TR(i)P Goes On: Auxiliary TRP Channel Subunits?

Transient receptor potential (TRP) cation channels are a diverse family of channels whose members play prominent roles as cellular sensors and effectors. The important role of TRP channels (and mechanosensitive piezo channels) in the complex interaction of our senses with the environment was underlined by the award of the Nobel Prize in Physiology or Medicine to 2 pioneers in this field, David Julius and Ardem Patapoutian. There are many competent and comprehensive reviews on many aspects of the TRP channels, and there is no intention to expand on them. Rather, after an introduction to the nomenclature, the molecular architecture of native TRP channel/protein complexes in vivo will be summarized using TRP channels of the canonical transient receptor potential subfamily as an example. This molecular architecture provides the basis for the signatures of native canonical transient receptor potential currents and their control by endogenous modulators and potential drugs.

Cavß3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalitis.

BACKGROUND: Tight control of cytoplasmic Ca2+ in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavß3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. METHODS: We investigated the function of Cavß3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavß3-/- (Cav ß3-deficient) mice as controls. RESULTS: We identified Cavß3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavß3. After induction of experimental autoimmune encephalomyelitis, Cavß3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavß3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavß3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavß3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavß3 with inositol 1,4,5-trisphosphate receptor proteins. CONCLUSIONS: Independent of its function as a subunit of Cav channels, Cavß3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavß3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.

TRPC4 and GIRK channels underlie neuronal coding of firing patterns that reflect Gq/11-Gi/o coincidence signals of variable strengths.

Transient receptor potential canonical 4 (TRPC4) is a receptor-operated cation channel codependent on both the Gq/11­phospholipase C signaling pathway and Gi/o proteins for activation. This makes TRPC4 an excellent coincidence sensor of neurotransmission through Gq/11- and Gi/o-coupled receptors. In whole-cell slice recordings of lateral septal neurons, TRPC4 mediates a strong depolarizing plateau that shuts down action potential firing, which may or may not be followed by a hyperpolarization that extends the firing pause to varying durations depending on the strength of Gi/o stimulation. We show that the depolarizing plateau is codependent on Gq/11-coupled group I metabotropic glutamate receptors and on Gi/o-coupled γ-aminobutyric acid type B receptors. The hyperpolarization is mediated by Gi/o activation of G protein­activated inwardly rectifying K+ (GIRK) channels. Moreover, the firing patterns, elicited by either electrical stimulation or receptor agonists, encode information about the relative strengths of Gq/11 and Gi/o inputs in the following fashion. Pure Gq/11 input produces weak depolarization accompanied by firing acceleration, whereas pure Gi/o input causes hyperpolarization that pauses firing. Although coincident Gq/11­Gi/o inputs also pause firing, the pause is preceded by a burst, and both the pause duration and firing recovery patterns reflect the relative strengths of Gq/11 versus Gi/o inputs. Computer simulations demonstrate that different combinations of TRPC4 and GIRK conductances are sufficient to produce the range of firing patterns observed experimentally. Thus, concurrent neurotransmission through the Gq/11 and Gi/o pathways is converted to discernible electrical responses by the joint actions of TRPC4 and GIRK for communication to downstream neurons.

Reconstitution of ß-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms.

L-type voltage-gated CaV1.2 channels crucially regulate cardiac muscle contraction. Activation of ß-adrenergic receptors (ß-AR) augments contraction via protein kinase A (PKA)-induced increase of calcium influx through CaV1.2 channels. To date, the full ß-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a CaV1.2 inhibitory protein, as essential for PKA regulation of CaV1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of ß1-AR or ß2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of CaV1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of ß-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified CaV1.2 variants and the distinct features of ß1-AR versus ß2-AR activity. This system allows for the addressing of central unresolved issues in the ß-AR-CaV1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Englerin A Inhibits T-Type Voltage-Gated Calcium Channels at Low Micromolar Concentrations.

Englerin A (EA) is a potent agonist of tetrameric transient receptor potential canonical (TRPC) ion channels containing TRPC4 and TRPC5 subunits. TRPC proteins form cation channels that are activated by plasma membrane receptors. They convert extracellular signals such as angiotensin II into cellular responses, whereupon Na+ and Ca2+ influx and depolarization of the plasma membrane occur. Via depolarization, voltage-gated Ca2+ (CaV) channels can be activated, further increasing Ca2+ influx. We investigated the extent to which EA also affects the functions of CaV channels using the high-voltage-activated L-type Ca2+ channel CaV1.2 and the low-voltage-activated T-type Ca2+ channels CaV3.1, CaV3.2, and CaV3.3. After expression of cDNAs in human embryonic kidney (HEK293) cells, EA inhibited currents through all T-type channels at half-maximal inhibitory concentrations (IC50) of 7.5 to 10.3 µM. In zona glomerulosa cells of the adrenal gland, angiotensin II-induced elevation of cytoplasmic Ca2+ concentration leads to aldosterone release. We identified transcripts of low- and high-voltage-activated CaV channels and of TRPC1 and TRPC5 in the human adrenocortical (HAC15) zona glomerulosa cell line. Although no EA-induced TRPC activity was measurable, Ca2+ channel blockers distinguished T- and L-type Ca2+ currents. EA blocked 60% of the CaV current in HAC15 cells and T- and L-type channels analyzed at -30 mV and 10 mV were inhibited with IC50 values of 2.3 and 2.6 µM, respectively. Although the T-type blocker Z944 reduced basal and angiotensin II-induced 24-hour aldosterone release, EA was not effective. In summary, we show here that EA blocks CaV1.2 and T-type CaV channels at low-micromolar concentrations. SIGNIFICANCE STATEMENT: In this study we showed that englerin A (EA), a potent agonist of tetrameric transient receptor potential canonical (TRPC)4- or TRPC5-containing channels and currently under investigation to treat certain types of cancer, also inhibits the L-type voltage-gated Ca2+ (CaV) channel CaV1.2 and the T-type CaV channels CaV3.1, CaV3.2, and CaV3.3 channels at low micromolar concentrations.

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.

Outside the limit: questioning the distance restrictions for cooperative miRNA binding sites.

Among the concepts in biology that are widely taken granted is a potentiated cooperative effect of multiple miRNAs on the same target. This strong hypothesis contrasts insufficient experimental evidence. The quantity as well as the quality of required side constraints of cooperative binding remain largely hidden. For miR-21-5p and miR-155-5p, two commonly investigated regulators across diseases, we selected 15 joint target genes. These were chosen to represent various neighboring 3'UTR binding site constellations, partially exceeding the distance rules that have been established for over a decade. We identified different cooperative scenarios with the binding of one miRNA enhancing the binding effects of the other miRNA and vice versa. Using both, reporter assays and whole proteome analyses, we observed these cooperative miRNA effects for genes that bear 3'UTR binding sites at distances greater than the previously defined limits. Astonishingly, the experiments provide even stronger evidence for cooperative miRNA effects than originally postulated. In the light of these findings the definition of targetomes specified for single miRNAs need to be refined by a concept that acknowledges the cooperative effects of miRNAs.

Activity of the yeast vacuolar TRP channel TRPY1 is inhibited by Ca2+-calmodulin binding.

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca2+ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1-6 yeast strain, carrying a non-Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1-6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+-CaM required the cytoplasmic amino acid stretch E33-Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+-CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+-CaM.

Stimulation of the EP3 receptor causes lung oedema by activation of TRPC6 in pulmonary endothelial cells.

BACKGROUND: Prostaglandin E2 (PGE2) increases pulmonary vascular permeability by activation of the PGE2 receptor 3 (EP3), which may explain adverse pulmonary effects of the EP1/EP3 receptor agonist sulprostone in patients. In addition, PGE2 contributes to pulmonary oedema in response to platelet-activating factor (PAF). PAF increases endothelial permeability by recruiting the cation channel transient receptor potential canonical 6 (TRPC6) to endothelial caveolae via acid sphingomyelinase (ASMase). Yet, the roles of PGE2 and EP3 in this pathway are unknown. We hypothesised that EP3 receptor activation may increase pulmonary vascular permeability by activation of TRPC6, and thus, synergise with ASMase-mediated TRPC6 recruitment in PAF-induced lung oedema. METHODS: In isolated lungs, we measured increases in endothelial calcium (ΔCa2+) or lung weight (Δweight), and endothelial caveolar TRPC6 abundance as well as phosphorylation. RESULTS: PAF-induced ΔCa2+ and Δweight were attenuated in EP3-deficient mice. Sulprostone replicated PAF-induced ΔCa2+ and Δweight which were blocked by pharmacological/genetic inhibition of TRPC6, ASMase or Src-family kinases (SrcFK). PAF, but not sulprostone, increased TRPC6 abundance in endothelial caveolae. Immunoprecipitation revealed PAF- and sulprostone-induced tyrosine-phosphorylation of TRPC6 that was prevented by inhibition of phospholipase C (PLC) or SrcFK. PLC inhibition also blocked sulprostone-induced ΔCa2+ and Δweight, as did inhibition of SrcFK or inhibitory G-protein (Gi) signalling. CONCLUSIONS: EP3 activation triggers pulmonary oedema via Gi-dependent activation of PLC and subsequent SrcFK-dependent tyrosine phosphorylation of TRPC6. In PAF-induced lung oedema, this TRPC6 activation coincides with ASMase-dependent caveolar recruitment of TRPC6, resulting in rapid endothelial Ca2+ influx and barrier failure.

TRPC channels regulate Ca2+-signaling and short-term plasticity of fast glutamatergic synapses.

Transient receptor potential (TRP) proteins form Ca2+-permeable, nonselective cation channels, but their role in neuronal Ca2+ homeostasis is elusive. In the present paper, we show that TRPC channels potently regulate synaptic plasticity by changing the presynaptic Ca2+-homeostasis of hippocampal neurons. Specifically, loss of TRPC1/C4/C5 channels decreases basal-evoked secretion, reduces the pool size of readily releasable vesicles, and accelerates synaptic depression during high-frequency stimulation (HFS). In contrast, primary TRPC5 channel-expressing neurons, identified by a novel TRPC5-τ-green fluorescent protein (τGFP) knockin mouse line, show strong short-term enhancement (STE) of synaptic signaling during HFS, indicating a key role of TRPC5 in short-term plasticity. Lentiviral expression of either TRPC1 or TRPC5 turns classic synaptic depression of wild-type neurons into STE, demonstrating that TRPCs are instrumental in regulating synaptic plasticity. Presynaptic Ca2+ imaging shows that TRPC activity strongly boosts synaptic Ca2+ dynamics, showing that TRPC channels provide an additional presynaptic Ca2+ entry pathway, which efficiently regulates synaptic strength and plasticity.

A proteomic atlas of the neointima identifies novel druggable targets for preventive therapy.

AIMS: In-stent restenosis is a complication after coronary stenting associated with morbidity and mortality. Here, we sought to investigate the molecular processes underlying neointima formation and to identify new treatment and prevention targets. METHODS AND RESULTS: Neointima formation was induced by wire injury in mouse femoral arteries. High-accuracy proteomic measurement of single femoral arteries to a depth of about 5000 proteins revealed massive proteome remodelling, with more than half of all proteins exhibiting expression differences between injured and non-injured vessels. We observed major changes in the composition of the extracellular matrix and cell migration processes. Among the latter, we identified the classical transient receptor potential channel 6 (TRPC6) to drive neointima formation. While Trpc6-/- mice presented reduced neointima formation compared to wild-type mice (1.44 ± 0.39 vs. 2.16 ± 0.48, P = 0.01), activating or repressing TRPC6 in human vascular smooth muscle cells resulted in increased [vehicle 156.9 ± 15.8 vs. 1-oleoyl-2-acetyl-sn-glycerol 179.1 ± 8.07 (103 pixels), P = 0.01] or decreased migratory capacity [vehicle 130.0 ± 26.1 vs. SAR7334 111.4 ± 38.0 (103 pixels), P = 0.04], respectively. In a cohort of individuals with angiographic follow-up (n = 3068, males: 69.9%, age: 59 ± 11 years, follow-up 217.1 ± 156.4 days), homozygous carriers of a common genetic variant associated with elevated TRPC6 expression were at increased risk of restenosis after coronary stenting (adjusted odds ratio 1.49, 95% confidence interval 1.08-2.05; P = 0.01). CONCLUSIONS: Our study provides a proteomic atlas of the healthy and injured arterial wall that can be used to define novel factors for therapeutic targeting. We present TRPC6 as an actionable target to prevent neointima formation secondary to vascular injury and stent implantation.

L-type CaV1.2 calcium channels: from in vitro findings to in vivo function.

The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca(2+) itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that ß-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVß2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca(2+)-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.

Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart.

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under ß-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.

Heteromeric channels formed by TRPC1, TRPC4 and TRPC5 define hippocampal synaptic transmission and working memory.

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high-resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5-triple-knockout (Trpc1/4/5-/-) mice, lacking any TRPC1-, TRPC4-, or TRPC5-containing channels, action potential-triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo, Trpc1/4/5-/- mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5-/- animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.

Defective survival of naive CD8+ T lymphocytes in the absence of the beta3 regulatory subunit of voltage-gated calcium channels.

The survival of T lymphocytes requires sustained, Ca(2+) influx-dependent gene expression. The molecular mechanism that governs sustained Ca(2+) influx in naive T lymphocytes is unknown. Here we report an essential role for the beta3 regulatory subunit of voltage-gated calcium (Ca(v)) channels in the maintenance of naive CD8(+) T cells. Deficiency in beta3 resulted in a profound survival defect of CD8(+) T cells. This defect correlated with depletion of the pore-forming subunit Ca(v)1.4 and attenuation of T cell antigen receptor (TCR)-mediated global Ca(2+) entry in CD8(+) T cells. Ca(v)1.4 and beta3 associated with T cell signaling machinery and Ca(v)1.4 localized in lipid rafts. Our data demonstrate a mechanism by which Ca(2+) entry is controlled by a Ca(v)1.4-beta3 channel complex in T cells.

Domain zipping and unzipping modulates TRPM4's properties in human cardiac conduction disease.

The transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cation channel linked to human cardiac diseases. The human mutation K914R within TRPM4's S4-S5 linker was identified in patients with atrioventricular block. During UV-flash-mediated Ca2+ transients, TRPM4K914R  generated a threefold augmented membrane current concomitant with 2 to 3-fold slowed down activation and deactivation kinetics resulting in excessive membrane currents during human cardiac action potentials. Mutagenesis of K914 paired with molecular modeling suggested the importance of the nanoscopic interface between the S4-S5 linker, the MHR4-, and TRP-domain as a major determinant for TRPM4's behavior. Rational mutagenesis of an interacting amino acid (R1062Q) in the TRP domain was able to offset K914R`s gain-of-function by zipping and unzipping of this nanoscopic interface. In conclusion, repulsion and attraction between the amino acids at positions 914 and 1062 alters the flexibility of the nanoscopic interface suggesting a zipping and unzipping mechanism that modulates TRPM4's functions. Pharmacological modulation of this intramolecular mechanism might represent a novel therapeutic strategy for the management of TRPM4-mediated cardiac diseases.

Tracheal brush cells release acetylcholine in response to bitter tastants for paracrine and autocrine signaling.

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.

Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems.

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography-high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely.

Toxicometabolomics of the new psychoactive substances α-PBP and α-PEP studied in HepaRG cell incubates by means of untargeted metabolomics revealed unexpected amino acid adducts.

Toxicometabolomics, essentially applying metabolomics to toxicology of endogenous compounds such as drugs of abuse or new psychoactive substances (NPS), can be investigated by using different in vitro models and dedicated metabolomics techniques to enhance the number of relevant findings. The present study aimed to study the toxicometabolomics of the two NPS α-pyrrolidinobutiophenone (1-phenyl-2-(pyrrolidin-1-yl)butan-1-one, α-PBP) and α-pyrrolidinoheptaphenone (1-phenyl-2-(pyrrolidin-1-yl)heptan-1-one, α-PEP, PV8) in HepaRG cell line incubates. Evaluation was performed using reversed-phase and normal-phase liquid chromatography coupled with high-resolution mass spectrometry in positive and negative ionization mode, respectively, to analyze cells and cell media. Statistical evaluation was performed using one-way ANOVA, principal component discriminant function analysis, as well as hierarchical clustering. In general, the analysis of cells did not mainly reveal any features, but the parent compounds of the drugs of abuse. For α-PBP an increase in N-methylnicotinamide was found, which may indicate hepatotoxic potential of the substance. After analysis of cell media, significant features led to the identification of several metabolites of both compounds. Amino acid adducts with glycine and alanine were found, and these have not been described in any study before and are likely to appear in vivo. Additionally, significant changes in the metabolism of cholesterol were revealed after incubation with α-PEP. In summary, the application of metabolomics techniques after HepaRG cells exposure to NPS did not lead to an increased number of identified drug metabolites compared to previously published studies, but gave a wider perspective on the physiological effect of the investigated compounds on human liver cells.

Transient Receptor Potential Vanilloid 6 (TRPV6) Proteins Control the Extracellular Matrix Structure of the Placental Labyrinth.

Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto-maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts upon functional deletion of TRPV6. Trpv6-/- trophoblasts show a distinct calcium-dependent phenotype. Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases, including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes are more abundantly expressed in Trpv6-/- trophoblast lysates, whereas the extracellular matrix protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly reduced. Trpv6-/-placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin degradation. Our results show that the extracellular matrix formation of the placental labyrinth depends on TRPV6; its deletion in trophoblasts correlates with the increased expression of proteases controlling the extracellular matrix in the labyrinth during pregnancy.