Blocker Amiloride Research Articles

Amiloride lowers plasma TNF and interleukin-6 but not interleukin-17A in patients with hypertension and type 2 diabetes.

Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na+ channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1β, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: 1) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (n = 69, amiloride 5-10 mg/day for 8 wk) and 2) patients with hypertension and type 1 diabetes mellitus (T1DM) (n = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K+ increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1β. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.NEW & NOTEWORTHY ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.

Mechanisms of carbon dioxide detection in the earthworm Dendrobaena veneta

IntroductionCarbon dioxide (CO2) is a critical biological signal that is noxious to many animals at high concentrations. The earthworm Dendrobaena veneta lives in subterranean burrows containing high levels of CO2 and respires through its skin. Despite the ecological and agricultural importance of earthworms, relatively little is known about how they make decisions in their environment, including their response to elevated levels of CO2.MethodsTo examine CO2 detection in this species, we designed the exudate assay, in which we placed an earthworm in a sealed container, exposed it to varying concentrations of CO2 for one minute, and recorded the amount of exudate secreted. Because earthworms excrete exudate in response to noxious stimuli, we hypothesized that the amount of exudate produced was proportional to the amount of irritation. We repeated these experiments after treatment with several blockers for molecules with potential involvement in CO2 detection, including carbonic anhydrases, guanylate cyclase, TRPA1, ASICs, and OTOP channels. We also confirmed the presence of homologous transcripts for each of these gene families in an epithelial transcriptome for D. veneta. Additionally, since organisms often detect CO2 levels indirectly by monitoring the conversion to carbonic acid (a weak acid), we used the exudate assay to evaluate aversion to additional weak acids (formic acid, acetic acid, and propionic acid).ResultsEarthworms excreted significantly more exudate in response to CO2 in a dosage-dependent manner, and this response was muted by the general carbonic anhydrase inhibitor acetazolamide, the carbonic anhydrase IX/XII inhibitor indisulam, the calcium channel blocker ruthenium red, the sodium channel blocker amiloride, and the acid-sensing ion channel blocker diminazene aceturate.DiscussionThese data provide evidence of the role of carbonic anhydrase and epithelial sodium channels in earthworm CO2 detection, establish that, similar to other subterranean-dwelling animals, earthworms are extremely tolerant of CO2, and contribute to our understanding of the mechanisms used by earthworms to detect and react to weak acids in their environment.

Study of Amiloride Binding to Human Serum Albumin: Insights from Thermodynamic, Spectroscopic, and Molecular Docking Investigations

This study was undertaken to investigate the interaction between the sodium channel blocker amiloride (AML) and human serum albumin (HSA). A combination of multi-spectroscopic techniques and computational methods were employed to identify the AML binding site on HSA and the forces responsible for the formation of the HSA–AML complex. Our findings revealed that AML specifically binds to Sudlow’s site II, located in subdomain IIIA of HSA, and that the complex formed is stabilized using van der Waals hydrogen-bonding and hydrophobic interactions. FRET analysis showed that the distance between AML and Trp214 was optimal for efficient quenching. UV-Vis spectroscopy and circular dichroism indicated minor changes in the structure of HSA after AML binding, and molecular dynamics simulations (MDS) conducted over 100 ns provided additional evidence of stable HSA–AML-complex formation. This study enhances understanding of the interaction between AML and HSA and the mechanism responsible.

Function and phylogeny support the independent evolution of an ASIC-like Deg/ENaC channel in the Placozoa

ASIC channels are bilaterian proton-gated sodium channels belonging to the large and functionally-diverse Deg/ENaC family that also includes peptide- and mechanically-gated channels. Here, we report that the non-bilaterian invertebrate Trichoplax adhaerens possesses a proton-activated Deg/ENaC channel, TadNaC2, with a unique combination of biophysical features including tachyphylaxis like ASIC1a, reduced proton sensitivity like ASIC2a, biphasic macroscopic currents like ASIC3, as well as low sensitivity to the Deg/ENaC channel blocker amiloride and Ca2+ ions. Structural modeling and mutation analyses reveal that TadNaC2 proton gating is different from ASIC channels, lacking key molecular determinants, and involving unique residues within the palm and finger regions. Phylogenetic analysis reveals that a monophyletic clade of T. adhaerens Deg/ENaC channels, which includes TadNaC2, is phylogenetically distinct from ASIC channels, instead forming a clade with BASIC channels. Altogether, this work suggests that ASIC-like channels evolved independently in T. adhaerens and its phylum Placozoa. Our phylogenetic analysis also identifies several clades of uncharacterized metazoan Deg/ENaC channels, and provides phylogenetic evidence for the existence of Deg/ENaC channels outside of Metazoa, present in the gene data of select unicellular heterokont and filasterea-related species.

Abstract P300: Blood Pressure Increase Upon High Dietary Potassium Supplementation Is Not Associated With Renal Sodium Retention Or Plasma Volume Expansion

Background: High dietary K + supplementation increases arterial blood pressure in humans and in mice. The mechanisms underlying this unexpected response are still not fully understood. Recent studies in mice reported an activation of the epithelial sodium channel (ENaC) suggesting that renal Na + retention may prominently contribute to the blood pressure increase caused by a high KCl intake. Here, we tested whether a high K + intake causes renal Na + retention and plasma volume expansion. Methods: Na + -replete C57BL/6J mice were subjected to either a normal (0.93%, NK) or a high (5%, HK) KCl or KCitrate diet for up to 14 days. Blood pressure responses to diets and to the additional administration of the ENaC blocker amiloride were measured using radiotelemetry in unrestrained animals. Plasma aldosterone concentrations were quantified by ELISA. Renal Na + retention was assessed by determining urinary Na + excretion. To detect plasma volume expansion, we used a dilution method (Evans blue), determined blood hematocrit, and estimated end diastolic left ventricular volume by echocardiography after 10 days of diet. Results: Both high K + diets increased arterial blood pressure selectively in the dark phase by a nearly identical extent (ΔMAP dark: HKCl +12.7±2.3 mm Hg, p=0.0045, HKCit +12.1±3.0 mm Hg, p=0.0211; ΔMAP light: HKCl -0.2±1.5 mm Hg, p>0.9999, HKCit +2.4±2.2 mm Hg, p=0.9692, 1-way ANOVA, n=7). Even though plasma aldosterone levels were markedly elevated (HKCl 9.9-fold, p=0.0001, HKCit 4.6-fold, p=0.0116, 1-way ANOVA, n=11-12), neither a reduction of renal Na + excretion (urinary [Na + ]/[creatinine] NK 1.0±0.14, HKCl 1.40±0.23, p=0.3873, HKCit 0.94±0.20, p<0.9999, 1-way ANOVA, n=7-13) nor an expansion of plasma volume (NK 898±23 μl, HKCl 918±57 μl, p>0.9999, HKCit 870±62 μl, p>0.9999, 1-way ANOVA, n=6-13) was detected upon the two high K + diets. Amiloride did not normalize arterial blood pressure in mice fed a HKCl or HKCit diet (ΔMAP dark: HKCl +1.2±2.3 mm Hg, p=0.6391 HKCit +2.8±2.0 mm Hg, p=0.2472, 2-tailed paired t-test, n=4). Conclusions: Our findings indicate that excessive K + intake causes elevated blood pressure independent of renal Na + retention and volume expansion in Na + -replete mice.

The Epithelial Sodium Channel-An Underestimated Drug Target.

Epithelial sodium channels (ENaC) are part of a complex network of interacting biochemical pathways and as such are involved in several disease states. Dependent on site and type of mutation, gain- or loss-of-function generated symptoms occur which span from asymptomatic to life-threatening disorders such as Liddle syndrome, cystic fibrosis or generalized pseudohypoaldosteronism type 1. Variants of ENaC which are implicated in disease assist further understanding of their molecular mechanisms in order to create models for specific pharmacological targeting. Identification and characterization of ENaC modifiers not only furthers our basic understanding of how these regulatory processes interact, but also enables discovery of new therapeutic targets for the disease conditions caused by ENaC dysfunction. Numerous test compounds have revealed encouraging results in vitro and in animal models but less in clinical settings. The EMA- and FDA-designated orphan drug solnatide is currently being tested in phase 2 clinical trials in the setting of acute respiratory distress syndrome, and the NOX1/ NOX4 inhibitor setanaxib is undergoing clinical phase 2 and 3 trials for therapy of primary biliary cholangitis, liver stiffness, and carcinoma. The established ENaC blocker amiloride is mainly used as an add-on drug in the therapy of resistant hypertension and is being studied in ongoing clinical phase 3 and 4 trials for special applications. This review focuses on discussing some recent developments in the search for novel therapeutic agents.

Abstract LB156: Identified the ISG15 mediated extracellular vesicles drives ovarian cancer progression and metastasis: extracellular vesicular ISG15 is a potential biomarker and therapeutic target

Abstract Background & Objective: High-grade serous ovarian cancer (HGSOC) accounts for over 80% of all epithelial ovarian cancer (OC) diagnoses, and the majority of patients with HGSOC are diagnosed with advanced metastatic disease. Aberrant expression of interferon stimulated gene 15 (ISG15) has been demonstrated in human malignancies, and we have identified that ISG15 is highly expressed in HGSOC tumors and peritoneal ascites. This study seeks to determine whether ISG15 contributes to HGSOC progression and metastasis through extracellular vesicle (EVs) secretion. Method: ISG15 expression was analyzed in ascites samples and primary ovarian cancer cells (POCC) from different patients by ELISA and WB. Cell surface biotinylation assay was done to show the modulation of endo and exocytosis by ISG15 and STAT3. Immunoprecipitation pull down assay was done to demonstrate the interaction of ISG15 with activated STAT3. Confocal microscopy showed the co-localization of STAT3 with the endosome marker TSG101. In-vivo studies were done using bio-luminescence imaging in orthotopic ovarian tumor mouse models to measure tumor progression and metastasis. Results: ISG15 was found to be significantly elevated in HGSOC metastases (pelvic, mesenteric and diaphragmatic samples) as compared to primary ovarian tumors or benign samples. We observed that ISGylation was increased in POCC cells derived from ascites with increased USP18 expression. Our results confirmed a significant decrease in EV’s secretion in ISG15 KD POCC cells. Mice injected with ISG15 OE -POCC cells showed increased tumor burden when compared to the ISG15Kd cells in mice. Furthermore, we observed a significant reduction of ovarian tumor growth and metastasis in an orthotopic mouse model treated with a small molecule inhibitor-DAP5 that targeted ISG15 or exosome blocker (Amiloride) compared to untreated mice. In addition, we found the expression of ISG15 within the EVs represents a promising development in elucidating prognostic markers for HGSOC patients. Conclusion: Based on our results, ISG15 expression is elevated in HGSOC patient ascites and metastatic disease sites. The aberrant expression of ISG15 in patient ascites plays a key role in the secretion of EVs carrying ISG15, which contributes to HGSOC progression and metastases. Our study provides the pre-clinical evidence regarding new molecular targets, novel prognostic markers, and innovative therapeutic strategies for HGSOC, aiming to ultimately improve the survival of patients suffering from this disease. Citation Format: Kalpana Deepa Priya Dorayappan, Vincent Wagner, Dongju Park, Meghan M. Newcomer, Michelle DS Lightfoot, Deepika Kalaiyarasan, Takahiko Sakaue, Wafa Khadraoui, Casey Cosgrove, Larry J. Maxwell, Qi-En Wang Wang, David O’Malley, Raphael E. Pollock, David E. Cohn, Selvendiran Karuppaiyah. Identified the ISG15 mediated extracellular vesicles drives ovarian cancer progression and metastasis: extracellular vesicular ISG15 is a potential biomarker and therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB156.

Abstract P335: Contribution Of The Gut To The Salt-sensitive Phenotype Of Dahl S Rats Is Reflected In Increased Sodium Absorption In The Distal Colon

Salt-sensitive hypertension in Dahl sensitive (Dahl S) rats is primarily attributed to sodium mishandling associated with kidney dysfunction, and very few studies interrogated sodium absorption in the gut of Dahl S rats and its potential contribution to the salt-sensitive phenotype. To assess this, we compared sodium channel (ENaC)-driven sodium (Na + ) currents, as a measure of Na + transport across the distal colon, the primary sodium absorption site in the GI tract, between Dahl S and Dahl salt-resistant (Dahl R) rats ex vivo . In parallel, we evaluated fecal sodium levels to assess overall fecal sodium loss in Dahl S and R rats. Male adult (12-14wo) Dahl S and R were administered a low sodium diet (0.3% NaCl, Teklad) ad libitum from weaning. Prior to the experiment, food intake/rat was measured every day for three consecutive days. On the day of experiment, rats were euthanized by an overdose of isoflurane. Distal colons were dissected and transferred to the Ussing chamber with oxygenated warm Krebs solution containing d-glucose. Increasing concentrations of the ENaC blocker amiloride (0.01-1000uM) were applied to the Ussing chamber on the apical side of the distal colon in 10-minute intervals. Maximum decreases in total ionic current across the distal colon were measured in real time following addition of amiloride to quantify ENaC Na + current across the distal colon. Fecal samples were homogenized in distilled water, and sodium levels per gram of fecal mass were determined by flame photometry. Despite no differences in food intake, we observed a dose-dependent 2 to 4-fold higher responses to amiloride in the distal colon of Dahl S compared to Dahl R rats, suggesting elevated Na + current in the Dahl S. Conversely, a ~2-fold lower Na + concentration was detected in the feces of Dahl S compared to Dahl R rats. In conclusion, elevated ENaC-driven Na + current in the distal colon, coupled with reduced fecal Na + concentration, suggest higher absorption/transport of Na + across the distal colon of Dahl S compared to Dahl R rats, which was independent of salt intake. Considering that the circulating Na + levels are reportedly maintained in the Dahl S by the overworked kidney, elevated colonic sodium absorption may contribute to kidney disease and salt-sensitive phenotype in Dahl S.

Deficient regulation of the epithelial Na+ channel (ENaC) activity and impaired urinary conservation of Na+ in Epac1 ‐/‐ and Epac2 ‐/‐ mice

Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1‐/‐ and Epac2‐/‐ mice exhibit a delayed anti‐natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split‐opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the knockout mice fed with Na+ deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker, ESI‐05 and Epac1&2 blocker, ESI‐09 decreased ENaC activity in EpacWT mice kept on Na+ deficient diet but not on the regular diet. ESI‐09 injections led to natriuresis in EpacWT mice on Na+ deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate non‐redundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC over‐activation.

Epac1-/- and Epac2-/- mice exhibit deficient epithelial Na+ channel regulation and impaired urinary Na+ conservation.

Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological, and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1–/– and Epac2–/– mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the KO mice fed with a Na+-deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker ESI-05 and Epac1&2 blocker ESI-09 decreased ENaC activity in Epac WT mice kept on the Na+-deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in Epac WT mice on the Na+-deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate similar but nonredundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC overactivation.

CAMP triggers Na+ absorption by distal airway surface epithelium in cystic fibrosis swine.

A controversial hypothesis pertaining to cystic fibrosis (CF) lung disease is that the CF transmembrane conductance regulator (CFTR) channel fails to inhibit the epithelial Na+ channel (ENaC), yielding increased Na+ reabsorption and airway dehydration. We use a non-invasive self-referencing Na+-selective microelectrode technique to measure Na+ transport across individual folds of distal airway surface epithelium preparations from CFTR-/- (CF) and wild-type (WT) swine. We show that, under unstimulated control conditions, WT and CF epithelia exhibit similar, low rates of Na+ transport that are unaffected by the ENaC blocker amiloride. However, in the presence of the cyclic AMP (cAMP)-elevating agents forskolin+IBMX (isobutylmethylxanthine), folds of WT tissues secrete large amounts of Na+, while CFTR-/- tissues absorb small, but potentially important, amounts of Na+. In cAMP-stimulated conditions, amiloride inhibits Na+ absorption in CFTR-/- tissues but does not affect secretion in WT tissues. Our results are consistent with the hypothesis that ENaC-mediated Na+ absorption may contribute to dehydration of CF distal airways.

WITHDRAWN: Liddle syndrome: a case report

Liddle syndrome is an autosomal dominant hereditary disease caused by a single gene mutation. Typical clinical manifestations are early-onset hypertension and hypokalaemia and can be treated using ENaC blockers (amiloride and aminopterin). This report describes a 17-year-old male with hypertension and hypokalaemia. We performed a Captopril inhibition test and a postural stimulation test for the diagnosis and typing of primary aldosteronism. The serum renin was low, and aldosterone was high, so the patient was initially misdiagnosed as primary aldosteronism. After a genetic analysis, a diagnosis of Liddle syndrome was made due to the presence of an SCNN1B p.Pro617Ser mutation. After diagnosis, the patient was administered one tablet of amiloride twice a day (each tablet contains 2.5mg of amiloride hydrochloride and 25mg of hydrochlorothiazide 25mg). The patient's blood pressure (average of 120-135/70-80mmHg) and serum potassium levels (3.6-4.0mmol/L) returned to normal and were well-controlled after treatment. The patient is an atypical case of Liddle syndrome; genetic analysis is helpful and essential for diagnosis.

LIDDLE SYNDROME – DETECTION OF NOVEL MUTATION IN A FAMILY WITH HYPERTENSION

Abstract Objective: Liddle syndrome is a rare monogenic form of arterial hypertension with autosomal dominant inheritance. It is caused by mutation of genes coding one of three subunits of epithelial sodium channel (ENaC), which is located in distal nephron. Under physiological conditions, its expression and activity is regulated to maintain fluid and electrolytes homeostasis by reabsorbing Na+. Mutated channel cannot be degraded and therefore, it is accumulated on cell membrane; this results in enhanced sodium reabsorption. The most common clinical presentation of Liddle syndrome is early onset of hypertension, hypokalemia, suppressed plasma renin activity and low plasma aldosterone level. Up to now, 36 mutations causing Liddle syndrome were described. Design and method: Genetic assessment was performed in a Czech family of three generations (n = 14, see Figure) where hypertension and tendency to hypokalemia was present in several members. We used PCR amplification of specific alleles of SCNN1B and SCNN1G genes which was followed by Sanger sequencing. Results: We present a novel mutation causing Liddle syndrome: Tyr604∗ in beta-subunit of epithelial sodium channel. This mutation is supposed to generate a premature stop codon at position 604, resulting in truncated beta-subunit of EnaC. It was found in 7 members of the family: I-1, II-1 and 2, and III-1 to 4. Phenotypes of these subjects were different: from severe hypertension and hypokalemia (subjects I-1 and III-3, manifestation in 10 and 5 years, respectively) to normotension with normal potassium levels (III-1 and 2, now aged 8 and 10 years, respectively); however, all the seven subjects with mutation had hypoaldosteronism (serum level < 0.13 ng/ml). Conclusions: A new mutation causing Liddle syndrome is described. It is manifested with different severity and mild forms that are not manifested in early childhood exist; thus, Liddle syndrome may be more frequent among adult hypertensive subjects than currently supposed. Proper diagnosis is important as the patients respond well to treatment with ENaC blocker amiloride.

Instant activation of TRP channels by NH4 + promotes neuronal bursting and glutamate spikes in CA1 neurons.

Ammonia () is a by-product of cell metabolism and may elicit subcellular effects with specific physiological responses. Chronic effects have been implicated in several neurological diseases and attributed to persistent elevation in blood ammonia levels transferred to the brain. In previous studies the activities of neurons and astrocytes have been examined at ammonia concentrations an order of magnitude higher than measured in the blood. The effects developed within several minutes. Here we focused upon acute responses of neurons to ammonia and whether they may occur at much lower doses. To this end, we combined patch-clamp in CA1 neurons with glutamate imaging in hippocampal slices. Particular attention was paid to the Rett syndrome that has been originally attributed to hyperammonemia. We compared the responses in the wild-type (WT) and model Rett mice (MECP2-null, RTT) to ammonia doses from 0.3 mM on. In both preparations promptly depolarized neurons and increased the ambient glutamate. The bursting activity emerged in WT and it was augmented in RTT. Searching for subcellular mechanisms we examined possible modulation of ion channels by ammonia. We did not find any changes in HCN- and Ca2+ currents, which substantially contribute to the bursting activity. The non-selective cation channels were markedly potentiated by ammonia. ASIC channels had a major contribution to the augmentation of neuronal activity by ammonia. Interestingly, their general blocker amiloride (100 μM) moderately excited CA1 cells akin to . In its presence subsequent ammonia effects were markedly compromised. Blockade of TRPC1 channels partially occluded effects. ASIC and TRPC1 blockers decreased the amplitude of excitatory postsynaptic currents (EPSC) and neuronal bursts, congruent with a postsynaptic location of the channels. Inhibition of TRPV1 channels potentiated the responses to . EPSC amplitudes did not change, but the frequency decreased, indicating presynaptic effects. All extracellular actions were observed at concentrations as low as 0.3 mM and the neurons reacted immediately after ammonia arrived the slice. We propose that a brief augmentation of neuronal activity by may occur either spontaneously during arousal or induced by inhalation of smelling salts.

Mechanisms of sodium retention in nephrotic syndrome.

Proteinuria in nephrotic syndrome is associated with sodium retention and edema. Recent studies from mice, rats and humans have shown that the sodium retention depends on urinary serine proteases and that it can be mitigated by blockers (amiloride, triamterene) of the epithelial sodium channel ENaC. The present review outlines the mechanisms of protease-stimulated sodium retention during proteinuric diseases. Inhibition of protease activity in nephrotic mice using aprotinin alleviates sodium retention. From both human and mice studies, an increased proteolytic cleavage of the γENaC subunit plays a role in ENaC activation. In animal models, urokinase-plasmin contributes but not as sole mediators of sodium retention. Across experimental models, human case reports and small intervention trials, amiloride alleviates nephrotic sodium retention and low-renin hypertension with high efficacy. Although the exact mechanisms for proteolytic ENaC activation are not resolved, multiple, redundant proteases are involved. Experimental and clinical evidence indicate that aberrant proteolytic ENaC activation is a primary driver of sodium retention in nephrotic syndrome and contributes to hypertension in conditions with low-grade proteinuria. Thus, we foresee increased and personalized use of amiloride treatment of nephrotic and other proteinuric disease patients with associated sodium retention and hypertension.

Segregated Expression of ENaC Subunits in Taste Cells.

Salt taste is one of the 5 basic taste qualities. Depending on the concentration, table salt is perceived either as appetitive or aversive, suggesting the contribution of several mechanisms to salt taste, distinguishable by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. A taste-specific knockout of the α-subunit of the ENaC revealed the relevance of this polypeptide for low-salt transduction, whereas the response to other taste qualities remained normal. The fully functional ENaC is composed of α-, β-, and γ-subunits. In taste tissue, however, the precise constitution of the channel and the cell population responsible for detecting table salt remain uncertain. In order to examine the cells and subunits building the ENaC, we generated mice carrying modified alleles allowing the synthesis of green and red fluorescent proteins in cells expressing the α- and β-subunit, respectively. Fluorescence signals were detected in all types of taste papillae and in taste buds of the soft palate and naso-incisor duct. However, the lingual expression patterns of the reporters differed depending on tongue topography. Additionally, immunohistochemistry for the γ-subunit of the ENaC revealed a lack of overlap between all potential subunits. The data suggest that amiloride-sensitive recognition of table salt is unlikely to depend on the classical ENaCs formed by α-, β-, and γ-subunits and ask for a careful investigation of the channel composition.

A Na+ leak channel cloned from Trichoplax adhaerens extends extracellular pH and Ca2+ sensing for the DEG/ENaC family close to the base of Metazoa

Acid-sensitive ion channels belonging to the degenerin/epithelial sodium channel (DEG/ENaC) family activate in response to extracellular protons and are considered unique to deuterostomes. However, sensitivity to pH/protons is more widespread, where, for example, human ENaC Na+ leak channels are potentiated and mouse BASIC and Caenorhabditis elegans ACD-1 Na+ leak channels are blocked by extracellular protons. For many DEG/ENaC channels, extracellular Ca2+ ions modulate gating, and in some cases, the binding of protons and Ca2+ is interdependent. Here, we functionally characterize a DEG/ENaC channel from the early-diverging animal Trichoplax adhaerens, TadNaC6, that conducts Na+-selective leak currents in vitro sensitive to blockade by both extracellular protons and Ca2+. We determine that proton block is enhanced in low external Ca2+ concentration, whereas calcium block is enhanced in low external proton concentration, indicative of competitive binding of these two ligands to extracellular sites of the channel protein. TadNaC6 lacks most determinant residues for proton and Ca2+ sensitivity in other DEG/ENaC channels, and a mutation of one conserved residue (S353A) associated with Ca2+ block in rodent BASIC channels instead affected proton sensitivity, all indicative of independent evolution of H+ and Ca2+ sensitivity. Strikingly, TadNaC6 was potently activated by the general DEG/ENaC channel blocker amiloride, a rare feature only reported for the acid-activated channel ASIC3. The sequence and structural divergence of TadNaC6, coupled with its noncanonical functional features, provide unique opportunities for probing the proton, Ca2+, and amiloride regulation of DEG/ENaC channels and insight into the possible core-gating features of ancestral ion channels.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500μM), 60% by ZD7288 (100μM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

Involvement of the epithelial sodium channel in initiation of mechanically evoked swallows in anaesthetized rats.

Afferents carried by the superior laryngeal nerve play a primary role in the initiation of laryngeal mechanically evoked swallows in anaesthetized rats. Amiloride and its analogues inhibit swallowing evoked by mechanical stimulation, but not swallowing evoked by chemical and electrical stimulation. The epithelial sodium channel is probably involved in the initiation of laryngeal mechanically evoked swallows. The swallowing reflex plays a critical role in airway protection. Because impaired laryngeal mechanosensation is associated with food bolus aspiration, it is important to know how the laryngeal sensory system regulates swallowing initiation. This study was performed to clarify the neuronal mechanism of mechanically evoked swallows. Urethane-anaesthetized Sprague-Dawley male rats were used. A swallow was identified by activation of the suprahyoid and thyrohyoid muscles on electromyography. The swallowing threshold was measured by von Frey filament and electrical stimulation of the larynx. The number of swallows induced by upper airway distension and capsaicin application (0.03nmol, 3μl) to the vocal folds was counted. The effects of topical application (0.3-30nmol, 3μl) of the epithelial sodium channel (ENaC) blocker amiloride and its analogues (benzamil and dimethylamiloride), acid-sensing ion channel (ASIC) inhibitors (mambalgine-1 and diminazene) and gadolinium to the laryngeal mucosa on swallowing initiation were evaluated. A nerve transection study indicated that afferents carried by the superior laryngeal nerve play a primary role in the initiation of laryngeal mechanically evoked swallows. The mechanical threshold of swallowing was increased in a dose-dependent manner by amiloride and its analogues and gadolinium, but not by ASIC inhibitors. The number of swallows by upper airway distension was significantly decreased by benzamil application. However, the initiation of swallows evoked by capsaicin and electrical stimulation was not affected by benzamil application. We speculate that the ENaC is involved in the initiation of laryngeal mechanically evoked swallows.

The Na+/H+-Exchanger NHE1 Regulates Extra- and Intracellular pH and Nimodipine-sensitive [Ca2+]i in the Suprachiasmatic Nucleus

The central clock in the suprachiasmatic nucleus (SCN) has higher metabolic activity than extra-SCN areas in the anterior hypothalamus. Here we investigated whether the Na+/H+ exchanger (NHE) may regulate extracellular pH (pHe), intracellular pH (pHi) and [Ca2+]i in the SCN. In hypothalamic slices bathed in HEPES-buffered solution a standing acidification of ~0.3 pH units was recorded with pH-sensitive microelectrodes in the SCN but not extra-SCN areas. The NHE blocker amiloride alkalinised the pHe. RT-PCR revealed mRNA for plasmalemmal-type NHE1, NHE4, and NHE5 isoforms, whereas the NHE1-specific antagonist cariporide alkalinised the pHe. Real-time PCR and western blotting failed to detect day-night variation in NHE1 mRNA and protein levels. Cariporide induced intracellular acidosis, increased basal [Ca2+]i, and decreased depolarisation-induced Ca2+ rise, with the latter two effects being abolished with nimodipine blocking the L-type Ca2+ channels. Immunofluorescent staining revealed high levels of punctate colocalisation of NHE1 with serotonin transporter (SERT) or CaV1.2, as well as triple staining of NHE1, CaV1.2, and SERT or the presynaptic marker Bassoon. Our results indicate that NHE1 actively extrudes H+ to regulate pHi and nimodipine-sensitive [Ca2+]i in the soma, and along with CaV1.2 may also regulate presynaptic Ca2+ levels and, perhaps at least serotonergic, neurotransmission in the SCN.