OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

Experimental capture of miRNA targetomes: disease-specific 3'UTR library-based miRNA targetomics for Parkinson's disease.

The identification of targetomes remains a challenge given the pleiotropic effect of miRNAs, the limited effects of miRNAs on individual targets, and the sheer number of estimated miRNA-target gene interactions (MTIs), which is around 44,571,700. Currently, targetome identification for single miRNAs relies on computational evidence and functional studies covering smaller numbers of targets. To ensure that the targetome analysis could be experimentally verified by functional assays, we employed a systematic approach and explored the targetomes of four miRNAs (miR-129-5p, miR-129-1-3p, miR-133b, and miR-873-5p) by analyzing 410 predicted target genes, both of which were previously associated with Parkinson's disease (PD). After performing 13,536 transfections, we validated 442 of the 705 putative MTIs (62,7%) through dual luciferase reporter assays. These analyses increased the number of validated MTIs by at least 2.1-fold for miR-133b and by a maximum of 24.3-fold for miR-873-5p. Our study contributes to the experimental capture of miRNA targetomes by addressing i) the ratio of experimentally verified MTIs to predicted MTIs, ii) the sizes of disease-related miRNA targetomes, and iii) the density of MTI networks. A web service to support the analyses on the MTI level is available online ( https://ccb-web.cs.uni-saarland.de/utr-seremato ), and all the data have been added to the miRATBase database ( https://ccb-web.cs.uni-saarland.de/miratbase ).

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.

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.

Calcium channel ß3 subunit regulates ATP-dependent migration of dendritic cells.

Migratory dendritic cells (migDCs) continuously patrol tissues and are activated by injury and inflammation. Extracellular adenosine triphosphate (ATP) is released by damaged cells or actively secreted during inflammation and increases migDC motility. However, the underlying molecular mechanisms by which ATP accelerates migDC migration is not understood. Here, we show that migDCs can be distinguished from other DC subsets and immune cells by their expression of the voltage-gated calcium channel subunit ß3 (Cavß3; CACNB3), which exclusively facilitates ATP-dependent migration in vitro and during tissue damage in vivo. By contrast, CACNB3 does not regulate lipopolysaccharide-dependent migration. Mechanistically, CACNB3 regulates ATP-dependent inositol 1,4,5-trisphophate receptor-controlled calcium release from the endoplasmic reticulum. This, in turn, is required for ATP-mediated suppression of adhesion molecules, their detachment, and initiation of migDC migration. Thus, Cacnb3-deficient migDCs have an impaired migration after ATP exposure. In summary, we identified CACNB3 as a master regulator of ATP-dependent migDC migration that controls tissue-specific immunological responses during injury and inflammation.

Maternal Epidermal Growth Factor Promotes Neonatal Claudin-2 Dependent Increases in Small Intestinal Calcium Permeability.

A higher concentration of calcium in breast milk than blood favors paracellular calcium absorption enabling growth during postnatal development. We aimed to determine whether suckling animals have greater intestinal calcium permeability to maximize absorption and to identify the underlying molecular mechanism. We examined intestinal claudin expression at different ages in mice and in human intestinal epithelial (Caco-2) cells in response to hormones or human milk. We also measured intestinal calcium permeability in wildtype, Cldn2 and Cldn12 KO mice and Caco-2 cells in response to hormones or human milk. Bone mineralization in mice was assessed by µCT. Calcium permeability across the jejunum and ileum of mice were 2-fold greater at 2 wk than 2 mo postnatal age. At 2 wk, Cldn2 and Cldn12 expression were greater, but only Cldn2 KO mice had decreased calcium permeability compared to wildtype. This translated to decreased bone volume, cross-sectional thickness, and tissue mineral density of femurs. Weaning from breast milk led to a 50% decrease in Cldn2 expression in the jejunum and ileum. Epidermal growth factor (EGF) in breast milk specifically increased only CLDN2 expression and calcium permeability in Caco-2 cells. These data support intestinal permeability to calcium, conferred by claudin-2, being greater in suckling mice and being driven by EGF in breast milk. Loss of the CLDN2 pathway leads to suboptimal bone mineralization at 2 wk of life. Overall, EGF-mediated control of intestinal claudin-2 expression contributes to maximal intestinal calcium absorption in suckling animals.

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.

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.

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.

Subunit composition, molecular environment, and activation of native TRPC channels encoded by their interactomes.

In the mammalian brain TRPC channels, a family of Ca2+-permeable cation channels, are involved in a variety of processes from neuronal growth and synapse formation to transmitter release, synaptic transmission and plasticity. The molecular appearance and operation of native TRPC channels, however, remained poorly understood. Here, we used high-resolution proteomics to show that TRPC channels in the rodent brain are macro-molecular complexes of more than 1 MDa in size that result from the co-assembly of the tetrameric channel core with an ensemble of interacting proteins (interactome). The core(s) of TRPC1-, C4-, and C5-containing channels are mostly heteromers with defined stoichiometries for each subtype, whereas TRPC3, C6, and C7 preferentially form homomers. In addition, TRPC1/C4/C5 channels may co-assemble with the metabotropic glutamate receptor mGluR1, thus guaranteeing both specificity and reliability of channel activation via the phospholipase-Ca2+ pathway. Our results unveil the subunit composition of native TRPC channels and resolve the molecular details underlying their activation.

Ca2+ transport via TRPV6 is regulated by rapid internalization of the channel.

Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca2+-selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane. In TRPV6-expressing cells, brefeldin A, a toxin that blocks the transit between the endoplasmic reticulum and the Golgi apparatus, caused a drop in [Ca2+]i with a half time in the range of 0.5-1 h. Upon wash-out of the toxin, the [Ca2+]i rose to a steady-state level within 2-3 h. Consistently, the synchronized forward trafficking of TRPV6VL-eGFP after brefeldin A wash-out led to a visible accumulation of the protein within the plasma membrane, as shown by confocal and total internal reflection microscopy. Analysis of the internalization route and differentiation of vesicle populations provided evidence for a clathrin-dependent internalization pathway. Most TRPV6VL-bearing vesicles co-stained with Rab5a, a marker protein for early endosomes. Fewer vesicles were co-localized with Rab7a (late endosomes) or with Rab11 (recycling endosomes). From these data, we propose that the lack of plasma membrane visibility of the channel results from a rapid internalization, which in addition to transcriptional regulation, adds a layer of functional channel regulation to modulate transepithelial Ca2+ transport.

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.

A simplified strategy to assess the cytotoxicity of new psychoactive substances in HepG2 cells using a high content screening assay - Exemplified for nine compounds.

New psychoactive substances (NPS) are an issue of global concern posing a serious threat to the healthcare systems. Consumption of some NPS has been associated with toxic effects on the liver amongst others. However, data concerning their (cyto-)toxicity are usually not available. For a straightforward assessment of their cytotoxic potential, a simplified strategy measuring six different cytotoxicity indicating parameters simultaneously by a high content screening assay (HCSA) was developed. Its applicability was further investigated using nine NPS from heterogeneous chemical classes. HepG2 cells were incubated with NPS for 48 h at a low and high concentration (7.81 and 125 µM), respectively. To study metabolism-mediated effects on their cytotoxicity, cells were additionally incubated with the unspecific cytochrome (CYP) P450 inhibitor 1-aminobenzotriazole. Four fluorescence dyes were used to monitor cell count, nuclear size, and nuclear intensity (all Hoechst33342), mitochondrial membrane potential (TMRM), cytoplasmic calcium levels (CAL-520), and plasma membrane integrity (TOTO-3). Amongst the investigated NPS, ephylone, CUMYL-CBMICA, and dibutylone showed a strong cytotoxic potential, affecting two parameters at 7.81 µM. 5-MeO-MiPT showed moderate effects by impairing one parameter at 7.81 and one at 125 µM. Furthermore, at the high concentration of 5-MeO-MiPT, an effect of metabolism on cytotoxicity was observed. The HCSA confirmed the cytotoxic potential of ephylone and 5-MeO-MiPT, as the investigated concentrations were in the range of their published blood concentrations which induced liver damages after intake. The mitochondrial membrane potential was the parameter with the highest sensitivity and thus considered as suitable "cytobiomarker". In turn, parameters showing a high variability or unexpected effects such as cytosolic calcium levels and plasma membrane integrity might be omitted in the future. Even though 5-MeO-MiPT showed metabolism-based effects, HepG2 are known to have limited metabolic activity compared to cell lines such as HepaRG. Therefore, in further experiments cell lines with higher CYP-expression needs to be included and findings compared. Nevertheless, the simplified HCSA-based strategy allowed to screen NPS from diverse chemical groups for a first assessment of the cytotoxic properties of the parent compound. This information is crucial for a thorough risk assessment of NPS not only for public health authorities.

Localization of the Priming Factors CAPS1 and CAPS2 in Mouse Sensory Neurons Is Determined by Their N-Termini.

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using - in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses.

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.

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.

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.

IP3-dependent Ca2+ signals are tightly controlled by Cavß3, but not by Cavß1, 2 and 4.

Independent of its function as a subunit of voltage-gated Ca2+ channels, the Cavß3 subunit desensitizes fibroblasts and pancreatic ß-cells to low concentrations of inositol-1,4,5-trisphosphate (IP3). This alters agonist-induced Ca2+ signaling and cellular functions, for example, insulin secretion and wound healing. A total of four Cavß subunits exist, Cavß1, Cavß2, Cavß3, and Cavß4. To investigate whether the other Cavß subunits, like Cavß3, can desensitize cells to IP3 and thereby modulate Ca2+ signaling, we expressed the cDNAs of Cavß1, Cavß2, Cavß3, and Cavß4 in COS-7 cells lacking endogenous Cavß proteins. ATP stimulation of these cells results in the release of Ca2+ from intracellular stores. This receptor-mediated Ca2+ release is significantly decreased by Cavß3 but not by Cavß1, Cavß2, and Cavß4. Electrophysiological recordings of voltage-dependent Ca2+ currents from fibroblasts show a small Ca2+ current, the amplitude of which is slightly but not significantly smaller in fibroblasts from Cavß2 gene-deficient animals than in fibroblasts from wild-type animals. Compared with fibroblasts from wild-type animals, Ca2+ release is not significantly increased in Cavß2-deficient fibroblasts, in contrast to Ca2+ release in Cavß3-deficient fibroblasts. In summary, our results show that desensitization of cells to low concentrations of IP3 is a specific property of Cavß3 that is not shared by other Cavß subunits.

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.

Is the proteomic composition of the salivary pellicle dependent on the substrate material?

PURPOSE: The use of dental restorative materials is a routine task in clinical dentistry. Upon exposure to the oral cavity, continuous adsorption of salivary proteins and other macromolecules to all surfaces occurs, representing the first step in dental biofilm formation. Different physico-chemical properties of substrate materials potentially influence the composition of the initial biofilm, termed pellicle. This study aimed at characterizing and comparing the individual proteomic composition of the 3-min pellicle formed on bovine enamel and six restorative materials. EXPERIMENTAL DESIGN: After chemical elution, pellicle proteins were identified by nano-LC-HR-MS/MS. Proteomic profiles were analyzed in terms of molecular weights, isoelectric points, molecular functions and compared to saliva to reveal substrate material-specific adsorption patterns. RESULTS: A total of 1348 different pellicle proteins were identified, with 187-686 proteins in individual 3-min pellicles. Unexpectedly, this yielded quite similar distribution patterns independent of the substrate materials. Furthermore, overall similar fold changes were obtained for the major part of commonly enriched or depleted proteins in the pellicles. CONCLUSIONS AND CLINICAL RELEVANCE: The current results point to a minor role of the substrate material on the proteomic composition of the 3-min pellicle and represent core data for understanding the complex surface interactions in the oral cavity.