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.

Luteapyrone, a Novel Æ´-Pyrone Isolated from the Filamentous Fungus Metapochonia lutea.

In the process of screening for new bioactive microbial metabolites we found a novel Æ´-pyrone derivative for which we propose the trivial name luteapyrone, in a recently described microscopic filamentous fungus, Metapochonia lutea BiMM-F96/DF4. The compound was isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structure was elucidated by NMR and LC-MS. The new compound was found to be non-cytotoxic against three mammalian cell lines (HEK 263, KB-3.1 and Caco-2). Similarly, no antimicrobial activity was observed in tested microorganisms (gram positive and negative bacteria, yeast and fungi).

The role of the nAChR subunits α5, ß2, and ß4 on synaptic transmission in the mouse superior cervical ganglion.

Our previous immunoprecipitation analysis of nicotinic acetylcholine receptors (nAChRs) in the mouse superior cervical ganglion (SCG) revealed that approximately 55%, 24%, and 21% of receptors are comprised of α3ß4, α3ß4α5, and α3ß4ß2 subunits, respectively. Moreover, mice lacking ß4 subunits do not express α5-containing receptors but still express a small number of α3ß2 receptors. Here, we investigated how synaptic transmission is affected in the SCG of α5ß4-KO and α5ß2-KO mice. Using an ex vivo SCG preparation, we stimulated the preganglionic cervical sympathetic trunk and measured compound action potentials (CAPs) in the postganglionic internal carotid nerve. We found that CAP amplitude was unaffected in α5ß4-KO and α5ß2-KO ganglia, whereas the stimulation threshold for eliciting CAPs was significantly higher in α5ß4-KO ganglia. Moreover, intracellular recordings in SCG neurons revealed no difference in EPSP amplitude. We also found that the ganglionic blocking agent hexamethonium was the most potent in α5ß4-KO ganglia (IC50 : 22.1 µmol/L), followed by α5ß2-KO (IC50 : 126.7 µmol/L) and WT ganglia (IC50 : 389.2 µmol/L). Based on these data, we estimated an IC50 of 568.6 µmol/L for a receptor population consisting solely of α3ß4α5 receptors; and we estimated that α3ß4α5 receptors comprise 72% of nAChRs expressed in the mouse SCG. Similarly, by measuring the effects of hexamethonium on ACh-induced currents in cultured SCG neurons, we found that α3ß4α5 receptors comprise 63% of nAChRs. Thus, in contrast to our results obtained using immunoprecipitation, these data indicate that the majority of receptors at the cell surface of SCG neurons consist of α3ß4α5.

Phosphorylation of cardiac voltage-gated sodium channel: Potential players with multiple dimensions.

Cardiomyocytes are highly coordinated cells with multiple proteins organized in micro domains. Minor changes or interference in subcellular proteins can cause major disturbances in physiology. The cardiac sodium channel (NaV 1.5) is an important determinant of correct electrical activity in cardiomyocytes which are localized at intercalated discs, T-tubules and lateral membranes in the form of a macromolecular complex with multiple interacting protein partners. The channel is tightly regulated by post-translational modifications for smooth conduction and propagation of action potentials. Among regulatory mechanisms, phosphorylation is an enzymatic and reversible process which modulates NaV 1.5 channel function by attaching phosphate groups to serine, threonine or tyrosine residues. Phosphorylation of NaV 1.5 is implicated in both normal physiological and pathological processes and is carried out by multiple kinases. In this review, we discuss and summarize recent literature about the (a) structure of NaV 1.5 channel, (b) formation and subcellular localization of NaV 1.5 channel macromolecular complex, (c) post-translational phosphorylation and regulation of NaV 1.5 channel, and (d) how these phosphorylation events of NaV 1.5 channel alter the biophysical properties and affect the channel during disease status. We expect, by reviewing these aspects will greatly improve our understanding of NaV 1.5 channel biology, physiology and pathology, which will also provide an insight into the mechanism of arrythmogenesis at molecular level.

Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxins.

Destruxins, secondary metabolites of entomopathogenic fungi, exert a wide variety of interesting characteristics ranging from antiviral to anticancer effects. Although their mode of action was evaluated previously, the molecular mechanisms of resistance development are unknown. Hence, we have established destruxin-resistant sublines of HCT116 colon carcinoma cells by selection with the most prevalent derivatives, destruxin (dtx)A, dtxB and dtxE. Various cell biological and molecular techniques were applied to elucidate the regulatory mechanisms underlying these acquired and highly stable destruxin resistance phenotypes. Interestingly, well-known chemoresistance-mediating ABC efflux transporters were not the major players. Instead, in dtxA- and dtxB-resistant cells a hyper-activated mevalonate pathway was uncovered resulting in increased de-novo cholesterol synthesis rates and elevated levels of lanosterol, cholesterol as well as several oxysterol metabolites. Accordingly, inhibition of the mevalonate pathway at two different steps, using either statins or zoledronic acid, significantly reduced acquired but also intrinsic destruxin resistance. Vice versa, cholesterol supplementation protected destruxin-sensitive cells against their cytotoxic activity. Additionally, an increased cell membrane adhesiveness of dtxA-resistant as compared to parental cells was detected by atomic force microscopy. This was paralleled by a dramatically reduced ionophoric capacity of dtxA in resistant cells when cultured in absence but not in presence of statins. Summarizing, our results suggest a reduced ionophoric activity of destruxins due to cholesterol-mediated plasma membrane re-organization as molecular mechanism underlying acquired destruxin resistance in human colon cancer cells. Whether this mechanism might be valid also in other cell types and organisms exposed to destruxins e.g. as bio-insecticides needs to be evaluated.

Identification of phosphorylation sites and binding pockets for modulation of NaV 1.5 channel by Fyn tyrosine kinase.

Cardiac sodium channel NaV 1.5 is the predominant form of sodium channels in cardiomyocytes, which exists as a macromolecular complex and interacts with multiple protein partners. Fyn kinase is one of the interacting proteins which colocalize, phosphorylate and modulate the NaV 1.5 channel. To elaborate this interaction we created expression vectors for the N-terminal, intracellular loop, and C-terminal regions of the NaV 1.5 channel, to express in HEK-293 cells. By co-immunoprecipitation and anti-phosphotyrosine blotting, we identified proline-rich binding sites for Fyn kinase in the N-terminal, IC-loopi-ii and C-terminal. After binding, Fyn kinase phosphorylates tyrosine residues present in the N- and C-terminal, which produce a depolarizing shift of 7 mV in fast inactivation. The functional relevance of these binding and phosphorylation sites was further underpinned by creating full length mutants masking these sites sequentially. An activation and inactivation curves were recorded with or without co-expressed Fyn kinase which indicates that phosphorylation of tyrosine residues at positions 68, 87, 112 in the N-terminal and at positions 1811 and 1889 in the C-terminal creates a depolarizing shift in fast inactivation of NaV 1.5 channel.

The Natural Fungal Metabolite Beauvericin Exerts Anticancer Activity In Vivo: A Pre-Clinical Pilot Study.

Recently, in vitro anti-cancer properties of beauvericin, a fungal metabolite were shown in various cancer cell lines. In this study, we assessed the specificity of this effect by comparing beauvericin cytotoxicity in malignant versus non-malignant cells. Moreover, we tested in vivo anticancer effects of beauvericin by treating BALB/c and CB-17/SCID mice bearing murine CT-26 or human KB-3-1-grafted tumors, respectively. Tumor size and weight were measured and histological sections were evaluated by Ki-67 and H/E staining as well as TdT-mediated-dUTP-nick-end (TUNEL) labeling. Beauvericin levels were determined in various tissues and body fluids by LC-MS/MS. In addition to a more pronounced activity against malignant cells, we detected decreased tumor volumes and weights in beauvericin-treated mice compared to controls in both the allo- and the xenograft model without any adverse effects. No significant differences were detected concerning percentages of proliferating and mitotic cells in tumor sections from treated and untreated mice. However, a significant increase of necrotic areas within whole tumor sections of beauvericin-treated mice was found in both models corresponding to an enhanced number of TUNEL-positive, i.e., apoptotic, cells. Furthermore, moderate beauvericin accumulation was detected in tumor tissues. In conclusion, we suggest beauvericin as a promising novel natural compound for anticancer therapy.

TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B.

Previous in vitro studies have indicated that tumor necrosis factor (TNF) activates amiloride-sensitive epithelial sodium channel (ENaC) current through its lectin-like (TIP) domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide), showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the α-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only ß-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αßγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B), a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αßγ-ENaC, whereas membrane abundance varied between mutants. Solnatide restored function in α-ENaC frameshift mutants to current density levels of wild type ENaC or higher despite their lacking a binding site for solnatide, previously located to the region between TM2 and the C-terminus of the α-subunit. TNF similarly restored current density to wild type levels in the mutant αR448fs. Activation of ßγ-ENaC may contribute to this moderate current enhancement, but whatever the mechanism, experimental data indicate that solnatide could be a new strategy to treat PHA1B.

Voltage gated ion channels blockade is the underlying mechanism of BIMU8 induced cardiotoxicity.

BIMU8 is a 5-HT4a receptor agonist and used as an experimental drug to counteract opioid induced respiratory depression. In preliminary experiments serious disturbances in ECG were observed in anesthetized rabbits which prompted us to explore the underlying cause of BIMU8 induced abnormal changes in ECG recordings. Electrophysiological experiments were performed on HEK-293 cells expressing hERG, CaV1.2 and NaV1.5 ion channels. In whole-cell recordings BIMU8 effectively blocked these three channels, with IC50 values of 0.06±0.05, 1.46±0.26 and 4.66±0.58µM for hERG, NaV1.5 and CaV1.2, respectively. Additionally it also produced a hyperpolarizing shift of 3.27mV in half maximal activation and 12.87mV in fast inactivation of NaV1.5 channel. These experimental findings indicate that BIMU8 is a potent blocker of hERG, NaV1.5 and CaV1.2 cardiac ion channels thus revealing its proarrhythmic potential.

Restoration of Epithelial Sodium Channel Function by Synthetic Peptides in Pseudohypoaldosteronism Type 1B Mutants.

The synthetically produced cyclic peptides solnatide (a.k.a. TIP or AP301) and its congener AP318, whose molecular structures mimic the lectin-like domain of human tumor necrosis factor (TNF), have been shown to activate the epithelial sodium channel (ENaC) in various cell- and animal-based studies. Loss-of-ENaC-function leads to a rare, life-threatening, salt-wasting syndrome, pseudohypoaldosteronism type 1B (PHA1B), which presents with failure to thrive, dehydration, low blood pressure, anorexia and vomiting; hyperkalemia, hyponatremia and metabolic acidosis suggest hypoaldosteronism, but plasma aldosterone and renin activity are high. The aim of the present study was to investigate whether the ENaC-activating effect of solnatide and AP318 could rescue loss-of-function phenotype of ENaC carrying mutations at conserved amino acid positions observed to cause PHA1B. The macroscopic Na+ current of all investigated mutants was decreased compared to wild type ENaC when measured in whole-cell patch clamp experiments, and a great variation in the membrane abundance of different mutant ENaCs was observed with Western blotting experiments. However, whatever mechanism leads to loss-of-function of the studied ENaC mutations, the synthetic peptides solnatide and AP318 could restore ENaC function up to or even higher than current levels of wild type ENaC. As therapy of PHA1B is only symptomatic so far, the peptides solnatide and AP318, which directly target ENaC, are promising candidates for the treatment of the channelopathy-caused disease PHA1B.

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

Differential modulation of fast inactivation in cardiac sodium channel splice variants by Fyn tyrosine kinase.

BACKGROUND/AIMS: Post-translational modifications such as phosphorylation and dephosphorylation can finely tune the function of ion channels. Nav1.5 is the main sodium channel in human hearts and alternative splicing of the transcript generates two major splice variants, characterized by the presence (Q-pre) or absence (Q-del) of glutamine at position 1077. In the heart, both the Nav1.5 channel and Fyn tyrosine kinase are colocalized at adherens junctions. This study aimed to investigate the modulation of the aforementioned splice variants by Fyn tyrosine kinase. METHODS AND RESULTS: Q-del and Q-pre were transiently expressed alone, with catalytically active Fyn kinase (FynKa) or with a catalytically dead Fyn kinase (FynKd). Co-expression of Nav1.5 channel splice variants and Fyn kinase was confirmed by Western blotting and their Interaction was established by co-immunoprecipitation experiments. The enzymatic activity of Fyn kinase and phosphorylation of Nav1.5 channel were ascertained by immunoprecipitation and anti-phosphotyrosine immunoblotting. Whole-cell ionic currents were recorded in patch clamp experiments to examine the modulation of Nav1.5 channel variants by Fyn kinase, which indicated a hyperpolarizing shift of 9.68 mV in fast inactivation of Q-del. In contrast, a depolarizing shift of 8.77 mV in fast inactivation was observed in the case of Q-pre, while activation curves remained unaltered for both splice variants. This differential modulation in fast inactivation was further assessed by mutating tyrosine 1495 to phenylalanine in the inactivation loop, which completely removed the modulatory effect of Fyn kinase in Q-pre splice variant, while in Q-del variant hyperpolarizing shift in fast inactivation was reduced to 4.74 mV. Finally, the modulatory effect of Fyn kinase was compensated at a mid-value of 94.63 ± 0.34, when both splice variants were co-expressed at a normal physiological ratio. CONCLUSION: Q-del and Q-pre were differentially modulated by Fyn kinase, and this fine modification resulted in smooth electrical activity in the heart.

Glycosylation-dependent activation of epithelial sodium channel by solnatide.

Dysfunction of the epithelial sodium channel (ENaC), which regulates salt and water homeostasis in epithelia, causes several human pathological conditions, including pulmonary oedema. This is a potentially lethal complication of acute lung injury at least partially caused by dysfunctional alveolar liquid clearance, which in turn impairs alveolar gas exchange. Solnatide (named TIP-peptide, AP301), a 17 residue peptide mimicking the lectin-like domain of TNF has been shown to activate ENaC in several experimental animal models of acute lung injury and is being evaluated as a potential therapy for pulmonary oedema. The peptide has recently completed phase 1 and 2a clinical trials. In this study, we identify a glycosylation-dependent mechanism that preserves ENaC function and expression. Since our previous data suggested that the pore-forming subunits of ENaC are essential for maximal current activation by solnatide, we performed single- and multi-N-glycosylation site mutations in αN232,293,312,397,511Q- and δN166,211,384Q-subunits, in order to identify crucial residues for interaction with solnatide within the extracellular loop of the channel. Additionally, we generated αL576X and αN232,293,312,397,511Q,L576X deletion mutants of ENaC-α, since we have previously demonstrated that the carboxy terminal domain of this subunit is also involved in its interaction with solnatide. In cells expressing αN232,293,312,397,511Q,L576Xßγ-hENaC or δN166,311,384Q,D552Xßγ-hENaC activation by solnatide, as measured in whole cell patch clamp mode, was completely abolished, whereas it was attenuated in αL576Xßγ-hENaC- and δD552Xßγ-hENaC-expressing cells. Taken together, our findings delineate an N-glycan dependent interaction between the TIP-peptide and ENaC leading to normalization of both sodium and fluid absorption in oedematous alveoli to non-oedematous levels.

The naturally born fusariotoxin enniatin B and sorafenib exert synergistic activity against cervical cancer in vitro and in vivo.

During the last decades substantial progress has been made in developing systemic cancer therapy. However, tumors are frequently intrinsically resistant against structurally and mechanistically unrelated drugs. Thus, it is of predominant interest to overcome drug resistance and to encourage the research for novel chemotherapeutic approaches. Recently, we have introduced enniatins, naturally occurring cyclohexadepsipeptides produced by filamentous fungi of the genus Fusarium, as potential anticancer drugs. Here, we expend this approach by demonstrating antiangiogenic properties for enniatin B (Enn B) indicated by a strong inhibition of human endothelial cell migration and tube formation. Moreover, combination of Enn B with the clinically approved multi-kinase inhibitor sorafenib (Sora) displayed profound synergistic in vitro and in vivo anticancer effects against cervical cancer. Subsequent studies showed that this strong synergism is accompanied by a marked increase in mitochondrial injury and apoptosis induction reflected by mitochondrial membrane depolarization, caspase-7 activation, and subsequent cleavage of PARP. Additionally, cells were shown to stop DNA synthesis and accumulate in S and G2/M phase of the cell cycle. The multifaceted characteristics underlying this strong synergism were suggested to be based on interference with the p38 MAPK as well as the ERK signaling pathways. Finally, also in vivo studies revealed that the combination treatment is distinctly superior to single drug treatments against the KB-3-1 cervix carcinoma xenograft model. Taken together, our data confirm the anticancer benefits of the naturally occurring fusariotoxin Enn B and further present Enn B/Sora as a novel combination strategy especially for the treatment of cervical cancer.

A C-terminal membrane anchor affects the interactions of prion proteins with lipid membranes.

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP(C) into pathogenic PrP(Sc). Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23-231, FL_PrP), N-terminally truncated PrP (residues 90-231, T_PrP), and PrP missing its central hydrophobic region (Δ105-125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.

Calpain-mediated integrin deregulation as a novel mode of action for the anticancer gallium compound KP46.

On the basis of enhanced tumor accumulation and bone affinity, gallium compounds are under development as anticancer and antimetastatic agents. In this study, we analyzed molecular targets of one of the lead anticancer gallium complexes [KP46, Tris(8-quinolinolato)gallium(III)] focusing on colon and lung cancer. Within a few hours, KP46 treatment at low micromolar concentrations induced cell body contraction and loss of adhesion followed by prompt cell decomposition. This rapid KP46-induced cell death lacked classic apoptotic features and was insensitive toward a pan-caspase inhibitor. Surprisingly, however, it was accompanied by upregulation of proapoptotic Bcl-2 family members. Furthermore, a Bax- but not a p53-knockout HCT-116 subline exhibited significant KP46 resistance. Rapid KP46-induced detachment was accompanied by downregulation of focal adhesion proteins, including several integrin subunits. Loss of integrin-ß1 and talin plasma membrane localization corresponded to reduced binding of RGD (Arg-Gly-Asp) peptides to KP46-treated cells. Accordingly, KP46-induced cell death and destabilization of integrins were enhanced by culture on collagen type I, a major integrin ligand. In contrast, KP46-mediated adhesion defects were partially rescued by Mg(2+) ions, promoting integrin-mediated cell adhesion. Focal adhesion dynamics are regulated by calpains via cleavage of multiple cell adhesion molecules. Cotreatment with the cell-permeable calpain inhibitor PD150606 diminished KP46-mediated integrin destabilization and rapid cell death induction. KP46 treatment distinctly inhibited HCT-116 colon cancer xenograft in vivo by causing reduced integrin plasma membrane localization, tissue disintegration, and intense tumor necrosis. This study identifies integrin deregulation via a calpain-mediated mechanism as a novel mode of action for the anticancer gallium compound KP46.

A novel tumor necrosis factor-mediated mechanism of direct epithelial sodium channel activation.

RATIONALE: Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS: TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Mechanism of action of novel lung edema therapeutic AP301 by activation of the epithelial sodium channel.

AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human tumor necrosis factor lectin-like domain (TIP domain) sequence, is currently being developed as a treatment for lung edema and has been shown to reduce extravascular lung water and improve lung function in mouse, rat, and pig models. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial Na⁺ channel (ENaC) and nonselective cyclic-nucleotide-gated cation channels creates the major driving force for reabsorption of water through the alveolar epithelium in addition to other ion channels such as potassium and chloride channels. AP301 can increase amiloride-sensitive current in A549 cells as well as in freshly isolated type II alveolar epithelial cells from different species. ENaC is expressed endogenously in all of these cell types. Consequently, this study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells as well as in human embryonic kidney cells and Chinese hamster ovary cells heterologously expressing human ENaC subunits (α, ß, γ, and δ) was measured in patch clamp experiments. The congener TIP peptide AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] activated ENaC by increasing single-channel open probability. AP301 increased current in proteolytically activated (cleaved) but not near-silent (uncleaved) ENaC in a reversible manner. αßγ- or δßγ-ENaC coexpression was required for maximal activity. No increase in current was observed after deglycosylation of extracellular domains of ENaC. Thus, our data suggest that the specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).

Sex-specific-differences in cardiometabolic risk in type 1 diabetes: a cross-sectional study.

BACKGROUND: Little is known about the impact of sex-specific differences in the management of type 1 diabetes (T1DM). Thus, we evaluated the influence of gender on risk factors, complications, clinical care and adherence in patients with T1DM. METHODS: In a cross-sectional study, sex-specific disparities in glycaemic control, cardiovascular risk factors, diabetic complications, concomitant medication use and adherence to treatment recommendations were evaluated in 225 consecutive patients (45.3% women) who were comparable with respect to age, diabetes duration, and body mass index. RESULTS: Although women with T1DM had a higher total cholesterol than men, triglycerides were higher in obese men and males with HbA1c>7% than in their female counterparts. No sex differences were observed in glycaemic control and in micro- or macrovascular complications. However, the subgroup analysis showed that nephropathy was more common in obese men, hyperlipidaemic women and all hypertensive patients, whereas peripheral neuropathy was more common in hyperlipidaemic women. Retinopathy was found more frequently in women with HbA1c>7%, obese men and in both sexes with a long duration of diabetes. The multivariate analysis revealed that microvascular complications were associated with the duration of disease and BMI in both sexes and with hyperlipidaemia in males. The overall adherence to interventions according to the guidelines was higher in men than in women. This adherence was concerned particularly with co-medication in patients diagnosed with hypertension, aspirin prescription in elderly patients and the achievement of target lipid levels following the prescription of statins. CONCLUSIONS: Our data showed sex differences in lipids and overweight in patients with T1DM. Although glycaemic control and the frequency of diabetic complications were comparable between the sexes, the overall adherence to guidelines, particularly with respect to the prescription of statins and aspirin, was lower in women than in men.

AP301, a synthetic peptide mimicking the lectin-like domain of TNF, enhances amiloride-sensitive Na(+) current in primary dog, pig and rat alveolar type II cells.

Pulmonary permeability oedema is a frequent complication in a number of life-threatening lung conditions, such as ALI and ARDS. Apart from ventilation strategies, no specific therapy yet exists for treatment of these potentially fatal illnesses. The oedema-reducing capacity of the lectin-like domain of TNF (TIP) and of synthetic peptides, mTIP and hTIP, which mimic the TIP domain of mouse and human TNF, have been demonstrated in various studies in rodents. Cell-based electrophysiological studies have revealed that the alveolar fluid clearing capacity of TNF and the TIP peptides is due to activation of the amiloride-sensitive Na(+) current in alveolar epithelial cells and that the primary site of action is on the apical side of these cells. AP301, a synthetic cyclic peptide mimicking the TIP domain of human TNF is currently undergoing clinical trials as a therapy for pulmonary permeability oedema. AP301 has been shown to improve alveolar liquid clearance and lung function in a porcine model of ALI. For non-clinical regulatory assessment, dog, pig and rat are standard animal models; accordingly, pre-clinical toxicological and pharmacological safety studies have been conducted with AP301 in dogs and rats. Hitherto, no studies have assessed the pharmacodynamic effect of AP301 on primary canine or porcine type II AEC. The current study describes the effect of AP301 on the amiloride-sensitive Na(+) current in type II AEC isolated from dog, pig and rat lungs. In whole cell patch clamp experiments with dog type II AEC, an increase in the amiloride-sensitive Na(+) current from 3.7 pA to 49.4 pA was observed in the presence of AP301; in pig type II AEC, an increase from 10.0 pA to 159.6 pA was observed, and in rat AEC, from 6.9 pA to 62.4 pA. In whole cell patch clamp experiments in A549 cells, AP301-induced enhancement of the amiloride-sensitive current was eliminated when Na(+) in the bath solution was replaced with N-methyl-d-glucamine (NMDG), and when the cells were pre-incubated with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), an inhibitor of ENaC, but enhancement was unaffected by addition of cyclic nucleotide-gated (CNG) channel inhibitors Zn(2+) or l-cis-diltiazem prior to AP301. These results provide strong evidence that AP301 activates the amiloride-sensitive Na(+) current through ENaC in type II AEC from dog, pig and rat. To our knowledge, this is the first cell-based analysis of the oedema-clearing effect of AP301 observed in the porcine model of pulmonary oedema. Furthermore, the results validate the dog and pig models in non-clinical assessment of AP301.