New insights in the renal distribution profile of TRPC3 - Of mice and men.

Several reports previously investigated the Transient Receptor Potential Canonical subfamily channel 3 (TRPC3) in the kidney. However, most of the conclusions are based on animal samples or cell cultures leaving the door open for human tissue investigations. Moreover, results often disagreed among investigators. Histological description is lacking since most of these studies focused on functional aspects. Nevertheless, the same reports highlighted the potential key-role of TRPC3 in renal disorders. Hence, our interest to investigate the localization of TRPC3 in human kidneys. For this purpose, both healthy mouse and human kidney samples that were originated from tumor nephrectomies have been prepared for immunohistochemical staining using a knockout-validated antibody. A blocking peptide was used to confirm antibody specificity. A normalized weighted diaminobenzidine (DAB) area score between 0 and 3 comparable to a pixelwise H-score was established and employed for semiquantitative analysis. Altogether, our results suggest that glomeruli only express little TRPC3 compared to several segments of the tubular system. Cortical and medullary proximal tubules are stained, although intracortical differences in staining exist in mice. Intermediate tubules, however, are only weakly stained. The distal tubule was studied in three localizations and staining was marked although slightly varying throughout the different subsegments. Finally, the collecting duct was also immunolabeled in both human and mouse tissue. We therefore provide evidence that TRPC3 is expressed in various localizations of both human and mouse samples. We verify results of previous studies and propose until now undescribed localizations of TRPC3 in the mouse but especially and of greater interest in the human kidney. We thereby not only support the translational concept of the TRPC3 channel as key-player in physiology and pathophysiology of the human kidney but also present new potential targets to functional analysis.

Human airway tuft cells influence the mucociliary clearance through cholinergic signalling.

BACKGROUND: Airway tuft cells, formerly called brush cells have long been described only morphologically in human airways. More recent RNAseq studies described a chemosensory cell population, which includes tuft cells, by a distinct gene transcription signature. Yet, until which level in the tracheobronchial tree in native human airway epithelium tuft cells occur and if they function as regulators of innate immunity, e.g., by regulating mucociliary clearance, remained largely elusive. METHODS: We performed immunohistochemistry, RT-PCR and immunoblotting analyses for various tuft cell markers to confirm the presence of this cell type in human tracheal samples. Immunohistochemistry was conducted to study the distribution of tuft cells along the intrapulmonary airways in humans. We assessed the influence of bitter substances and the taste transduction pathway on mucociliary clearance in mouse and human tracheal samples by measuring particle transport speed. RESULTS: Tuft cells identified by the expression of their well-established marker POU class 2 homeobox 3 (POU2F3) were present from the trachea to the bronchioles. We identified choline acetyltransferase in POU2F3 expressing cells as well as the transient receptor potential melastatin 5 (TRPM5) channel in a small population of tracheal epithelial cells with morphological appearance of tuft cells. Application of bitter substances, such as denatonium, led to an increase in mucociliary clearance in human tracheal preparations. This was dependent on activation of the TRPM5 channel and involved cholinergic and nitric oxide signalling, indicating a functional role for human tuft cells in the regulation of mucociliary clearance. CONCLUSIONS: We were able to detect tuft cells in the tracheobronchial tree down to the level of the bronchioles. Moreover, taste transduction and cholinergic signalling occur in the same cells and regulate mucociliary clearance. Thus, tuft cells are potentially involved in the regulation of innate immunity in human airways.

Caveolin-3 loss linked with the P104L LGMD-1C mutation modulates skeletal muscle mTORC1 signalling and cholesterol homeostasis.

BACKGROUND: Caveolins are the principal structural components of plasma membrane caveolae. Dominant pathogenic mutations in the muscle-specific caveolin-3 (Cav3) gene isoform, such as the limb girdle muscular dystrophy type 1C (LGMD-1C) P104L mutation, result in dramatic loss of the Cav3 protein and pathophysiological muscle weakness/wasting. We hypothesize that such muscle degeneration may be linked to disturbances in signalling events that impact protein turnover. Herein, we report studies assessing the effects of Cav3 deficiency on mammalian or mechanistic target of rapamycin complex 1 (mTORC1) signalling in skeletal muscle cells. METHODS: L6 myoblasts were stably transfected with Cav3P104L or expression of native Cav3 was abolished by CRISPR/Cas9 genome editing (Cav3 knockout [Cav3KO]) prior to performing subcellular fractionation and immunoblotting, analysis of real-time mitochondrial respiration or fixed cell immunocytochemistry. Skeletal muscle from wild-type and Cav3-/- mice was processed for immunoblot analysis of downstream mTORC1 substrate phosphorylation. RESULTS: Cav3 was detected in lysosomal-enriched membranes isolated from L6 myoblasts and observed by confocal microscopy to co-localize with lysosomal-specific markers. Cav3P104L expression, which results in significant (~95%) loss of native Cav3, or CRISPR/Cas9-mediated Cav3KO, reduced amino acid-dependent mTORC1 activation. The decline in mTORC1-directed signalling was detected by immunoblot analysis of L6 muscle cells and gastrocnemius Cav3-/- mouse muscle as judged by reduced phosphorylation of mTORC1 substrates that play key roles in the initiation of protein synthesis (4EBP1S65 and S6K1T389 ). S6K1T389 and 4EBP1S65 phosphorylation reduced by over 75% and 80% in Cav3KO muscle cells and by over 90% and 30% in Cav3-/- mouse skeletal muscle, respectively. The reduction in protein synthetic capacity in L6 muscle cells was confirmed by analysis of puromycylated peptides using the SUnSET assay. Cav3 loss was also associated with a 26% increase in lysosomal cholesterol, and pharmacological manipulation of lysosomal cholesterol was effective in replicating the reduction in mTORC1 activity observed in Cav3KO cells. Notably, re-expression of Cav3 in Cav3KO myoblasts normalized lysosomal cholesterol content, which coincided with a recovery in protein translation and an associated increase in mTORC1-directed phosphorylation of downstream targets. CONCLUSIONS: Our findings indicate that Cav3 can localize on lysosomal membranes and is a novel regulator of mTORC1 signalling in muscle. Cav3 deficiency associated with the Cav3P104L mutation impairs mTORC1 activation and protein synthetic capacity in skeletal muscle cells, which may be linked to disturbances in lysosomal cholesterol trafficking and contribute to the pathology of LGMD-1C.

Chemosensory cells in the respiratory tract as crucial regulators of innate immune responses.

During recent years chemosensory cells in extraoral tissues have been established as mediators for the detection and regulation of innate immune processes in response to pathogens. Under physiological conditions, chemosensory cells are present throughout the respiratory epithelium of the upper and lower airways as well as in the main olfactory epithelium. Additionally, they emerge in the alveolar region of the lung upon viral infections. Chemosensory cells in the upper and the lower airways detect signalling molecules from gram-positive and gram-negative bacteria as well as aeroallergens and fungi. Upon stimulation they release multiple molecules, such as the transmitter acetylcholine, the cysteinyl leukotriene E4 and the cytokine interleukin-25, which act as autocrine and paracrine signals and thereby orchestrate the innate immune responses in the respiratory system. Activation of chemosensory cells stimulates various immune cells, e.g. type 2 innate lymphoid cells, modulates mucociliary clearance and induces a protective neurogenic inflammation. This review compiles and discusses recent findings regarding chemosensory cell function in the respiratory tract.

Establishing an experimental Pseudomonas aeruginosa keratitis model in mice - Challenges and solutions.

BACKGROUND: With the ongoing increase in antimicrobial resistances seen in bacterial isolates causing a keratitis in humans, animal models have become an important tool to study new antimicrobial therapies. Nevertheless, the establishment of experimental keratitis is difficult. Here, we discuss the impact of different arrangements, including animal age, bacterial strain and dose as well as epithelium removal on the outcome of experimental keratitis. We therefore present the methods and results of our establishing experiments. METHODS: Bacterial load determination and flow cytometry were performed using eye homogenate gained from a 72 h lasting murine Pseudomonas aeruginosa keratitis model. Additionally, the intensity of the infection was scored from 0 to 5, the mice weighed, and blood immune cells counted. RESULTS: We found that older C57BL/6 N mice (8-11 months) are more susceptible to develop a keratitis than younger mice (5-6 weeks). Epithelium removal has no major impact on infectivity and disease progression in aged mice. P. aeruginosa exoU+ strains, such as PA54, should preferentially be used and highly concentrated (∼ 5 ×107 colony forming units CFU). Establishing an infection with the exoU- PAO1 derivative DSM 19880 was not possible. CONCLUSIONS: We present a replicable method to achieve a successful experimental P. aeruginosa keratitis in C57BL/6 N mice that is sustained or aggravated over the observation period of 3 days in 80 % of all animals tested. Our work is of particular interest to all researchers planning the establishment of such experimental models. We show some key aspects that can simplify and quicken the procedure, ultimately saving costs and animal life.

Pitfalls of using sequence databases for heterologous expression studies - a technical review.

Synthesis of DNA fragments based on gene sequences that are available in public resources has become an efficient and affordable method that has 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 characterisation 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.

Combining mass spectrometry and genetic labeling in mice to report TRP channel expression.

Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction. Detailed characterization of the expression pattern of the individual TRP channels throughout the organism is thus essential to interpret data such as those derived from systemic phenotyping of global TRP knockout mice. Murine TRP channel reporter strains enable reliable labeling of TRP expression with a fluorescent marker. Here we present an optimized method to visualize primary TRP-expressing cells with single cell resolution throughout the entire organism. In parallel, we methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest. The TRP protein expression data are then correlated with the GFP reporter expression data. The combined methodological approach presented here can be adopted to generate expression data for other genes of interest and reporter mice.•We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution.•We methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest in mice.

Characterization of the Secreted Acid Phosphatase SapS Reveals a Novel Virulence Factor of Staphylococcus aureus That Contributes to Survival and Virulence in Mice.

Staphylococcus aureus possesses a large arsenal of immune-modulating factors, enabling it to bypass the immune system's response. Here, we demonstrate that the acid phosphatase SapS is secreted during macrophage infection and promotes its intracellular survival in this type of immune cell. In animal models, the SA564 sapS mutant demonstrated a significantly lower bacterial burden in liver and renal tissues of mice at four days post infection in comparison to the wild type, along with lower pathogenicity in a zebrafish infection model. The SA564 sapS mutant elicits a lower inflammatory response in mice than the wild-type strain, while S. aureus cells harbouring a functional sapS induce a chemokine response that favours the recruitment of neutrophils to the infection site. Our in vitro and quantitative transcript analysis show that SapS has an effect on S. aureus capacity to adapt to oxidative stress during growth. SapS is also involved in S. aureus biofilm formation. Thus, this study shows for the first time that SapS plays a significant role during infection, most likely through inhibiting a variety of the host's defence mechanisms.

Taste Receptor Activation in Tracheal Brush Cells by Denatonium Modulates ENaC Channels via Ca2+, cAMP and ACh.

Mucociliary clearance is a primary defence mechanism of the airways consisting of two components, ciliary beating and transepithelial ion transport (ISC). Specialised chemosensory cholinergic epithelial cells, named brush cells (BC), are involved in regulating various physiological and immunological processes. However, it remains unclear if BC influence ISC. In murine tracheae, denatonium, a taste receptor agonist, reduced basal ISC in a concentration-dependent manner (EC50 397 µM). The inhibition of bitter taste signalling components with gallein (Gßγ subunits), U73122 (phospholipase C), 2-APB (IP3-receptors) or with TPPO (Trpm5, transient receptor potential-melastatin 5 channel) reduced the denatonium effect. Supportively, the ISC was also diminished in Trpm5-/- mice. Mecamylamine (nicotinic acetylcholine receptor, nAChR, inhibitor) and amiloride (epithelial sodium channel, ENaC, antagonist) decreased the denatonium effect. Additionally, the inhibition of Gα subunits (pertussis toxin) reduced the denatonium effect, while an inhibition of phosphodiesterase (IBMX) increased and of adenylate cyclase (forskolin) reversed the denatonium effect. The cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh172 and the KCNQ1 potassium channel antagonist chromanol 293B both reduced the denatonium effect. Thus, denatonium reduces ISC via the canonical bitter taste signalling cascade leading to the Trpm5-dependent nAChR-mediated inhibition of ENaC as well as Gα signalling leading to a reduction in cAMP-dependent ISC. Therefore, BC activation contributes to the regulation of fluid homeostasis.

Additional data for the mouse TRPV6 expression atlas.

To identify TRPV6 expression in the whole mouse with a cellular resolution we took advantage of TRPV6-IRES-Cre knock-in mice crossed with the enhanced ROSA26-τGFP reporter line. In the resulting TRPV6-IC/eR26-τGFP animals, TRPV6-expressing cells are labeled with τGFP. Data were collected from organs prepared from fixed experimental adult and juvenile TRPV6-IC/eR26τGFP and Cre-negative eR26-τGFP control animals of both sexes. Organ cryosections from each age and sex were stained for GFP and imaged with a slide scanner. Here, we describe reporter gene expression in a large number of tissues. We also document the absence of τGFP signal in the corresponding Cre-negative control tissues, including controls for the TRPV6 expression data described in [1]. The data reported here and in [1] constitute the TRPV6 expression atlas for the mouse. Our data offer a wealth of information to enable investigation of the functional role of TRPV6 channels in different tissues.

Bitter taste signaling in tracheal epithelial brush cells elicits innate immune responses to bacterial infection.

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.

Of Humans and Gerbils- Independent Diversification of Neuroligin-4 Into X- and Y-Specific Genes in Primates and Rodents.

The neural cell adhesion protein neuroligin-4 has puzzled neuroscientists and geneticist alike for almost two decades. Its clinical association with autism spectrum disorders (ASD) is well established, however, its diversification into sex chromosome-specific copies, NLGN4X and NLGN4Y, remains uncharted territory. Just recently, the presence of substantial neuroligin-4 sequence differences between humans and laboratory mice, in which Nlgn4 is a pseudoautosomal gene, could be explained as a consequence of dramatic changes affecting the pseudoautosomal region on both sex chromosomes in a subset of rodents, the clade eumuroida. In this study, we describe the presence of sex chromosome-specific copies of neuroligin-4 genes in the Mongolian gerbil (Meriones unguiculatus) marking the first encounter of its kind in rodents. Gerbils are members of the family Muridae and are closely related to mice and rats. Our results have been incorporated into an extended evolutionary analysis covering primates, rodents, lagomorphs, treeshrews and culogos comprising together the mammalian superorder euarchontoglires. We gathered evidence that substantial changes in neuroligin-4 genes have also occurred outside eumuroida in other rodent species as well as in lagomorphs. These changes feature, e.g., a general reduction of its gene size, an increase in its average GC-content as well as in the third position (GC3) of synonymous codons, and the accumulation of repetitive sequences in line with previous observations. We further show conclusively that the diversification of neuroligin-4 in sex chromosome-specific copies has happened multiple times independently during mammal evolution proving that Y-chromosomal NLGN4Y genes do not originate from a single common NLGN4Y ancestor.

The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation.

Staphylococcus epidermidis is a common cause of device related infections on which pathogens form biofilms (i.e., multilayered cell populations embedded in an extracellular matrix). Here, we report that the transcription factor SpoVG is essential for the capacity of S. epidermidis to form such biofilms on artificial surfaces under in vitro conditions. Inactivation of spoVG in the polysaccharide intercellular adhesin (PIA) producing S. epidermidis strain 1457 yielded a mutant that, unlike its parental strain, failed to produce a clear biofilm in a microtiter plate-based static biofilm assay. A decreased biofilm formation capacity was also observed when 1457 ΔspoVG cells were co-cultured with polyurethane-based peripheral venous catheter fragments under dynamic conditions, while the cis-complemented 1457 ΔspoVG::spoVG derivative formed biofilms comparable to the levels seen with the wild-type. Transcriptional studies demonstrated that the deletion of spoVG significantly altered the expression of the intercellular adhesion (ica) locus by upregulating the transcription of the ica operon repressor icaR and down-regulating the transcription of icaADBC. Electrophoretic mobility shift assays (EMSA) revealed an interaction between SpoVG and the icaA-icaR intergenic region, suggesting SpoVG to promote biofilm formation of S. epidermidis by modulating ica expression. However, when mice were challenged with the 1457 ΔspoVG mutant in a foreign body infection model, only marginal differences in biomasses produced on the infected catheter fragments between the mutant and the parental strain were observed. These findings suggest that SpoVG is critical for the PIA-dependent biofilm formation of S. epidermis under in vitro conditions, but is largely dispensable for biofilm formation of this skin commensal under in vivo conditions.

Nicotinic Acetylcholine Receptors in the Respiratory Tract.

Nicotinic acetylcholine receptors (nAChR) are widely distributed in neuronal and non-neuronal tissues, where they play diverse physiological roles. In this review, we highlight the recent findings regarding the role of nAChR in the respiratory tract with a special focus on the involvement of nAChR in the regulation of multiple processes in health and disease. We discuss the role of nAChR in mucociliary clearance, inflammation, and infection and in airway diseases such as asthma, chronic obstructive pulmonary disease, and cancer. The subtype diversity of nAChR enables differential regulation, making them a suitable pharmaceutical target in many diseases. The stimulation of the α3ß4 nAChR could be beneficial in diseases accompanied by impaired mucociliary clearance, and the anti-inflammatory effect due to an α7 nAChR stimulation could alleviate symptoms in diseases with chronic inflammation such as chronic obstructive pulmonary disease and asthma, while the inhibition of the α5 nAChR could potentially be applied in non-small cell lung cancer treatment. However, while clinical studies targeting nAChR in the airways are still lacking, we suggest that more detailed research into this topic and possible pharmaceutical applications could represent a valuable tool to alleviate the symptoms of diverse airway diseases.

A TRPV6 expression atlas for the mouse.

The transient receptor potential vanilloid 6 (TRPV6) channel is highly Ca2+-selective and has been implicated in mediating transcellular Ca2+ transport and thus maintaining the Ca2+ balance in the body. To characterize its physiological function(s), a detailed expression profile of the TRPV6 channel throughout the body is essential. Capitalizing on a recently established murine Trpv6-reporter strain, we identified primary TRPV6 channel-expressing cells in an organism-wide manner. In a complementary experimental approach, we characterized TRPV6 expression in different tissues of wild-type mice by TRPV6 immunoprecipitation (IP) followed by mass spectrometry analysis and correlated these data with the reporter gene expression. Taken together, we present a TRPV6 expression atlas throughout the entire body of juvenile and adult mice, providing a novel resource to investigate the role of TRPV6 channels in vivo.

Two Functional Epithelial Sodium Channel Isoforms Are Present in Rodents despite Pronounced Evolutionary Pseudogenization and Exon Fusion.

The epithelial sodium channel (ENaC) plays a key role in salt and water homeostasis in tetrapod vertebrates. There are four ENaC subunits (α, ß, γ, δ), forming heterotrimeric αßγ- or δßγ-ENaCs. Although the physiology of αßγ-ENaC is well understood, for decades the field has stalled with respect to δßγ-ENaC due to the lack of mammalian model organisms. The SCNN1D gene coding for δ-ENaC was previously believed to be absent in rodents, hindering studies using standard laboratory animals. We analyzed all currently available rodent genomes and discovered that SCNN1D is present in rodents but was independently lost in five rodent lineages, including the Muridae (mice and rats). The independent loss of SCNN1D in rodent lineages may be constrained by phylogeny and taxon-specific adaptation to dry habitats, however habitat aridity does not provide a selection pressure for maintenance of SCNN1D across Rodentia. A fusion of two exons coding for a structurally flexible region in the extracellular domain of δ-ENaC appeared in the Hystricognathi (a group that includes guinea pigs). This conserved pattern evolved at least 41 Ma and represents a new autapomorphic feature for this clade. Exon fusion does not impair functionality of guinea pig (Cavia porcellus) δßγ-ENaC expressed in Xenopus oocytes. Electrophysiological characterization at the whole-cell and single-channel level revealed conserved biophysical features and mechanisms controlling guinea pig αßγ- and δßγ-ENaC function as compared with human orthologs. Guinea pigs therefore represent commercially available mammalian model animals that will help shed light on the physiological function of δ-ENaC.

Of mice and men - and guinea pigs?

This year marks the twentieth anniversary of the publication of the first draft of the human genome and its broad availability to the scientific community. In parallel, the annotation of the mouse genome led to the identification and analysis of countless genes by means of genetic manipulation. Today, when comparing both genomes, it might surprise that some genes are still seeking their respective homologs in either species. In this review, we aim at raising awareness for the remarkable differences between the researcher's favorite rodents, i.e., mice and rats, when it comes to the generation of rodent research models regarding genes with a particular delicate localization, namely the pseudoautosomal region on both sex chromosomes. Many of these genes are of utmost clinical relevance in humans and still miss a rodent disease model giving their absence in mice and rats or low sequence similarity compared to humans. The abundance of rodents within mammals prompted us to investigate different branches of rodents leading us to the re-discovery of the guinea pig as a mammalian research model for a distinct group of genes.

Time is bone - Quantitative comparison of decalcification solvents in human femur samples using dual-X-ray-absorptiometry and computed tomography.

INTRODUCTION: Bone decalcification is a necessary preprocessing step in histological and anatomical studies. Several solutions for decalcification with different claimed times for full decalcification are commercially available. Current literature lacks direct, quantitative measurement of calcium hydrocyapatite degradation during decalcification to compare different solutions. Therefore, the aim of this study was to test the performance of three different decalcification solutions in human bone by direct measurement of calcium hydroxyapatite using dual-X-ray-absorptiometry (DEXA) and volumetric computed tomography (CT). METHODS: Four femur slices were acquired from the proximal femur of a 76-year-old body donor. The slices were submerged in formaldehyde (control), EDTA, Osteosoft (Merck, Darmstadt, Germany) and "Rapid Bone Decalcifier" (RBD) (American MasterTech Scientific, Lodi, USA). Consecutive DEXA and CT scans were performed at 2 h, 4 h, 8 h, 11 h, 20 h, 44 h and 77 h after solutions were added. Besides the calcium hydroxyapatite concentration, the bone volume was measured each time. RESULTS: Fastest decline in volume was seen in the RBD probe. Further, RBD was the only solution, being able to fully decalcify the bone slice after 77 h. Although a steady decline in volume and hydroxyapatite concentration was seen for EDTA and Osteosoft as well, both were not able to decalcify the slices. CONCLUSION: Overall, the purely qualititve acquired literature data on bone decalcifiers was verified by our quantitative data for human, cortical-rich bones. Hydrochloric-acid based solutions seem to be preferable in order to rapidly dissolve the calcium hydroxyapatite.

Nicotine stimulates ion transport via metabotropic ß4 subunit containing nicotinic ACh receptors.

BACKGROUND AND PURPOSE: Mucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown. EXPERIMENTAL APPROACH: Transepithelial short circuit currents (ISC ) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca2+ ]i were studied on freshly dissociated mouse tracheal epithelial cells. KEY RESULTS: Apical application of the nAChR agonist nicotine transiently increased ISC . The nicotine effect was abolished by the nAChR antagonist mecamylamine. α-Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3ß2, α4ß2, α4ß4 and α3ß4) and A-85380 (α4ß2 and α3ß4) increased ISC . The antagonists dihydro-ß-erythroidine (α4ß2, α3ß2, α4ß4 and α3ß4), α-conotoxin MII (α3ß2) and α-conotoxin PnIA (α3ß2) reduced the nicotine effect. Nicotine- and epibatidine-induced currents were unaltered in ß2-/- mice, but in ß4-/- mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+ -ATPase inhibitor) or the ryanodine receptor antagonists JTV-519 and dantrolene there was a reduction in the nicotine-effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H-89 and the TMEM16A (Ca2+ -activated chloride channel) inhibitor T16Ainh-A01 significantly reduced the nicotine-effect. CONCLUSION AND IMPLICATIONS: α3ß4 nAChRs are responsible for the nicotine-induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A.