Tas1R3 Dependent and Independent Recognition of Sugars in the Urethra and the Role of Tuft Cells in this Process.

Increased sugar concentrations on mucosal surfaces display risk factors for infections. This study aims to clarify sugar monitoring in the urethra. Urethral tuft cells (UTC) are known sentinels monitoring the urethral lumen for potentially harmful substances and initiating protective mechanisms. Next-generation sequencing (NGS), RT-PCR, and immunohistochemistry show expression of the taste receptor Tas1R3 in murine UTC, a crucial component of the classical sweet detection pathway. Isolated UTC respond to various sugars with an increase of intracellular [Ca2+]. The Tas1R3 inhibitor gurmarin and Tas1R3 deletion reduces these responses. Utilizing mice lacking UTC, glibenclamide, a K+-ATP channel antagonist, and phlorizin, a SGLT1 inhibitor, reveal an additional Tas1R3 independent sweet detection pathway. Inhibition of both pathways abrogates the sugar responses. Rat cystometry shows that intraurethral application of sucrose and glucose increases detrusor muscle activity Tas1R3 dependently. Sugar monitoring in the urethra occurs via two distinct pathways. A Tas1R3 dependent pathway, exclusive to UTC, and a Tas1R3 independent sweet detection pathway, which can be found both in UTC and in other urethral epithelial cells.

A succinate/SUCNR1-brush cell defense program in the tracheal epithelium.

Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cß2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.

Secondary Metabolites in Allergic Plant Pollen Samples Modulate Afferent Neurons and Murine Tracheal Rings.

Plant pollens are strong airborne elicitors of asthma. Their proteinaceous allergens have been studied intensively, but little is known about a possible contribution of pollen secondary metabolites to the nonallergic exacerbation of asthma. Pollen samples originating from 30 plant species were analyzed by HPLC coupled to PDA, ESIMS, and ELSD detectors and off-line NMR spectroscopy. Polyamine conjugates, flavonoids, and sesquiterpene lactones were identified. Polyamine conjugates were characteristic of all Asteraceae species. The presence of sesquiterpene lactones in Asteraceae pollen varied between species and pollen lots. All plant pollen, including those from non-Asteraceae species, contained to some extent electrophiles as determined by their reaction with N-acetyl-l-cysteine. Selected pollen extracts and pure compounds were tested in murine afferent neurons and in murine tracheal preparations. Tetrahydrofuran extracts of Ambrosia artemisiifolia and Ambrosia psilostachya pollen and a mixture of sesquiterpene lactones coronopilin/parthenin increased the intracellular Ca2+ concentration in 15%, 32%, and 37% of cinnamaldehyde-responsive neurons, respectively. In organ bath experiments, only the sesquiterpene lactones tested induced a weak dilatation of naïve tracheas and strongly lowered the maximal methacholine-induced tracheal constriction. A tetrahydrofuran extract of A. psilostachya and coronopilin/parthenin led to a time-dependent relaxation of the methacholine-preconstricted trachea. These results provide the first evidence for a potential role of pollen secondary metabolites in the modulation of the tracheal tone.

Caveolin-1: Functional Insights into Its Role in Muscarine- and Serotonin-Induced Smooth Muscle Constriction in Murine Airways.

An increased bronchoconstrictor response is a hallmark in the progression of obstructive airway diseases. Acetylcholine and 5-hydroxytryptamine (5-HT, serotonin) are the major bronchoconstrictors. There is evidence that both cholinergic and serotonergic signaling in airway smooth muscle (ASM) involve caveolae. We hypothesized that caveolin-1 (cav-1), a structural protein of caveolae, plays an important regulatory role in ASM contraction. We analyzed airway contraction in different tracheal segments and extra- and intrapulmonary bronchi in cav-1 deficient (cav-1-/-) and wild-type mice using organ bath recordings and videomorphometry of methyl-beta-cyclodextrin (MCD) treated and non-treated precision-cut lung slices (PCLS). The presence of caveolae was investigated by electron microscopy. Receptor subtypes driving 5-HT-responses were studied by RT-PCR and videomorphometry after pharmacological inhibition with ketanserin. Cav-1 was present in tracheal epithelium and ASM. Muscarine induced a dose dependent contraction in all airway segments. A significantly higher Emax was observed in the caudal trachea. Although, caveolae abundancy was largely reduced in cav-1-/- mice, muscarine-induced airway contraction was maintained, albeit at diminished potency in the middle trachea, in the caudal trachea and in the bronchus without changes in the maximum efficacy. MCD-treatment of PLCS from cav-1-/- mice reduced cholinergic constriction by about 50%, indicating that cholesterol-rich plasma domains account for a substantial portion of the muscarine-induced bronchoconstriction. Notably, cav-1-deficiency fully abrogated 5-HT-induced contraction of extrapulmonary airways. In contrast, 5-HT-induced bronchoconstriction was fully maintained in cav-1-deficient intrapulmonary bronchi, but desensitization upon repetitive stimulation was enhanced. RT-PCR analysis revealed 5-HT1B, 5-HT2A, 5-HT6, and 5-HT7 receptors as the most prevalent subtypes in the airways. The 5-HT-induced-constriction in PCLS could be antagonized by ketanserin, a 5-HT2A receptor inhibitor. In conclusion, the role of cav-1, caveolae, and cholesterol-rich plasma domains in regulation of airway tone are highly agonist-specific and dependent on airway level. Cav-1 is indispensable for serotonergic contraction of extrapulmonary airways and modulates cholinergic constriction of the trachea and main bronchus. Thus, cav-1/caveolae shall be considered in settings such as bronchial hyperreactivity in common airway diseases and might provide an opportunity for modulation of the constrictor response.

Cholinergic activation of the murine trachealis muscle via non-vesicular acetylcholine release involving low-affinity choline transporters.

In addition to quantal, vesicular release of acetylcholine (ACh), there is also non-quantal release at the motor endplate which is insufficient to evoke postsynaptic responses unless acetylcholinesterase (AChE) is inhibited. We here addressed potential non-quantal release in the mouse trachea by organ bath experiments and (immuno)histochemical methods. Electrical field stimulation (EFS) of nerve terminals elicited tracheal constriction that is largely due to ACh release. Classical enzyme histochemistry demonstrated acetylcholinesterase (AChE) activity in nerve fibers in the muscle and butyrylcholinesterase (BChE) activity in the smooth muscle cells. Acute inhibition of both esterases by eserine significantly raised tracheal tone which was fully sensitive to atropine. This effect was reduced, but not abolished, in AChE, but not in BChE gene-deficient mice. The eserine-induced increase in tracheal tone was unaffected by vesamicol (10(-5)M), an inhibitor of the vesicular acetylcholine transporter, and by corticosterone (10(-4)M), an inhibitor of organic cation transporters. Hemicholinium-3, in low concentrations an inhibitor of the high-affinity choline transporter-1 (CHT1), completely abrogated the eserine effects when applied in high concentrations (10(-4)M) pointing towards an involvement of low-affinity choline transporters. To evaluate the cellular sources of non-quantal ACh release in the trachea, expression of low-affinity choline transporter-like family (CTL1-5) was evaluated by RT-PCR analysis. Even though these transporters were largely abundant in the epithelium, denudation of airway epithelial cells had no effect on eserine-induced tracheal contraction, indicating a non-quantal release of ACh from non-epithelial sources in the airways. These data provide evidence for an epithelium-independent non-vesicular, non-quantal ACh release in the mouse trachea involving low-affinity choline transporters.

Mental stress in atopic dermatitis--neuronal plasticity and the cholinergic system are affected in atopic dermatitis and in response to acute experimental mental stress in a randomized controlled pilot study.

RATIONALE: In mouse models for atopic dermatitis (AD) hypothalamus pituitary adrenal axis (HPA) dysfunction and neuropeptide-dependent neurogenic inflammation explain stress-aggravated flares to some extent. Lately, cholinergic signaling has emerged as a link between innate and adaptive immunity as well as stress responses in chronic inflammatory diseases. Here we aim to determine in humans the impact of acute stress on neuro-immune interaction as well as on the non-neuronal cholinergic system (NNCS). METHODS: Skin biopsies were obtained from 22 individuals (AD patients and matched healthy control subjects) before and after the Trier social stress test (TSST). To assess neuro-immune interaction, nerve fiber (NF)-density, NF-mast cell contacts and mast cell activation were determined by immunohistomorphometry. To evaluate NNCS effects, expression of secreted mammal Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP) 1 and 2 (endogenous nicotinic acetylcholine receptor ligands) and their main corresponding receptors were assessed by quantitative RT-PCR. RESULTS: With respect to neuro-immune interaction we found higher numbers of NGF+ dermal NF in lesional compared to non-lesional AD but lower numbers of Gap43+ growing NF at baseline. Mast cell-NF contacts correlated with SCORAD and itch in lesional skin. With respect to the NNCS, nicotinic acetylcholine receptor α7 (α7nAChR) mRNA was significantly lower in lesional AD skin at baseline. After TSST, PGP 9.5+ NF numbers dropped in lesional AD as did their contacts with mast cells. NGF+ NF now correlated with SCORAD and mast cell-NF contacts with itch in non-lesional skin. At the same time, SLURP-2 levels increased in lesional AD skin. CONCLUSIONS: In humans chronic inflammatory and highly acute psycho-emotional stress interact to modulate cutaneous neuro-immune communication and NNCS marker expression. These findings may have consequences for understanding and treatment of chronic inflammatory diseases in the future.

Adrenomedullin and the calcitonin receptor-like receptor system mRNA expressions in the rat heart and sensory ganglia in experimentally-induced long-term diabetes.

Both adrenomedullin and calcitonin gene-related peptide (CGRP) regulate vascular tone in the heart, being cardioprotective in hypoxia. Additionally, adrenomedullin exhibits antiproliferative and antiapoptotic functions in the myocardium, while CGRP exerts positive chronotropic effect. Their actions are mediated through the specific G protein-coupled receptor, CRLR, whose ligand affinity is determined by receptor activity modifying proteins RAMP1-3. CGRP binds to the complex formed by CRLR/RAMP1, whereas CRLR/RAMP2 and CRLR/RAMP3 serve as receptors for adrenomedullin. Here, we quantified expression of this signaling system in the rat heart and supplying sensory ganglia (dorsal root ganglia T1-T4 and vagal nodose ganglia) in streptozotocin-induced diabetes. In the course of diabetes, an increase of CRLR mRNA was noticed in the right ventricle 8 weeks and of RAMP3 mRNA in the left ventricle and right atrium 26 weeks after induction of diabetes. Relative expressions of other tested genes were not significantly altered. In the nodose vagal supplying specific cardiac afferents, but not in dorsal root ganglia which provide cardiac pain fibres, a small upregulation of CGRP expression was detected. In summary, the shifts observed in diabetes may favour a trend of a pronounced adrenomedullin signaling. These observations may provide a new possible therapeutic strategy for diabetic cardiomyopathy.

Intermedin: a skin peptide that is downregulated in atopic dermatitis.

Intermedin (IMD), also called adrenomedullin-2, is a peptide that belongs to the calcitonin/calcitonin gene-related peptide/amylin peptide family. IMD exerts many effects on the cardiovascular system, gastrointestinal tract, and central nervous system. Here, we analyzed the expression of the IMD peptide in human skin of healthy controls, in biopsies from lesional and non-lesional areas of atopic dermatitis (AD) skin, in cultured human keratinocytes, and in the HaCaT keratinocyte cell line at the transcriptional (quantitative reverse transcription-PCR) and translational (immunohistochemistry) level. IMD messenger RNA (mRNA) and protein could be detected in keratinocytes and human skin. Keratinocytes, nerve fibers, periglandular cells, arterial/arteriolar smooth muscle cells, and pericytes of dermal microvessels were intensely IMD-immunoreactive. The IMD mRNA was, compared to healthy skin, significantly reduced in lesional and non-lesional areas of AD skin. This was accompanied by a reduction of IMD immunoreactivity in pericytes of the upper dermis indicating that skin from AD patients is generally affected, and downregulation of IMD in AD skin is not a secondary phenomenon caused by acute inflammation but is a general characteristic of AD skin. These data further point to a role of IMD expressed by pericytes in conferring higher susceptibility of the skin of AD patients to inflammatory stimuli.

Role of acetylcholine and muscarinic receptors in serotonin-induced bronchoconstriction in the mouse.

For the murine trachea, it has been reported that constriction evoked by serotonin (5-HT) is largely dependent on acetylcholine (ACh) released from the epithelium, owing to the sensitivity of the 5-HT response to epithelium removal, sensitivity to atropine, and insensitivity to tetrodotoxin (Moffatt et al., 2003). Consistent with this assumption, the respiratory epithelium contains ACh, its synthesizing enzyme, and the high-affinity choline transporter CHT1 (Reinheimer et al., 1996; Pfeil et al., 2003; Proskocil et al., 2004). Recently, we demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells (Lips et al., 2005). Hence, we proposed that 5-HT evokes release of ACh from epithelial cells via OCTs and that this epithelial-derived ACh induces bronchoconstriction. We tested this hypothesis in a well-established model of videomorphometric analysis of bronchial diameter in precision-cut murine lung slices utilizing epithelium removal to assess the role of the epithelium, OCT mouse knockout (KO) strains to assess the role of OCT isoforms, and muscarinic receptor M2/M3 double-KO mice to assess the cholinergic component of 5-HT induced bronchoconstriction, as bronchi of this strain are entirely unresponsive to cholinergic stimulation(Struckmann et al., 2003).

Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse.

BACKGROUND: It has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs. METHODS: We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry. RESULTS: Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 +/- 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 +/- 3.6 %, n = 11). In wild-type mice, 5-HT (1 microM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 microM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 microM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle. CONCLUSION: The doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.

Activation of the SPHK/S1P signalling pathway is coupled to muscarinic receptor-dependent regulation of peripheral airways.

BACKGROUND: In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M2R and M3R). Cholinergic hypersensitivity is associated with chronic obstructive pulmonary disease and asthma, and therefore the identification of muscarinic signaling pathways are of great therapeutic interest. A pathway that has been shown to be activated via MR and to increase [Ca2+]i includes the activation of sphingosine kinases (SPHK) and the generation of the bioactive sphingolipid sphingosine 1-phosphate (S1P). Whether the SPHK/S1P signaling pathway is integrated in the muscarinic control of peripheral airways is not known. METHODS: To address this issue, we studied precision cut lung slices derived from FVB and M2R-KO and M3R-KO mice. RESULTS: In peripheral airways of FVB, wild-type, and MR-deficient mice, SPHK1 was mainly localized to smooth muscle. Muscarine induced a constriction in all investigated mouse strains which was reduced by inhibition of SPHK using D, L-threo-dihydrosphingosine (DHS) and N, N-dimethyl-sphingosine (DMS) but not by N-acetylsphingosine (N-AcS), a structurally related agent that does not affect SPHK function. The initial phase of constriction was nearly absent in peripheral airways of M3R-KO mice when SPHK was inhibited by DHS and DMS but was unaffected in M2R-KO mice. Quantitative RT-PCR revealed that the disruption of the M2R and M3R genes had no significant effect on the expression levels of the SPHK1-isoform in peripheral airways. CONCLUSION: These results demonstrate that the SPHK/S1P signaling pathway contributes to cholinergic constriction of murine peripheral airways. In addition, our data strongly suggest that SPHK is activated via the M2R. Given the important role of muscarinic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance.

Role of muscarinic receptor subtypes in the constriction of peripheral airways: studies on receptor-deficient mice.

In the airways, increases in cholinergic nerve activity and cholinergic hypersensitivity are associated with chronic obstructive pulmonary disease and asthma. However, the contribution of individual muscarinic acetylcholine receptor subtypes to the constriction of smaller intrapulmonary airways that are primarily responsible for airway resistance has not been analyzed. To address this issue, we used videomicroscopy and digital imaging of precision-cut lung slices derived from wild-type mice and mice deficient in either the M1 (mAChR1-/- mice), M2 (mAChR2-/- mice), or M3 receptor subtype (mAChR3-/- mice) or lacking both the M2 and M3 receptor subtypes (mAChR2/3-/- double-knockout mice). In peripheral airways from wild-type mice (mAChR+/+ mice), muscarine induced a triphasic concentration-dependent response, characterized by an initial constriction, a transient relaxation, and a sustained constriction. The bronchoconstriction was diminished by up to 60% in mAChR3-/- lungs and was completely abolished in mAChR2/3-/- lungs. The sustained bronchoconstriction was reduced in mAChR2-/- bronchi, and, interestingly, the transient relaxation was absent; the bronchoconstriction in response to 10-8 M muscarine was increased by 158% in mAChR1-/- mice. Quantitative reverse transcriptase-polymerase chain reaction analysis revealed that the disruption of specific mAChR genes had no significant effect on the expression levels of the remaining mAChR subtypes. These results demonstrate that cholinergic constriction of murine peripheral airways is mediated by the concerted action of the M2 and M3 receptor subtypes and suggest the existence of pulmonary M1 receptor activation, which counteracts cholinergic bronchoconstriction. Given the important role of muscarinic cholinergic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance.