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 M 1 (mAChR1 Ϫ/Ϫ mice), M 2 (mAChR2 Ϫ/Ϫ mice), or M 3 receptor subtype (mAChR3 Ϫ/Ϫ mice) or lacking both the M 2 and M 3 receptor subtypes (mAChR2/ 3 Ϫ/Ϫ double-knockout mice). In peripheral airways from wildtype mice (mAChR ϩ/ϩ 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 Ϫ/Ϫ lungs and was completely abolished in mAChR2/ 3 Ϫ/Ϫ lungs. The sustained bronchoconstriction was reduced in mAChR2 Ϫ/Ϫ bronchi, and, interestingly, the transient relaxation was absent; the bronchoconstriction in response to 10 Ϫ8 M muscarine was increased by 158% in mAChR1 Ϫ/Ϫ 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 M 2 and M 3 receptor subtypes and suggest the existence of pulmonary M 1 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.

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.

RationaleIn 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).MethodsSkin 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.ResultsWith 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.ConclusionsIn 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.

Background: In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M 2 R and M 3 R). 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 [Ca 2+ ] 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.

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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).

Targeted delivery of liposomal dexamethasone phosphate to the spleen provides a persistent therapeutic effect in rat antigeninduced arthritis Although long-term suppression of experimental arthritis has been reported for liposomal glucocorticoids, direct effects on subsequent flare-up reactions have not been investigated. The glucocorticoid dexamethasone phosphate (DXM-P) was

This supports the idea that the cholinergic system is dysregulated independently from inflammation in AD and that inflammation further modulates individual nAChR subunits.