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RELA haploinsufficiency is a recently described autoinflammatory condition presenting with intermittent fevers and mucocutaneous ulcerations. The RELA gene encodes the p65 protein, one of five NF-κB family transcription factors. As RELA is an essential regulator of mucosal homeostasis, haploinsufficiency leads to decreased NF-κB signaling which promotes TNF-driven mucosal apoptosis with impaired epithelial recovery. Thus far, only eight cases have been reported in the literature. Here, we report four families with three novel and one previously described pathogenic variant in RELA. These four families included 23 affected individuals for which genetic testing was available in 16. Almost half of these patients had been previously diagnosed with more common rheumatologic entities (such as Behcet's Disease; BD) prior to the discovery of their pathogenic RELA variants. The most common clinical features were orogenital ulcers, rash, joint inflammation, and fever. The least common were conjunctivitis and recurrent infections. Clinical variability was remarkable even among familial cases, and incomplete penetrance was observed. Patients in our series were treated with a variety of medications, and benefit was observed with glucocorticoids, colchicine, and TNF inhibitors. Altogether, our work adds to the current literature and doubles the number of reported cases with RELA-Associated Inflammatory Disease (RAID). It reaffirms the central importance of the NF-κB pathway in immunity and inflammation, as well as the important regulatory role of RELA in mucosal homeostasis. RELA associated inflammatory disease should be considered in all patients with BD, particularly those with early onset and/or with a strong family history.
The deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessively inherited disease that has undergone extensive phenotypic expansion since being first described in patients with fevers, recurrent strokes, livedo racemosa, and polyarteritis nodosa in 2014. It is now recognized that patients may develop multisystem disease that spans multiple medical subspecialties. Here, we describe the findings from a large single center longitudinal cohort of 60 patients, the broad phenotypic presentation, as well as highlight the cohort's experience with hematopoietic cell transplantation and COVID-19. Disease manifestations could be separated into three major phenotypes: inflammatory/vascular, immune dysregulatory, and hematologic, however, most patients presented with significant overlap between these three phenotype groups. The cardinal features of the inflammatory/vascular group included cutaneous manifestations and stroke. Evidence of immune dysregulation was commonly observed, including hypogammaglobulinemia, absent to low class-switched memory B cells, and inadequate response to vaccination. Despite these findings, infectious complications were exceedingly rare in this cohort. Hematologic findings including pure red cell aplasia (PRCA), immune-mediated neutropenia, and pancytopenia were observed in half of patients. We significantly extended our experience using anti-TNF agents, with no strokes observed in 2026 patient months on TNF inhibitors. Meanwhile, hematologic and immune features had a more varied response to anti-TNF therapy. Six patients received a total of 10 allogeneic hematopoietic cell transplant (HCT) procedures, with secondary graft failure necessitating repeat HCTs in three patients, as well as unplanned donor cell infusions to avoid graft rejection. All transplanted patients had been on anti-TNF agents prior to HCT and received varying degrees of reduced-intensity or non-myeloablative conditioning. All transplanted patients are still alive and have discontinued anti-TNF therapy. The long-term follow up afforded by this large single-center study underscores the clinical heterogeneity of DADA2 and the potential for phenotypes to evolve in any individual patient.
BACKGROUND
Biallelic loss-of-function variants in NCF1 lead to reactive oxygen species deficiency and chronic granulomatous disease (CGD). Heterozygosity for the p.Arg90His variant in NCF1 has been associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome in adult patients. This study demonstrates the association of the homozygous p.Arg90His variant with interferonopathy with features of autoinflammation and autoimmunity in a pediatric patient.
CASE PRESENTATION
A 5-year old female of Indian ancestry with early-onset recurrent fever and headache, and persistently elevated antinuclear, anti-Ro, and anti-La antibodies was found to carry the homozygous p.Arg90His variant in NCF1 through exome sequencing. Her unaffected parents and three other siblings were carriers for the mutant allele. Because the presence of two NCF1 pseudogenes, this variant was confirmed by independent genotyping methods. Her intracellular neutrophil oxidative burst and NCF1 expression levels were normal, and no clinical features of CGD were apparent. Gene expression analysis in peripheral blood detected an interferon gene expression signature, which was further supported by cytokine analyses of supernatants of cultured patient's cells. These findings suggested that her inflammatory disease is at least in part mediated by type I interferons. While her fever episodes responded well to systemic steroids, treatment with the JAK inhibitor tofacitinib resulted in decreased serum ferritin levels and reduced frequency of fevers.
CONCLUSION
Homozygosity for p.Arg90His in NCF1 should be considered contributory in young patients with an atypical systemic inflammatory antecedent phenotype that may evolve into autoimmunity later in life. The complex genomic organization of NCF1 poses a difficulty for high-throughput genotyping techniques and variants in this gene should be carefully evaluated when using the next generation and Sanger sequencing technologies. The p.Arg90His variant is found at a variable allele frequency in different populations, and is higher in people of South East Asian ancestry. In complex genetic diseases such as SLE, other rare and common susceptibility alleles might be necessary for the full disease expressivity.
Somatic Mutations in UBA1 Define a Distinct Subset of Relapsing Polychondritis Patients With VEXAS
(2021)
Neurodevelopmental disorder with dysmorphic facies and distal limb anomalies (NEDDFL), defined primarily by developmental delay/intellectual disability, speech delay, postnatal microcephaly, and dysmorphic features, is a syndrome resulting from heterozygous variants in the dosage-sensitive bromodomain PHD finger chromatin remodeler transcription factor BPTF gene. To date, only 11 individuals with NEDDFL due to de novo BPTF variants have been described. To expand the NEDDFL phenotypic spectrum, we describe the clinical features in 25 novel individuals with 20 distinct, clinically relevant variants in BPTF, including four individuals with inherited changes in BPTF. In addition to the previously described features, individuals in this cohort exhibited mild brain abnormalities, seizures, scoliosis, and a variety of ophthalmologic complications. These results further support the broad and multi-faceted complications due to haploinsufficiency of BPTF.
Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.
Mendelian diseases of dysregulated canonical NF-κB signaling: From immunodeficiency to inflammation
(2020)
For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter taste substances are most probably initiated by tracheal brush cells (BC). Our single-cell RNA-seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca2+](i) in BC and subsequent ACh-release. ACh-release is regulated in an autocrine manner. While the muscarinic ACh-receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca2+](i) in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5- and M3R-mediated. We show that ACh-release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.
Plant sap-feeding insects are widespread, having evolved to occupy diverse environmental niches despite exclusive feeding on an impoverished diet lacking in essential amino acids and vitamins. Success depends exquisitely on their symbiotic relationships with microbial symbionts housed within specialized eukaryotic bacteriocyte cells. Each bacteriocyte is packed with symbionts that are individually surrounded by a host-derived symbiosomal membrane representing the absolute host-symbiont interface. The symbiosomal membrane must be a dynamic and selectively permeable structure to enable bidirectional and differential movement of essential nutrients, metabolites, and biosynthetic intermediates, vital for growth and survival of host and symbiont. However, despite this crucial role, the molecular basis of membrane transport across the symbiosomal membrane remains unresolved in all bacteriocyte-containing insects. A transport protein was immuno-localized to the symbiosomal membrane separating the pea aphid Acyrthosiphon pisum from its intracellular symbiont Buchnera aphidicola. The transporter, A. pisum nonessential amino acid transporter 1, or ApNEAAT1 (gene: ACYPI008971), was characterized functionally following heterologous expression in Xenopus oocytes, and mediates both inward and outward transport of small dipolar amino acids (serine, proline, cysteine, alanine, glycine). Electroneutral ApNEAAT1 transport is driven by amino acid concentration gradients and is not coupled to transmembrane ion gradients. Previous metabolite profiling of hemolymph and bacteriocyte, alongside metabolic pathway analysis in host and symbiont, enable prediction of a physiological role for ApNEAAT1 in bidirectional host-symbiont amino acid transfer, supplying both host and symbiont with indispensable nutrients and biosynthetic precursors to facilitate metabolic complementarity.
The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. The epithelial sodium channel (ENaC) is present in the apical membrane of sodium-absorbing vertebrate epithelia and evolved as part of a machinery for efficient sodium conservation. ENaC belongs to the degenerin/ENaC protein family and is the only member that opens without an external stimulus. We hypothesized that ENaC evolved from a proton-activated sodium channel present in ionocytes of freshwater vertebrates and therefore investigated whether such ancestral traits are present in ENaC isoforms of the aquatic pipid frog Xenopus laevis. Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from alpha beta gamma- or delta beta gamma-subunit combinations, we demonstrate that Xenopus delta beta gamma-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges (pH 8.0-6.0). This effect was not observed in Xenopus alpha beta gamma-ENaC or human ENaC orthologs. We show that protons interfere with allosteric ENaC inhibition by extracellular sodium ions, thereby increasing the probability of channel opening. Using homology modeling of ENaC structure and site-directed mutagenesis, we identified a cleft region within the extracellular loop of the delta-subunit that contains several acidic amino acid residues that confer proton-sensitivity and enable allosteric inhibition by extracellular sodium ions. We propose that Xenopus delta beta gamma-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutively-active ion channel. Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water-to-land transition.
The pyrin inflammasome has evolved as an innate immune sensor to detect bacterial toxin-induced Rho guanosine triphosphatase (Rho GTPase)-inactivation, a process that is similar to the "guard" mechanism in plants. Rho GTPases act as molecular switches to regulate a variety of signal transduction pathways including cytoskeletal organization. Pathogens can modulate Rho GTPase activity to suppress host immune responses such as phagocytosis. Pyrin is encoded by MEFV, the gene that is mutated in patients with familial Mediterranean fever (FMF). FMF is the prototypic autoinflammatory disease characterized by recurring short episodes of systemic inflammation and is a common disorder in many populations in the Mediterranean basin. Pyrin specifically senses modifications in the activity of the small GTPase RhoA, which binds to many effector proteins including the serine/threonine-protein kinases PKN1 and PKN2 and actin-binding proteins. RhoA activation leads to PKN-mediated phosphorylation-dependent pyrin inhibition. Conversely, pathogen virulence factors downregulate RhoA activity in a variety of ways, and these changes are detected by the pyrin inflammasome irrespective of the type of modifications. MEFV pathogenic variants favor the active state of pyrin and elicit proinflammatory cytokine release and pyroptosis. They can be inherited either as a dominant or recessive trait depending on the variant's location and effect on the protein function. Mutations in the C-terminal B30.2 domain are usually considered recessive, although heterozygotes may manifest a biochemical or even a clinical phenotype. These variants are hypomorphic in regard to their effect on intramolecular interactions, but ultimately accentuate pyrin activity. Heterozygous mutations in other domains of pyrin affect residues critical for inhibition or protein oligomerization, and lead to constitutively active inflammasome. In healthy carriers of FMF mutations who have the subclinical inflammatory phenotype, the increased activity of pyrin might have been protective against endemic infections over human history. This finding is supported by the observation of high carrier frequencies of FMF-mutations in multiple populations. The pyrin inflammasome also plays a role in mediating inflammation in other autoinflammatory diseases linked to dysregulation in the actin polymerization pathway. Therefore, the assembly of the pyrin inflammasome is initiated in response to fluctuations in cytoplasmic homeostasis and perturbations in cytoskeletal dynamics.
Systemic autoinflammatory diseases (SAIDs) are a group of inflammatory disorders caused by dysregulation in the innate immune system that leads to enhanced immune responses. The clinical diagnosis of SAIDs can be difficult since individually these are rare diseases with considerable phenotypic overlap. Most SAIDs have a strong genetic background, but environmental and epigenetic influences can modulate the clinical phenotype. Molecular diagnosis has become essential for confirmation of clinical diagnosis. To date there are over 30 genes and a variety of modes of inheritance that have been associated with monogenic SAIDs. Mutations in the same gene can lead to very distinct phenotypes and can have different inheritance patterns. In addition, somatic mutations have been reported in several of these conditions. New genetic testing methods and databases are being developed to facilitate the molecular diagnosis of SAIDs, which is of major importance for treatment, prognosis and genetic counselling. The aim of this review is to summarize the latest advances in genetic testing for SAIDs and discuss potential obstacles that might arise during the molecular diagnosis of SAIDs.
The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits (, , and ), but a fourth -subunit may replace and form atypical -ENaCs. Using Xenopus laevis as a model, here we found that mRNAs of the - and -subunits are differentially expressed in different tissues and that -ENaC predominantly is present in the urogenital tract. Using whole-cell and single-channel electrophysiology of oocytes expressing Xenopus - or -ENaC, we demonstrate that the presence of the -subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the - and -subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike -ENaC, the -subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by -ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the -subunit prevented cleavage of -ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the Xenopus -ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution.
Evolutionary conservation of the antimicrobial function of mucus: a first defence against infection
(2018)
Mucus layers often provide a unique and multi-functional hydrogel interface between the epithelial cells of organisms and their external environment. Mucus has exceptional properties including elasticity, changeable rheology and an ability to self-repair by reannealing, and is therefore an ideal medium for trapping and immobilising pathogens and serving as a barrier to microbial infection. The ability to produce a functional surface mucosa was an important evolutionary step, which evolved first in the Cnidaria, which includes corals, and the Ctenophora. This allowed the exclusion of non-commensal microbes and the subsequent development of the mucus-lined digestive cavity seen in higher metazoans. The fundamental architecture of the constituent glycoprotein mucins is also evolutionarily conserved. Although an understanding of the biochemical interactions between bacteria and the mucus layer are important to the goal of developing new antimicrobial strategies, they remain relatively poorly understood. This review summarises the physicochemical properties and evolutionary importance of mucus, which make it so successful in the prevention of bacterial infection. In addition, the strategies developed by bacteria to counteract the mucus layer are also explored.
Cholinergic polymodal chemosensory cells in the mammalian urethra (urethral brush cells = UBC) functionally express the canonical bitter and umami taste transduction signaling cascade. Here, we aimed to determine whether UBC are functionally equipped for the perception of salt through ENaC (epithelial sodium channel). Cholinergic UBC were isolated from ChAT-eGFP reporter mice (ChAT = choline acetyltransferase). RT-PCR showed mRNA expression of ENaC subunits Scnn1a, Scnn1b, and Scnn1g in urethral epithelium and isolated UBC. Scnn1a could also be detected by next generation sequencing in 4/6 (66%) single UBC, two of them also expressed the bitter receptor Tas2R108. Strong expression of Scnn1a was seen in some urothelial umbrella cells and in 65% of UBC (30/46 cells) in a Scnn1a reporter mouse strain. Intracellular [Ca2+] was recorded in isolated UBC stimulated with the bitter substance denatonium benzoate (25 mM), ATP (0.5 mM) and NaCl (50 mM, on top of 145 mM Na+ and 153 mM Cl- baseline in buffer); mannitol (150 mM) served as osmolarity control. NaCl, but not mannitol, evoked an increase in intracellular [Ca2+] in 70% of the tested UBC. The NaCl-induced effect was blocked by the ENaC inhibitor amiloride (IC50 = 0.471 mu M). When responses to both NaCl and denatonium were tested, all three possible positive response patterns occurred in a balanced distribution: 42% NaCl only, 33% denatonium only, 25% to both stimuli. A similar reaction pattern was observed with ATP and NaCl as test stimuli. About 22% of the UBC reacted to all three stimuli. Thus, NaCl evokes calcium responses in several UBC, likely involving an amiloride-sensitive channel containing alpha-ENaC. This feature does not define a new subpopulation of UBC, but rather emphasizes their polymodal character. The actual function of alpha-ENaC in cholinergic UBC-salt perception, homeostatic ion transport, mechanoreception-remains to be determined.
An increased bronchoconstrictor response is a hallmark in the progression of obstructive airway diseases. Acetylcholine and 5-hydroxytryptamine (5-HT, serotonin) are the major bronchoconstrictors. There is evidence that both cholinergic and serotonergic signaling in airway smooth muscle (ASM) involve caveolae. We hypothesized that caveolin-1 (cav-1), a structural protein of caveolae, plays an important regulatory role in ASM contraction. We analyzed airway contraction in different tracheal segments and extra-and intrapulmonary bronchi in cav-1 deficient (cav-1-/-) and wild-type mice using organ bath recordings and videomorphometry of methyl-beta-cyclodextrin (MCD) treated and non-treated precision-cut lung slices (PCLS). The presence of caveolae was investigated by electron microscopy. Receptor subtypes driving 5-HT-responses were studied by RT-PCR and videomorphometry after pharmacological inhibition with ketanserin. Cav-1 was present in tracheal epithelium and ASM. Muscarine induced a dose dependent contraction in all airway segments. A significantly higher Emax was observed in the caudal trachea. Although, caveolae abundancy was largely reduced in cav-1-/- mice, muscarine-induced airway contraction was maintained, albeit at diminished potency in the middle trachea, in the caudal trachea and in the bronchus without changes in the maximum efficacy. MCD-treatment of PLCS from cav-1-/- mice reduced cholinergic constriction by about 50%, indicating that cholesterol-rich plasma domains account for a substantial portion of the muscarine-induced bronchoconstriction. Notably, cav-1-deficiency fully abrogated 5-HT-induced contraction of extrapulmonary airways. In contrast, 5-HT-induced bronchoconstriction was fully maintained in cav-1-deficient intrapulmonary bronchi, but desensitization upon repetitive stimulation was enhanced. RT-PCR analysis revealed 5-HT1B, 5-HT2A, 5-HT6, and 5-HT7 receptors as the most prevalent subtypes in the airways. The 5-HT-induced-constriction in PCLS could be antagonized by ketanserin, a 5-HT2A receptor inhibitor. In conclusion, the role of cav-1, caveolae, and cholesterol-rich plasma domains in regulation of airway tone are highly agonist-specific and dependent on airway level. Cav-1 is indispensable for serotonergic contraction of extrapulmonary airways and modulates cholinergic constriction of the trachea and main bronchus. Thus, cav-1/caveolae shall be considered in settings such as bronchial hyperreactivity in common airway diseases and might provide an opportunity for modulation of the constrictor response.
Recently, we discovered a cholinergic mechanism that inhibits the adenosine triphosphate (ATP)-dependent release of interleukin-1 beta (IL-1 beta) by human monocytes via nicotinic acetylcholine receptors (nAChRs) composed of alpha 7, alpha 9 and/or alpha 10 subunits. Furthermore, we identified phosphocholine (PC) and dipalmitoylphosphatidylcholine (DPPC) as novel nicotinic agonists that elicit metabotropic activity at monocytic nAChR. Interestingly, PC does not provoke ion channel responses at conventional nAChRs composed of subunits alpha 9 and alpha 10. The purpose of this study is to determine the composition of nAChRs necessary for nicotinic signaling in monocytic cells and to test the hypothesis that common metabolites of phosphatidylcholines, lysophosphatidylcholine (LPC) and glycerophosphocholine (G-PC), function as nAChR agonists. In peripheral blood mononuclear cells from nAChR gene-deficient mice, we demonstrated that inhibition of ATP-dependent release of IL-1 beta by acetylcholine (ACh), nicotine and PC depends on subunits alpha 7, alpha 9 and alpha 10. Using a panel of nAChR antagonists and siRNA technology, we confirmed the involvement of these subunits in the control of IL-1 beta release in the human monocytic cell line U937. Furthermore, we showed that LPC (C16:0) and G-PC efficiently inhibit ATP-dependent release of IL-1 beta. Of note, the inhibitory effects mediated by LPC and G-PC depend on nAChR subunits alpha 9 and alpha 10, but only to a small degree on alpha 7. In Xenopus laevis oocytes heterologously expressing different combinations of human alpha 7, alpha 9 or alpha 10 subunits, ACh induced canonical ion channel activity, whereas LPC, G-PC and PC did not. In conclusion, we demonstrate that canonical nicotinic agonists and PC elicit metabotropic nAChR activity in monocytes via interaction of nAChR subunits alpha 7, alpha 9 and alpha 10. For the metabotropic signaling of LPC and G-PC, nAChR subunits alpha 9 and alpha 10 are needed, whereas alpha 7 is virtually dispensable. Furthermore, molecules bearing a PC group in general seem to regulate immune functions without perturbing canonical ion channel functions of nAChR.
Hydrogen sulfide stimulates CFTR in Xenopus oocytes by activation of the cAMP/PKA signalling axis
(2017)
Hydrogen sulfide (H2S) has been recognized as a signalling molecule which affects the activity of ion channels and transporters in epithelial cells. The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial anion channel and a key regulator of electrolyte and fluid homeostasis. In this study, we investigated the regulation of CFTR by H2S. Human CFTR was heterologously expressed in Xenopus oocytes and its activity was electrophysiologically measured by microelectrode recordings. The H2S-forming sulphur salt Na2S as well as the slow-releasing H2S-liberating compound GYY4137 increased transmembrane currents of CFTR-expressing oocytes. Na2S had no effect on native, noninjected oocytes. The effect of Na2S was blocked by the CFTR inhibitor CFTR_inh172, the adenylyl cyclase inhibitor MDL 12330A, and the protein kinase A antagonist cAMPS-Rp. Na2S potentiated CFTR stimulation by forskolin, but not that by IBMX. Na2S enhanced CFTR stimulation by membranepermeable 8Br-cAMP under inhibition of adenylyl cyclase-mediated cAMP production by MDL 12330A. These data indicate that H2S activates CFTR in Xenopus oocytes by inhibiting phosphodiesterase activity and subsequent stimulation of CFTR by cAMP-dependent protein kinase A. In epithelia, an increased CFTR activity may correspond to a pro-secretory response to H2S which may be endogenously produced by the epithelium or H2S-generating microflora.
We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions.
Hydrogen sulfide (H2S) is a well-known environmental chemical threat with an unpleasant smell of rotten eggs. Aside from the established toxic effects of high-dose H2S, research over the past decade revealed that cells endogenously produce small amounts of H2S with physiological functions. H2S has therefore been classified as a gasotransmitter. A major challenge for cells and tissues is the maintenance of low physiological concentrations of H2S in order to prevent potential toxicity. Epithelia of the respiratory and gastrointestinal tract are especially faced with this problem, since these barriers are predominantly exposed to exogenous H2S from environmental sources or sulfur-metabolising microbiota. In this paper, we review the cellular mechanisms by which epithelial cells maintain physiological, endogenous H2S concentrations. Furthermore, we suggest a concept by which epithelia use their electrolyte and liquid transport machinery as defence mechanisms in order to eliminate exogenous sources for potentially harmful H2S concentrations.
Hydrogen sulfide contributes to hypoxic inhibition of airway transepithelial sodium absorption
(2016)
The generation and maintenance of intricate spatiotemporal patterns of gene expression in multicellular organisms requires the establishment of complex mechanisms of transcriptional regulation. Estimations that up to one million enhancers exist in the human genome accentuates the utmost importance of this type of cis-regulatory element for gene regulation. However, surprisingly little is known about the mechanisms used to temporarily or permanently activate or inactivate enhancers during cellular differentiation. The current work addresses the question how enhancer regulation can be achieved.
Using the chemokine (C-C motif) ligand gene Ccl22 as a model, the first example is based on the question how the activation of an enhancer can be prevented in a physiological context. Ccl22 is expressed by myeloid cells, such as dendritic cells, upon exposure to inflammatory stimuli. The expression in other cell types, such as fibroblasts, is prevented by the strong accumulation of H3K9me3 at the enhancer's proximal region. This accumulation is attenuated in myeloid cells through activity of the stimulus-induced demethylase Jmjd2d. To tease out which genomic fragment or fragments in the Ccl22 locus could be responsible for the maintenance of enhancer inactivity, potentially through the recruitment of H3K9 methyltransferases, the enhancer repressing capacity of 1 kb fragments of the gene locus was analysed in retroviral reporter assays. It was found that a fragment adjacent to the Ccl22 enhancer that overlaps with a member of a subfamily of long interspersed nuclear elements (LINEs) showed strong repressive potential on a model enhancer. Subsequent retroviral reporter assays with LINEs from loci of other stimulus-dependent genes identified additional LINE fragments that exhibit strong enhancer repressive capacity. These findings suggest a mechanism for enhancer silencing involving LINEs.
The second example concentrates on the inactivation of an enhancer during colorectal cancer (CRC) progression. The adenoma to carcinoma transition during CRC progression often is accompanied by a downregulation of the tumour suppressor gene EPHB2. The EMT inducing factor SNAIL1 strongly downregulated EPHB2 expression in a CRC cell model. To gain insights into the transcriptional regulation of EPHB2, potential cis-regulatory elements in the EPHB2 upstream region were analysed using reporter assays. A cell-type-specific enhancer was identified and subsequent chromatin analyses revealed a correlation between enhancer chromatin conformation and EPHB2 expression in different CRC cell lines. Additionally, the overexpression of the murine Snail1 induced chromatin changes at the EPHB2 enhancer towards a poised, transcriptionally silent chromatin conformation. Mutational analyses of the minimal enhancer region pinpointed three transcription factor binding motifs to be essential for full enhancer activity. Different binding patterns between CRC cell lines at the TCF/LEF motif were subsequently identified. Furthermore, a switch from TCF7L2 to LEF1 occupancy was found upon overexpression of Snail1 in vitro and in vivo. The generation of LS174T CRC cells overexpressing LEF1 confirmed the involvement of LEF1 in the downregulation of EPHB2 and the competitive displacement of TCF7L2. This part of the work demonstrated that the SNAIL1 induced downregulation of EPHB2 is dependent on the decommissioning of a transcriptional enhancer and led to a hypothetical model involving LEF1 and ZEB1.
In summary, this work highlighted two distinct mechanisms for enhancer regulation. One mechanism is based on enhancer repressive LINE fragments that might prevent stimulus-dependent enhancer activation. In the second, enhancer silencing was shown to be based on a competitive transcription factor binding mechanism.
The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel's selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17−/− mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions.
Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway.
Hydrogen sulfide (H2S) is well known as a highly toxic environmental chemical threat. Prolonged exposure to H2S can lead to the formation of pulmonary edema. However, the mechanisms of how H2S facilitates edema formation are poorly understood. Since edema formation can be enhanced by an impaired clearance of electrolytes and, consequently, fluid across the alveolar epithelium, it was questioned whether H2S may interfere with transepithelial electrolyte absorption. Electrolyte absorption was electrophysiologically measured across native distal lung preparations (Xenopus laevis) in Ussing chambers. The exposure of lung epithelia to H2S decreased net transepithelial electrolyte absorption. This was due to an impairment of amiloride-sensitive sodium transport. H2S inhibited the activity of the Na+/K+-ATPase as well as lidocaine-sensitive potassium channels located in the basolateral membrane of the epithelium. Inhibition of these transport molecules diminishes the electrochemical gradient which is necessary for transepithelial sodium absorption. Since sodium absorption osmotically facilitates alveolar fluid clearance, interference of H2S with the epithelial transport machinery provides a mechanism which enhances edema formation in H2S-exposed lungs.
Methylmalonic and propionic acidemia (MMA/PA) are inborn errors of metabolism characterized by accumulation of propionic acid and/or methylmalonic acid due to deficiency of methylmalonyl-CoA mutase (MUT) or propionyl-CoA carboxylase (PCC). MMA has an estimated incidence of ~ 1: 50,000 and PA of ~ 1:100'000 -150,000. Patients present either shortly after birth with acute deterioration, metabolic acidosis and hyperammonemia or later at any age with a more heterogeneous clinical picture, leading to early death or to severe neurological handicap in many survivors. Mental outcome tends to be worse in PA and late complications include chronic kidney disease almost exclusively in MMA and cardiomyopathy mainly in PA. Except for vitamin B12 responsive forms of MMA the outcome remains poor despite the existence of apparently effective therapy with a low protein diet and carnitine. This may be related to under recognition and delayed diagnosis due to nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity.
Propionic acidemia in a previously healthy adolescent with acute onset of dilated cardiomyopathy
(2014)
More than 25 years ago, it was a big surprise for physiologists that nitric oxide (NO) was identified as the endothelium derived relaxing factor which is responsible for endothelium-induced smooth muscle relaxation (Ignarro et al., 1987). Until then, small gaseous molecules were simply regarded as byproducts of cellular metabolism which were unlikely to be of any physiological relevance. The discovery that NO was synthesized by specific enzymes (NO-synthases), upon stimulation by specific, physiologically relevant stimuli (e.g., acetylcholine stimulation of endothelial cells), as well as the fact that it acted on specific cellular targets (e.g., soluble guanylate cyclase), set the course for numerous studies which investigated the physiological roles of gaseous signaling molecules—in other words, gasotransmitters (Wang, 2002).
Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle defect. The clinical presentation in female manifesting carriers varies both in onset and severity. We report on a female with insulin dependent diabetes mellitus and recurrent episodes of hyperammonemia. Since OTC activity measured in a liver biopsy sample was within normal limits, OTC deficiency was initially excluded from the differential diagnoses of hyperammonemia. Due to moderately elevated homocitrulline excretion, hyperornithinemia-hyperammonemia-homocitrullinuria-syndrome was suggested, but further assays in fibroblasts showed normal ornithine utilization. Later, when mutation analysis of the OTC gene became available, a known pathogenic missense mutation (c.533C>T) in exon 5 leading to an exchange of threonine-178 by methionine (p.Thr178Met) was detected. Skewed X-inactivation was demonstrated in leukocyte DNA. In the further clinical course the girl developed marked obesity. By initiating physical activities twice a week, therapeutic control of both diabetes and OTC deficiency improved, but obesity persisted. In conclusion, our case confirms that normal hepatic OTC enzyme activity measured in a single liver biopsy sample does not exclude a clinical relevant mosaic of OTC deficiency because of skewed X-inactivation. Mutation analysis of the OTC gene in whole blood may be a simple way to establish the diagnosis of OTC deficiency. The joint occurrence of OTC deficiency and diabetes in a patient has not been reported before.
BACKGROUND
Hyperlysinemia is an autosomal recessive inborn error of L-lysine degradation. To date only one causal mutation in the AASS gene encoding α-aminoadipic semialdehyde synthase has been reported. We aimed to better define the genetic basis of hyperlysinemia.
METHODS
We collected the clinical, biochemical and molecular data in a cohort of 8 hyperlysinemia patients with distinct neurological features.
RESULTS
We found novel causal mutations in AASS in all affected individuals, including 4 missense mutations, 2 deletions and 1 duplication. In two patients originating from one family, the hyperlysinemia was caused by a contiguous gene deletion syndrome affecting AASS and PTPRZ1.
CONCLUSIONS
Hyperlysinemia is caused by mutations in AASS. As hyperlysinemia is generally considered a benign metabolic variant, the more severe neurological disease course in two patients with a contiguous deletion syndrome may be explained by the additional loss of PTPRZ1. Our findings illustrate the importance of detailed biochemical and genetic studies in any hyperlysinemia patient.
BACKGROUND
Metabolic control and dietary management of patients with phenylketonuria (PKU) are based on single blood samples obtained at variable intervals. Sampling conditions are often not well-specified and intermittent variation of phenylalanine concentrations between two measurements remains unknown. We determined phenylalanine and tyrosine concentrations in blood over 24 hours. Additionally, the impact of food intake and physical exercise on phenylalanine and tyrosine concentrations was examined. Subcutaneous microdialysis was evaluated as a tool for monitoring phenylalanine and tyrosine concentrations in PKU patients.
METHODS
Phenylalanine and tyrosine concentrations of eight adult patients with PKU were determined at 60 minute intervals in serum, dried blood and subcutaneous microdialysate and additionally every 30 minutes postprandially in subcutaneous microdialysate. During the study period of 24 hours individually tailored meals with defined phenylalanine and tyrosine contents were served at fixed times and 20 min bicycle-ergometry was performed.
RESULTS
Serum phenylalanine concentrations showed only minor variations while tyrosine concentrations varied significantly more over the 24-hour period. Food intake within the patients' individual diet had no consistent effect on the mean phenylalanine concentration but the tyrosine concentration increased up to 300% individually. Mean phenylalanine concentration remained stable after short-term bicycle-exercise whereas mean tyrosine concentration declined significantly. Phenylalanine and tyrosine concentrations in dried blood were significantly lower than serum concentrations. No close correlation has been found between serum and microdialysis fluid for phenylalanine and tyrosine concentrations.
CONCLUSIONS
Slight diurnal variation of phenylalanine concentrations in serum implicates that a single blood sample does reliably reflect the metabolic control in this group of adult patients. Phenylalanine concentrations determined by subcutaneous microdialysis do not correlate with the patients' phenylalanine concentrations in serum/blood.
Reactive oxygen species and the bacteriostatic and bactericidal effects of isoconazole nitrate
(2013)
BACKGROUND
Propionic acidemia is an inherited disorder caused by deficiency of propionyl-CoA carboxylase. Although it is one of the most frequent organic acidurias, information on the outcome of affected individuals is still limited.
STUDY DESIGN/METHODS
Clinical and outcome data of 55 patients with propionic acidemia from 16 European metabolic centers were evaluated retrospectively. 35 patients were diagnosed by selective metabolic screening while 20 patients were identified by newborn screening. Endocrine parameters and bone age were evaluated. In addition, IQ testing was performed and the patients' and their families' quality of life was assessed.
RESULTS
The vast majority of patients (>85%) presented with metabolic decompensation in the neonatal period. Asymptomatic individuals were the exception. About three quarters of the study population was mentally retarded, median IQ was 55. Apart from neurologic symptoms, complications comprised hematologic abnormalities, cardiac diseases, feeding problems and impaired growth. Most patients considered their quality of life high. However, according to the parents' point of view psychic problems were four times more common in propionic acidemia patients than in healthy controls.
CONCLUSION
Our data show that the outcome of propionic acidemia is still unfavourable, in spite of improved clinical management. Many patients develop long-term complications affecting different organ systems. Impairment of neurocognitive development is of special concern. Nevertheless, self-assessment of quality of life of the patients and their parents yielded rather positive results.
A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency
(2013)
Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ~20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address:
http://www.thesgc.org/jimd/SCOT
Disorders of the degradation of branched chain amino acids: what is new in clinics and laboratories?
(2012)
Wesch D, Althaus M, Miranda P, Cruz-Muros I, Fronius M, Gonzalez-Hernandez T, Clauss WG, de la Rosa DA, Giraldez T. Differential N termini in epithelial Na+ channel delta-subunit isoforms modulate channel trafficking to the membrane. Am J Physiol Cell Physiol 302: C868-C879, 2012. First published December 7, 2011; doi: 10.1152/ajpcell.00255.2011.-The epithelial Na+ channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na+ reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, alpha, beta, and gamma. A fourth ENaC subunit, named delta, is expressed in the nervous system of primates, where its role is unknown. The human delta-ENaC gene generates at least two splice isoforms, delta(1) and delta(2), differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either delta(1) or delta(2), with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of delta(1) in Xenopus oocytes and HEK293 cells produces higher current levels than delta(2). Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of beta- and gamma-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in delta(2)-N terminus. Analysis of delta(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The delta(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that delta-ENaC undergoes dynamin-independent endocytosis as opposed to alpha beta gamma-channels.
The ability to breathe air represents a fundamental step in vertebrate evolution that was accompanied by several anatomical and physiological adaptations. The morphology of the air-blood barrier is highly conserved within air-breathing vertebrates. It is formed by three different plies, which are represented by the alveolar epithelium, the basal lamina, and the endothelial layer. Besides these conserved morphological elements, another common feature of vertebrate lungs is that they contain a certain amount of fluid that covers the alveolar epithelium. The volume and composition of the alveolar fluid is regulated by transepithelial ion transport mechanisms expressed in alveolar epithelial cells. These transport mechanisms have been reviewed extensively. Therefore, the present review focuses on the properties and functional significance of the alveolar fluid. How does the fluid enter the alveoli? What is the fate of the fluid in the alveoli? What is the function of the alveolar fluid in the lungs? The review highlights the importance of the alveolar fluid, its volume and its composition. Maintenance of the fluid volume and composition within certain limits is critical to facilitate gas exchange. We propose that the alveolar fluid is an essential element of the air-blood barrier. Therefore, it is appropriate to refer to this barrier as being formed by four plies, namely (1) the thin fluid layer covering the apical membrane of the epithelial cells, (2) the epithelial cell layer, (3) the basal membrane, and (4) the endothelial cells.
The vectorial transport of Na+ across epithelia is crucial for the maintenance of Na+ and water homeostasis in organs such as the kidneys, lung, or intestine. Dysregulated Na+ transport processes are associated with various human diseases such as hypertension, the salt-wasting syndrome pseudohypoaldosteronism type 1, pulmonary edema, cystic fibrosis, or intestinal disorders, which indicate that a precise regulation of epithelial Na+ transport is essential. Novel regulatory signaling molecules are gasotransmitters. There are currently three known gasotransmitters: nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S). These molecules are endogenously produced in mammalian cells by specific enzymes and have been shown to regulate various physiological processes. There is a growing body of evidence which indicates that gasotransmitters may also regulate Na+ transport across epithelia. This review will summarize the available data concerning NO, CO, and H2S dependent regulation of epithelial Na+ transport processes and will discuss whether or not these mediators can be considered as true physiological regulators of epithelial Na+ transport biology.
The gasotransmitter hydrogen sulphide decreases Na⁺ transport across pulmonary epithelial cells
(2012)
BACKGROUND AND PURPOSE The transepithelial absorption of Na(+) in the lungs is crucial for the maintenance of the volume and composition of epithelial lining fluid. The regulation of Na(+) transport is essential, because hypo- or hyperabsorption of Na(+) is associated with lung diseases such as pulmonary oedema or cystic fibrosis. This study investigated the effects of the gaseous signalling molecule hydrogen sulphide (H(2) S) on Na(+) absorption across pulmonary epithelial cells. EXPERIMENTAL APPROACH Ion transport processes were electrophysiologically assessed in Ussing chambers on H441 cells grown on permeable supports at air/liquid interface and on native tracheal preparations of pigs and mice. The effects of H(2)S were further investigated on Na(+) channels expressed in Xenopus oocytes and Na(+) /K(+)-ATPase activity in vitro. Membrane abundance of Na(+) /K(+)-ATPase was determined by surface biotinylation and Western blot. Cellular ATP concentrations were measured colorimetrically, and cytosolic Ca(2+) concentrations were measured with Fura-2. KEY RESULTS H(2)S rapidly and reversibly inhibited Na(+) transport in all the models employed. H(2)S had no effect on Na(+) channels, whereas it decreased Na(+) /K(+)-ATPase currents. H(2)S did not affect the membrane abundance of Na(+) /K(+)-ATPase, its metabolic or calcium-dependent regulation, or its direct activity. However, H(2)S inhibited basolateral calcium-dependent K(+) channels, which consequently decreased Na(+) absorption by H441 monolayers. CONCLUSIONS AND IMPLICATIONS H(2) S impairs pulmonary transepithelial Na(+) absorption, mainly by inhibiting basolateral Ca(2+)-dependent K(+) channels. These data suggest that the H(2)S signalling system might represent a novel pharmacological target for modifying pulmonary transepithelial Na(+) transport.
The osmolality of nonionic, iodinated contrast agents as an important factor for renal safety
(2012)
Molybdenum cofactor deficiency (MoCD) is a rare inherited metabolic disorder characterized by severe and progressive neurological damage mainly caused by the loss of sulfite oxidase activity. Elevated urinary levels of sulfite, thiosulfate, and S-sulfocysteine (SSC) are hallmarks in the diagnosis of MoCD and sulfite oxidase deficiency (SOD). Recently, a first successful treatment of a human MoCD type A patient based on a substitution therapy with the molybdenum cofactor precursor cPMP has been reported, resulting in nearly complete normalization of MoCD biomarkers. Knowing the rapid progression of the disease symptoms in nontreated patients, an early diagnosis of MoCD as well as a sensitive method to monitor daily changes in SSC levels, a key marker of sulfite toxicity, is crucial for treatment outcome. Here, we describe a fast and sensitive method for the analysis of SSC in human urine samples using high performance liquid chromatography (HPLC). The analysis is based on precolumn derivatization with O-phthaldialdehyde (OPA) and separation on a C18 reverse phase column coupled to UV detection. The method was extended to human serum analysis and no interference with endogenous amino acids was found. Finally, SSC values from 45 pediatric urine, 75 adult urine, and 24 serum samples from control individuals as well as MoCD patients are reported. Our method represents a cost-effective technique for routine diagnosis of MoCD and SOD, and can be used also to monitor treatment efficiency in those sulfite toxicity disorders on a daily basis.
Nitric oxide (NO) is an important regulator of Na+ reabsorption by pulmonary epithelial cells and therefore of alveolar fluid clearance. The mechanisms by which NO affects epithelial ion transport are poorly understood and vary from model to model. In this study, the effects of NO on sodium reabsorption by H441 cell monolayers were studied in an Ussing chamber. Two NO donors, (Z)-1-[N-(3-aminopropyl)-N-(n-propyl) amino]diazen-1-ium-1,2-diolate and diethylammonium(Z)-1-(N, N-diethylamino) diazen-1-ium-1,2-diolate, rapidly, reversibly, and dose-dependently reduced amiloride-sensitive, short-circuit currents across H441 cell monolayers. This effect was neutralized by the NO scavenger hemoglobin and was not observed with inactive NO donors. The effects of NO were not blocked by 8-bromoguanosine-3',5'-cyclic monophosphate or by soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one) and were therefore independent of soluble guanylate cyclase signaling. NO targeted apical, highly selective, amiloride-sensitive Na+ channels in basolaterally permeabilized H441 cell monolayers. NO had no effect on the activity of the human epithelial sodium channel heterologously expressed in Xenopus oocytes. NO decreased Na+/K+-ATPase activity in apically permeabilized H441 cell monolayers. The inhibition of Na+/K+-ATPase activity by NO was reversed by mercury and was mimicked by N-ethylmaleimide, which are agents that reverse and mimic, respectively, the reaction of NO with thiol groups. Consistent with these data, S-NO groups were detected on the Na+/K+-ATPase a subunit in response to NO-donor application, using a biotin-switch approach coupled to a Western blot. These data demonstrate that, in the H441 cell model, NO impairs Na+ reabsorption by interfering with the activity of highly selective Na+ channels and the Na+/K+-ATPase.
The development of pulmonary edema can be considered as a combination of alveolar flooding via increased fluid filtration, impaired alveolar-capillary barrier integrity, and disturbed resolution due to decreased alveolar fluid clearance. An important mechanism regulating alveolar fluid clearance is sodium transport across the alveolar epithelium. Transepithelial sodium transport is largely dependent on the activity of sodium channels in alveolar epithelial cells. This paper describes how sodium channels contribute to alveolar fluid clearance under physiological conditions and how deregulation of sodium channel activity might contribute to the pathogenesis of lung diseases associated with pulmonary edema. Furthermore, sodium channels as putative molecular targets for the treatment of pulmonary edema are discussed.