl (1-2000 microm). Both Cl(2) and HOCl-derived products decreased AFC in mice and whole cell and single channel I(Na) in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I(Na) in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of I(Na) by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the gamma-ENaC subunit restored the ability of HOCl to inhibit I(Na). Finally, I(Na) of oocytes expressing wild type alpha- and gamma-ENaC and a mutant form of beta ENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.

Regulation of epithelial sodium channels by cGMP/PKGII.

Airway and alveolar fluid clearance is mainly governed by vectorial salt movement via apically located rate-limiting Na(+) channels (ENaC) and basolateral Na(+)/K(+)-ATPases. ENaC is regulated by a spectrum of protein kinases, i.e. protein kinase A (PKA), C (PKC), and G (PKG). However, the molecular mechanisms for the regulation of ENaC by cGMP/PKG remain to be elucidated. In the present study, we studied the pharmacological responses of native epithelial Na(+) channels in human Clara cells and human alphabetagammadelta ENaCs expressed in oocytes to cGMP. 8-pCPT-cGMP increased amiloride-sensitive short-circuit current (I(sc)) across H441 monolayers and heterologously expressed alphabetagammadelta ENaC activity in a dose-dependent manner. Similarly, 8-pCPT-cGMP (a PKGII activator) but not 8-Br-cGMP (a PKGI activator) increased amiloride-sensitive whole cell currents in H441 cells in the presence of CFTRinh-172 and diltiazem. In all cases, the cGMP-activated Na(+) channel activity was inhibited by Rp-8-pCPT-cGMP, a specific PKGII inhibitor. This was substantiated by the evidence that PKGII was the sole isoform expressed in H441 cells at the protein level. Importantly, intratracheal instillation of 8-pCPT-cGMP in BALB/c mice increased amiloride-sensitive alveolar fluid clearance by approximately 30%, consistent with the in vitro results. We therefore conclude that PKGII is an activator of lung epithelial Na(+) channels, which may expedite the resolution of oedematous fluid in alveolar sacs.

Alpha(1)-antitrypsin inhibits epithelial Na+ transport in vitro and in vivo.

A variety of studies have shown that Na(+) reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which alpha(1)-antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloride-sensitive epithelial Na(+) channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml = 1 microM) decreased ENaC currents across Xenopus laevis oocytes injected with human alpha,beta,gamma-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC single-channel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO(-)), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO(-)-treated A1AT (1 microM) in C57BL/6 mice also decreased Na(+)-dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO(-)-treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na(+)-dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.

Membrane tension modulates the effects of apical cholesterol on the renal epithelial sodium channel.

We used patch-clamp techniques and A6 distal nephron cells as a model to determine how cholesterol regulates the renal epithelial sodium channel (ENaC). We found that luminal methyl-beta-cyclodextrin (mbetaCD, a cholesterol scavenger) did not acutely affect ENaC activity at a previously used concentration of 10 mM: but significantly decreased ENaC activity both when the cell membrane was stretched and at a higher concentration of 50 mM: Luminal cholesterol had no effect on ENaC activity at a concentration of 50 microg/ml but significantly increased ENaC activity both when the cell membrane was stretched and at a higher concentration of 200 microg/ml. Confocal microscopy data indicate that membrane tension facilitates both mbetaCD extraction of cholesterol and A6 cell uptake of exogenous cholesterol. Together with previous findings that cholesterol in the apical membrane is tightly packed with sphingolipids and that stretch can affect lipid distribution, our data suggest that membrane tension modulates the effects of mbetaCD and cholesterol on ENaC activity, probably by facilitating both extraction and enrichment of apical cholesterol.