Refine
Departments, institutes and facilities
- Institut für funktionale Gen-Analytik (IFGA) (584) (remove)
Document Type
- Article (493)
- Conference Object (56)
- Part of a Book (29)
- Contribution to a Periodical (2)
- Doctoral Thesis (2)
- Preprint (1)
- Working Paper (1)
Year of publication
Keywords
- ENaC (13)
- apoptosis (9)
- cytokine-induced killer cells (9)
- immunotherapy (7)
- DNA methylation (5)
- Organic aciduria (5)
- CD21 (4)
- Inborn error of metabolism (4)
- 5-Methylcytosine (3)
- Amiloride (3)
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 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.
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.
Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.
Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.
The development of whole-genome amplification (WGA) techniques has opened up new avenues for genetic analysis and genome research, in particular by facilitating the genome-wide analysis of few or even single copies of genomic DNA, such as from single cells (prokaryotic or eukaryotic) or virions. Using WGA, the few copies of genomic DNA obtained from such entities are unspecifically amplified using PCR or PCR-related processes in order to obtain higher DNA quantities that can then be successfully analysed further.
The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.
Cyanobacteria are gaining considerable interest as a method of supporting the long-term presence of humans on the Moon and settlements on Mars due to their ability to produce oxygen and their potential as bio-factories for space biotechnology/synthetic biology and other applications. Since many unknowns remain in our knowledge to bridge the gap and move cyanobacterial bioprocesses from Earth to space, we investigated cell division resumption on the rehydration of dried Chroococcidiopsis sp. CCMEE 029 accumulated DNA damage while exposed to space vacuum, Mars-like conditions, and Fe-ion radiation. Upon rehydration, the monitoring of the ftsZ gene showed that cell division was arrested until DNA damage was repaired, which took 48 h under laboratory conditions. During the recovery, a progressive DNA repair lasting 48 h of rehydration was revealed by PCR-stop assay. This was followed by overexpression of the ftsZ gene, ranging from 7.5- to 9-fold compared to the non-hydrated samples. Knowing the time required for DNA repair and cell division resumption is mandatory for deep-space experiments that are designed to unravel the effects of reduced/microgravity on this process. It is also necessary to meet mission requirements for dried-sample implementation and real-time monitoring upon recovery. Future experiments as part of the lunar exploration mission Artemis and the lunar gateway station will undoubtedly help to move cyanobacterial bioprocesses beyond low Earth orbit. From an astrobiological perspective, these experiments will further our understanding of microbial responses to deep-space conditions.
ESKAPEE Pathogen Biofilm Control on Surfaces with Probiotic Lactobacillaceae and Bacillus species
(2023)
Combatting the rapidly growing threat of antimicrobial resistance and reducing prevalence and transmission of ESKAPEE pathogens in healthcare settings requires innovative strategies, one of which is displacing these pathogens using beneficial microorganisms. Our review comprehensively examines the evidence of probiotic bacteria displacing ESKAPEE pathogens, with a focus on inanimate surfaces. A systematic search was conducted using the PubMed and Web of Science databases on 21 December 2021, and 143 studies were identified examining the effects of Lactobacillaceae and Bacillus spp. cells and products on the growth, colonization, and survival of ESKAPEE pathogens. While the diversity of study methods limits evidence analysis, results presented by narrative synthesis demonstrate that several species have the potential as cells or their products or supernatants to displace nosocomial infection-causing organisms in a variety of in vitro and in vivo settings. Our review aims to aid the development of new promising approaches to control pathogen biofilms in medical settings by informing researchers and policymakers about the potential of probiotics to combat nosocomial infections. More targeted studies are needed to assess safety and efficacy of different probiotic formulations, followed by large-scale studies to assess utility in infection control and medical practice.
ENaC channels
(2023)
Pitfalls of using sequence databases for heterologous expression studies - a technical review
(2023)
Synthesis of DNA fragments based on gene sequences available in public resources has become an efficient and affordable method that gradually replaced traditional cloning efforts such as PCR cloning from cDNA. However, database entries based on genome sequencing results are prone to errors which can lead to false sequence information and, ultimately, errors in functional characterization of proteins such as ion channels and transporters in heterologous expression systems. We have identified five common problems that repeatedly appear in public resources: 1) Not every gene has yet been annotated; 2) Not all gene annotations are necessarily correct; 3) Transcripts may contain automated corrections; 4) There are mismatches between gene, mRNA, and protein sequences; and 5) Splicing patterns often lack experimental validation. This technical review highlights and provides a strategy to bypass these issues in order to avoid critical mistakes that could impact future studies of any gene/protein of interest in heterologous expression systems. Abstract figure legend Projects involving heterologous gene expression are often characterised by similar steps. Initially, database research (A) is necessary to retrieve information of full of partial sequences of a gene of interest. A multitude of genome assemblies are annotated and deposited in public databases or that are available for refined search options using individual sequence information. The search results need to be scrutinised and compared to already available information (B). Once the sequence has been determined, DNA synthesis (C) by PCR or commercial synthesis are necessary for further cloning procedures (D). Eventually, the DNA needs to be transfected (E) and expressed in, e.g., eukaryotic cells (F). Finally, the expression of the gene of interest needs to be documented and its function analysed (G). This article is protected by copyright. All rights reserved.
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.
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.
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.
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.
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.
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.