Bile acids increase the activity of the epithelial Na+ channel

The epithelial Na⁺ channel (ENaC) is a key regulator of Na⁺ absorption in various epithelia including the distal nephron and the distal colon. ENaC is a constitutively active channel, but its activity is modulated by a number of mechanisms. These include proteolytic activation, ubiquitination and cell surface expression, phosphorylation, intracellular Na⁺ concentration, and shear stress. ENaC is related to the bile acid-sensitive ion channel (BASIC), a channel that is expressed in the epithelial cells of bile ducts. BASIC is activated by millimolar concentrations of extracellular bile acids. Bile acids are synthesized by the liver and secreted into the duodenum to aid lipolysis. A large fraction of the secreted bile acids is absorbed by the ileum and recirculated into the liver, but a small fraction passes the colon and is excreted. Bile acids can influence the ion transport processes in the intestinal tract including the colon. In this study, we show that various bile acids present in rat bile potently and reversibly increase the activity of rat ENaC expressed in Xenopus oocytes, suggesting that bile acids are natural modulators of ENaC activity.     

Inhibition of taste responses to Na+ salts by epithelial Na+ channel blockers in gerbil

The Na+ transport inhibitor amiloride blocks taste responses to NaCl by 60-70%. The purpose of the present study was to determine if greater inhibition could be achieved with three potent amiloride analogs that are specific for the epithelial Na+ channel: phenamil, 2',4'-dimethylbenzamil, and 3',4'-dichlorobenzamil. Application of phenamil (100 microM) to the anterior tongue blocked integrated responses to NaCl from the chorda tympani nerve by 98.04%, but had no significant effect on sucrose or NH4Cl. This finding suggests that the epithelial Na+ channel alone transduces the taste of NaCl in gerbil. The residual 30-40% of the response that is not blocked by amiloride can simply be explained by the fact that amiloride is less potent than phenamil. On average, 100 microM phenamil blocked responses to Na+ salts with a variety of anions by 94.2%; 100 microM 2',4'-dimethylbenzamil, by 89.83%; and 100 microM 3',4'-dichlorobenzamil, by 72.56%. Small residual responses to salts of glutamate and phosphate were not eliminated by the amiloride analogs; this suggests that other transduction mechanisms may account for a small portion of taste responses for these salts in the gerbil.     

Dual effect of temperature on the human epithelial Na+ channel

The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption in the distal segments of the nephron, in the colon and in the airways. Its activity is regulated by intracellular and extracellular factors but the mechanisms of this regulation are not yet completely understood. Recently, we have shown that the fast regulation of ENaC by the extracellular [Na⁺], a phenomenon termed self-inhibition, is temperature dependent. In the present study we examined the effects of temperature on the single-channel properties of ENaC. Single-channel recordings from excised patches showed that the channel open probability (P _o, estimated from the number of open channels N·P _o, where N is the total number of channels) increased on average two- to threefold while the single-channel conductance decreased by about half when the temperature of the perfusion solution was lowered from ~30 to ~15 °C. The effects of temperature on the single-channel conductance and P _o explain the changes of the macroscopic current that can be observed upon temperature changes and, in particular, the paradoxical effect of temperature on the current carried by ENaC.     

Genetic variation of the alpha subunit of the epithelial Na+ channel influences exhaled Na+ in healthy humans

Epithelial Na(+) channels (ENaC) are located in alveolar cells and are important in β(2)-adrenergic receptor-mediated lung fluid clearance through the removal of Na(+) from the alveolar airspace. Previous work has demonstrated that genetic variation of the alpha subunit of ENaC at amino acid 663 is important in channel function: cells with the genotype resulting in alanine at amino acid 663 (A663) demonstrate attenuated function when compared to genotypes with at least one allele encoding threonine (T663, AT/TT). We sought to determine the influence of genetic variation at position 663 of ENaC on exhaled Na(+) in healthy humans. Exhaled Na(+) was measured in 18 AA and 13 AT/TT subjects (age=27±8 years vs. 30±10 years; ht.=174±12 cm vs. 171±10 cm; wt.=68±12 kg vs. 73±14 kg; BMI=22±3 kg/m(2) vs. 25±4 kg/m(2), mean±SD, for AA and AT/TT, respectively). Measurements were made at baseline and at 30, 60 and 90 min following the administration of a nebulized β(2)-agonist (albuterol sulfate, 2.5 mg diluted in 3 ml normal saline). The AA group had a higher baseline level of exhaled Na(+) and a greater response to β(2)-agonist stimulation (baseline=3.1±1.8 mmol/l vs. 2.3±1.5 mmol/l; 30 min-post=2.1±0.7 mmol/l vs. 2.2±0.8 mmol/l; 60 min-post=2.0±0.5 mmol/l vs. 2.3±1.0 mmol/l; 90 min-post=1.8±0.8 mmol/l vs. 2.6±1.5 mmol/l, mean±SD, for AA and AT/TT, respectively, p<0.05). The results are consistent with the notion that genetic variation of ENaC influences β(2)-adrenergic receptor stimulated Na(+) clearance in the lungs, as there was a significant reduction in exhaled Na(+) over time in the AA group.     

Low-amiloride-affinity Na+ channel in the epithelial cells isolated from the endolymphatic sac of guinea-pigs

 By using the whole-cell patch-clamp technique, an amiloride-sensitive Na⁺-selective conductance was found in epithelial cells from the endolymphatic sac (ES) epithelia of guinea-pigs. In the current-clamp configuration, the average resting membrane potential was –41.7±8.4 mV (n = 22). Application of amiloride at a concentration of 20 μM elicited a decrease in cation conductance that was responsible for a membrane hyperpolarization by 17.9±6.0 mV (n = 22). Substitution of N-methyl d-glucamine chloride (NMDG-Cl) for external NaCl led to a more significant membrane hyperpolarization by 28.4±8.3 mV (n = 22). At holding potential of –70 mV, amiloride and ethylisopropylamiloride (EIPA) blocked the inward current in a concentration-dependent manner over the range of concentrations of between 0.1 μM and 50 μM, with an inhibitory constant (K _i) of 1.3±0.4 μM (n = 7) and 1.5±0.3 μM (n = 5), respectively. In the voltage-clamp configuration, substitution of NMDG-Cl for external NaCl significantly reduced the inward current (n = 9), indicating that the whole-cell conductance has a high permeability for Na⁺. Superfusion with 20 μM amiloride induced a significant reduction of the inward current, shifted the reversal potential from –39.4±8.8 mV to –60.4±10.5 mV (n = 12), and decreased the inward conductance from 5.0±1.3 nS to 3.7±1.5 nS (n = 12). The permeability ratio of Na⁺ over K⁺, calculated from the difference in reversal potential between the currents before and after application of amiloride, was approximately 5:1. Additionally, the conductance was not activated by application of forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo-cAMP (8-Br-cAMP). These findings suggest that a low-amiloride-affinity Na⁺ channel localized in the ES epithelial cells may be involved in uptake of Na⁺ in the ES. Received: 29 May 1996 / Received after revision: 1 August 1996 / Accepted: 2 August 1996     

An ATP- and Ca2+-regulated Na+ channel in isolated intestinal epithelial cells

When isolated intestinal epithelial cells are treated with 2 mM ATP, the unidirectional influx of Na+ to those cells increases from values near 50 to rates over 200 nmol . min-1 . mg protein-1. Calcium influx increases from 1 to 40 nmol . min-1 . mg protein-1. Within 2 min, the total cell Na+ increases two- to threefold, and total Ca+ increases about fivefold. The cells lose a major part of their capability for accumulating sugars during this interval. About 2 min after the time of ATP addition the normal permeability for Na+ and Ca2+ is restored, at which time the previously accumulated ions are rapidly extruded on a net basis until control levels are attained and the cells regain their usual sugar transport capability. The "repair" process requires Ca2+ in the incubation medium and is dependent on cellular uptake of Ca2+. Chlorpromazine (0.5 mM) blocks the Ca2+ entry route and the restoration of normal Na+ permeability. The Na+ entry route is selectively blocked by 4-acetamido-4'-isocyanostilbene-2,2'-disulfonic acid. The data show that ATP induces the influx of Na+ and Ca2+ by two different routes, which can be selectively inhibited. These ion flux routes may be involved in the events that allow intestinal tissue to convert from an absorptive state to a state in which net ion secretion occurs.     

A simulation study to rescue the Na+/Ca2+ exchanger knockout mice

The Na(+)/Ca(2+) exchanger (NCX) is the major Ca(2+) efflux system in cardiac myocytes, and thereby its global knockout is embryonically lethal. However, Henderson et al. (2004) found that mice with the cardiospecific knockout of NCX1 lived to adulthood. No adaptation was detected in expression levels of other proteins except for a 50% reduction in the L-type Ca(2+) current (I(CaL)) as revealed in electrophysiological studies. To predict mechanisms of survival, we simulated cardiac myocyte activity in the absence of NCX using a mathematical model of guinea pig ventricular myocytes. The NCX knockout resulted in contracture of the model cell because of a rise in the cytoplasmic Ca(2+) ([Ca(2+)](i)). However, up-regulation of the sarcolemmal Ca(2+) pump (PMCA) and/or down-regulation of I(CaL) enables steady rhythmic contractions even if NCX is totally excluded. The simulation predicted that the steady activities are maintained by a functional up-regulation of PMCA by about 2.3 times in addition to the down-regulation of I(CaL) to a half, as observed in the experiment. However, the model analysis predicted that the myocyte depending on PMCA for Ca(2+) extrusion is unstable against any changes in ionic fluxes and energetically unfavorable in comparison with the control. The reason for the instability is that the activity of PMCA driven by the ATP hydrolysis is hardly affected by changes in [Ca(2+)](i), but NCX has a reversal potential in the middle level of the action potential and is immediately affected by the Ca(2+) flux via NCX itself. The source code of the model is available at http://www.sim-bio.org/.     

Extracellular Na+ removal attenuates rundown of the epithelial Na+-channel (ENaC) by reducing the rate of channel retrieval

Regulation of the epithelial sodium channel (ENaC) is important for the long-term control of arterial blood pressure as evidenced by gain of function mutations of ENaC causing Liddles syndrome, a rare form of hereditary arterial hypertension. In Xenopus laevis oocytes expressing ENaC a spontaneous decline of ENaC currents over time, so-called rundown, is commonly observed. Mechanisms involved in rundown may be physiologically relevant and may be related to feedback regulation of ENaC by intra- or extracellular Na⁺. We tested the effect of extracellular Na⁺ removal on ENaC rundown. Spontaneous rundown of ENaC was largely prevented by extracellular Na⁺ removal and was partially prevented by primaquine suggesting that it is due to endocytic channel retrieval. Liddles syndrome mutation caused a reduced rate of rundown, and in oocytes expressing the mutated channel extracellular Na⁺ removal not only prevented rundown but even increased the ENaC currents (runup). Acute exposure to high extracellular Na⁺ drastically reduced whole-cell currents and surface expression of wild-type ENaC, while these effects were much smaller in ENaC with Liddles syndrome mutation consistent with a stabilization of the mutated channel in the plasma membrane. Interestingly, the apparent intracellular Na⁺ concentration [Na⁺]_i-app was high (>60 mM) in ENaC-expressing oocytes but rundown was not associated with a further increase in [Na⁺]_i-app. We conclude that the inhibitory effect of extracellular Na⁺ removal on rundown is due to an inhibition of endocytic ENaC retrieval.     

Mutations in the beta-subunit of the epithelial Na+ channel in patients with a cystic fibrosis-like syndrome

Cystic fibrosis (CF) is an autosomal recessive disorder of Cl(-) and Na(+) transport. The vast majority of CF patients have deleterious mutations in an epithelial Cl(-) channel called the CF transmembrane conductance regulator (CFTR). In contrast, defects in the epithelial Na(+) channel (SCNN1) have been associated with phenotypes dominated by renal disease (systemic pseudohypoaldosteronism type I and Liddle syndrome). We report two non-classic CF patients without CFTR mutations who have novel deleterious mutations in the beta-subunits of SCNN1 in the absence of overt renal disease.     

Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases

Mammalian airways are protected from infection by a thin film of airway surface liquid (ASL) which covers airway epithelial surfaces and acts as a lubricant to keep mucus from adhering to the epithelial surface. Precise regulation of ASL volume is essential for efficient mucus clearance and too great a reduction in ASL volume causes mucus dehydration and mucus stasis which contributes to chronic airway infection. The epithelial Na⁺ channel (ENaC) is the rate-limiting step that governs Na⁺ absorption in the airways. Recent in vitro and in vivo data have demonstrated that ENaC is a critical determinant of ASL volume and hence mucus clearance. ENaC must be cleaved by either intracellular furin-type proteases or extracellular serine proteases to be active and conduct Na⁺, and this process can be inhibited by protease inhibitors. ENaC can be regulated by multiple pathways, and once proteolytically cleaved ENaC may then be inhibited by intracellular second messengers such as cAMP and PIP₂. In the airways, however, regulation of ENaC by proteases seems to be the predominant mode of regulation since knockdown of either endogenous serine proteases such as prostasin, or inhibitors of ENaC proteolysis such as SPLUNC1, has large effects on ENaC activity in airway epithelia. In this review, we shall discuss how ENaC is proteolytically cleaved, how this process can regulate ASL volume, and how its failure to operate correctly may contribute to chronic airway disease.     

Influence of voltage and extracellular Na+ on amiloride block and transport kinetics of rat epithelial Na+ channel expressed in Xenopus oocytes

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.     

Regulation of alveolar epithelial Na+ channels by ERK1/2 in chlorine-breathing mice

The mechanisms by which the exposure of mice to Cl(2) decreases vectorial Na(+) transport and fluid clearance across their distal lung spaces have not been elucidated. We examined the biophysical, biochemical, and physiological changes of rodent lung epithelial Na(+) channels (ENaCs) after exposure to Cl(2), and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (I(amil)) across isolated alveolar Type II (ATII) cell monolayers and ENaC single-channel properties by patching ATII and ATI cells in situ. α-ENaC, γ-ENaC, total and phosphorylated extracellular signal-related kinase (ERK)1/2, and advanced products of lipid peroxidation in ATII cells were measured by Western blot analysis. Concentrations of reactive intermediates were assessed by electron spin resonance (ESR). Amiloride-sensitive Na(+) channels with conductances of 4.5 and 18 pS were evident in ATI and ATII cells in situ of air-breathing mice. At 1 hour and 24 hours after exposure to Cl(2), the open probabilities of these two channels decreased. This effect was prevented by incubating lung slices with inhibitors of ERK1/2 or of proteasomes and lysosomes. The exposure of ATII cell monolayers to Cl(2) increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased I(amil) and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl(2) decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in I(amil) and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl(2) activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity.     

雌激素受体α基因敲除对小鼠认知功能的影响机制

目的探讨ERα调节小鼠认知功能的作用及可能机制。方法将6只8～12周龄ERa基因敲除鼠和6只同周龄野生型小鼠分为2组：正常对照组和ERα基因敲除组。用水迷宫检测这2组小鼠的认知功能;ELISA法检测脑内Aβ含量;免疫组化法检测脑内Aβ沉淀情况。结果定位航行实验显示：与正常对照小鼠相比,ERα基因敲除鼠的逃避潜伏期显著延长（P〈O．01）;空间探索实验显示：ERα基因敲除鼠在目标象限的游泳距离占总的游泳距离的比例低于正常对照小鼠（P〈0．01）ELISA和免疫组化显示：与正常对照小鼠相比,ERα基因敲除鼠脑内Aβ生成增加（P〈0．01）。结论ERα基因敲除可导致认知功能下降,其机制可能与脑内Aβ生成增多有关。ERα基因敲除小鼠表现出类似阿尔芡海默病（AD）的病理以及行为学特征,提示其可以作为一种研究AD及其干预措施的新的动物模型。     

Restriction of the CD4+ T-cell receptor repertoire prevents immune pathology in TGF-beta1 knockout mice

Mice with a targeted deletion in TGF-beta1 spontaneously develop CD4+ T-cell-dependent multifocal inflammatory disease and autoimmune pathology. T cells from TGF-beta1-/- mice are strongly activated, but the mechanisms that lead to T-cell activation and organ pathology are not well understood. Recent work shows that TGF-beta1 raises the threshold for signaling through the TCR, suppressing the response of T cells to mitogenic stimuli. This suggests the possibility that CD4+ T cells in TGF-beta1-/- mice become aberrantly activated and cause damage in response to physiologic inputs that ordinarily are not sufficient for cell activation, such as homeostatic MHC-TCR interactions, cytokines, or adhesion molecules. This model predicts that pathology is largely antigen-independent, and that CD4+ T cells, regardless of antigen specificity, will become activated in TGF-beta1-/- mice, with subsequent organ pathology. To test this model, we crossed BALB/c-TGF-beta1-/- mice with the DO11.10 TCR transgenic mouse. To obviate the possible development of nonclonotypic TCRs, we also bred in a deficiency in RAG-1. Cohorts of highly inbred BALB/c background TGF-beta1-/- mice with an increasingly restricted CD4+ T-cell repertoire (TGF-beta1-/- mice; DO11.10-TGF-beta1-/- mice; DO11.10-RAG-1-/-TGF-beta1-/- mice) were then analyzed for inflammatory organ pathology and T-cell activation. The data show that progressively restricting the CD4+ T-cell repertoire improved survival, ameliorated target organ pathology, and reduced T-cell activation to control levels. Therefore, these results find no support for the involvement of atypical T-cell activation pathways in disease in TGF-beta1-/- mice. Rather, T-cell activation and pathology in TGF-beta1-/- mice appear to be functions of typical TCR activation pathways. This supports the hypothesis that immune pathology in TGF-beta1-/- mice is self-antigen triggered.     

Interaction between external Na+ and mexiletine on Na+ channel in guinea-pig ventricular myocytes

To assess the modulation of Na⁺ channel block with local anaesthetics by the change of external Na⁺ concentration ([Na⁺]_o), we examined the block by mexiletine at different [Na⁺]_o using the whole-cell and the cell-attached configurations of the patch-clamp technique. Lowering [Na⁺]_o increased the degree of use dependent block of the whole-cell Na⁺ current. The external Na⁺ dependence of the Na⁺ current block was caused by the interaction of mexiletine with the activated Na⁺ channel, but not with the inactivated channel. In single-Na⁺ channel current recordings at a reduced [Na⁺]_o of 70 mM, mexiletine shortened the mean open time of the channels (1.32±0.06 ms in the control vs. 0.86±0.12 ms with the drug, P<0.05) without changes in the unitary current amplitude, whereas the drug did not affect mean open time at a [Na⁺]_o of 140 mM. Moreover, the open time distributions during drug exposure at the reduced [Na⁺]_o were better fitted to a double exponential than to a single exponential in four out of six experiments. These data suggest that mexiletine induces two conductive states: the native open state and a state representing the first step of open channel block. The transition from the former to the latter is dependent on [Na⁺]_o, suggesting an antagonistic interaction of external Na⁺ with mexiletine.     

Meal patterns and foraging in melanocortin receptor knockout mice

We report the meal patterns of mice with the deletion of either the melanocortin type 3 or 4 receptors (MC3RKO or MC4RKO) compared with that of the wild type (WT) under conditions of varying foraging costs. Mice lived in two-lever operant chambers; the completion of a designated number of responses (termed procurement fixed ratio or PFR) on the "foraging" lever activated the other lever. On this second lever, the completion of a designated number of responses (termed consumatory fixed ratio or CFR) caused the delivery of a 20-mg food pellet. Animals could complete as many CFRs as they wished to constitute a meal, but whenever 10 min elapsed without pressing on this second lever, the meal was terminated and pressing on the "foraging" lever was again required to initiate a new meal. At lower PFRs, mice of all three genotypes took 5-7 well-defined meals per day of approximately 35 pellets/meal. At the highest PFR, mice of all three groups took about half this number of meals, with some increase in meal size, and total intake was slightly reduced. MC4RKO mice were obese compared with WT or MC3RKO but failed to eat more food in the operant chambers and, as a consequence, lost weight, regardless of PFR. Thus, changes in meal-taking strategies as a function of imposed foraging cost are not critically dependent on either MC3 or MC4 receptors, but these conditions did not allow us to study meal patterns in MC4RKO mice that are hyperphagic.     

Na+ channel expression and neuronal function in the Na+/H+ exchanger 1 null mutant mouse

Mice lacking Na(+)/H(+) exchanger 1 (NHE1) suffer from recurrent seizures and die early postnatally. Although the mechanisms for seizures are not well established, our previous electrophysiological work has shown that neuronal excitability and Na(+) current density are increased in hippocampal CA1 neurons of these mutant mice. However, it is unknown whether this increased density is related to altered expression or functional regulation of Na(+) channels. In this work, we asked three questions: is the increased excitability limited to CA1 neurons, is the increased Na(+) current density related to an increased Na(+) channel expression, and, if so, which Na(+) channel subtype(s) is upregulated? Using neurophysiological, autoradiographic, and immunoblotting techniques, we showed that both CA1 and cortical neurons have an increase in membrane excitability and Na(+) current density; Na(+) channel density is selectively upregulated in the hippocampus and cortex (P < 0.05); and Na(+) channel subtype I is significantly increased in the hippocampus and Na(+) channel subtype II is increased in the cortex. Our results demonstrate that mice lacking NHE1 upregulate their Na(+) channel expression in the hippocampal and cortical regions selectively; this leads to an increase in Na(+) current density and membrane excitability. We speculate that neuronal overexcitability due to Na(+) channel upregulation in the hippocampus and cortex forms the basis of epileptic seizures in NHE1 mutant mice.