Antagonism of m3 Alleviates Type 2 Inflammation in Allergic Rhinitis Mice.

BACKGROUND: Type 2 immune cells play a pivotal role in allergic rhinitis (AR). Increasing evidence shows that inhibition of cholinergic nerve activity decreases the severity of airway diseases including asthma and AR. However, the role of the cholinergic receptor muscarinic 3 (m3) in type 2 inflammation in AR is unknown. OBJECTIVE: We aimed to investigate the effect of m3 on the type 2 immune response, including both T helper 2 (Th2)-mediated and type 2 innate lymphocyte (ILC2)-mediated inflammation, in AR. METHODS: Peripheral blood mononuclear cells (PBMCs) from human were cultured in vitro. Treatment with the m3 antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) was used. The percentages of Th2 and ILC2 cells in PBMCs were evaluated by flow cytometry. AR mouse models were established by house dust mite (HDM) sensitization, and treated with tiotropium intranasally. The expression of Th2 cytokines, ILC2 cytokines and related factors in the nasal mucosa was assessed by immunohistochemistry and quantitative real-time polymerase chain reaction. Serum HDM-specific immunoglobulin E (sIgE) level was detected by enzyme-linked immunosorbent assay. RESULTS: Both Th2 and ILC2 percentages in PBMCs were decreased after 4-DAMP treatment. Similarly, the levels of Th2 cytokines (interleukin 4 [IL-4] and IL-13) and ILC2 cytokines and related factors (IL-25, IL-33, GATA3 and RORα) were significantly decreased in the nasal mucosa of AR mice after tiotropium treatment. Furthermore, tiotropium treatment decreased the nasal symptom score, the serum sIgE level and eosinophil infiltration in AR mice. In addition, tiotropium decreased phospholipase Cγ1 (PLCγ1), PLCγ2, nuclear factor of activated T cell 1 (NFATc1), and NFATc2 mRNA levels in AR mice. CONCLUSION: Antagonism of m3 alleviated type 2 inflammation in the nasal mucosa of AR mice.

Effects of 4-DAMP on allergic rhinitis in guinea pigs and its potential mechanism.

Background: Allergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa mainly mediated by immunoglobulin E (IgE), which seriously affects the life quality of affected patients. This study aimed to observe the effects of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a selective M3 receptor antagonist) on ovalbumin (OVA)-induced AR guinea pigs, and to explore its potential mechanism of involvement. Methods: An AR model was established by inducing male guinea pigs (4-6 weeks of age) with OVA. AR guinea pigs were randomly divided into a model group, 0.6 mg/kg ipratropium bromide (IB) group, 0.12 and 0.6 mg/kg 4-DAMP group (n=18). The 0.6 mg/kg IB group, 0.12 and 0.6 mg/kg 4-DAMP group animals were treated with IB (0.6 mg/kg) and 4-DAMP (0.12 or 0.6 mg/kg) by intranasal instillation per nostril daily. Animals in the model group and normal group were treated with saline as control. The AR symptom scores were counted and nasal secretion weights were measured. Histopathological methods were used to observe nasal mucosa. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of histamine and cytokines. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expressions of mucin 5AC (MUC5AC), matrix metalloproteinase 9 (MMP9), and epidermal growth factor receptor (EGFR). Results: Compared with model group animals, the AR symptom scores and nasal secretion weights of animals treated with 4-DAMP were reduced significantly, goblet cell metaplasia was reversed, and eosinophil infiltration was visibly alleviated. The levels of histamine and cytokines in nasal lavage fluid (NLF) were decreased, and the protein and messenger RNA (mRNA) expressions of MUC5AC, MMP9, and EGFR were inhibited. Conclusions: Treatment with 4-DAMP has a certain effect on AR, especially for mucus hypersecretion, which provides a new idea for clinical treatment of AR.

Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists.

Acetylcholine ameliorates endoplasmic reticulum stress in endothelial cells after hypoxia/reoxygenation via M3 AChR-AMPK signaling.

Endoplasmic reticulum (ER) stress is associated with various cardiovascular diseases. However, its pathophysiological relevance and the underlying mechanisms in the context of hypoxia/reoxygenation (H/R) in endothelial cells are not fully understood. Previous findings have suggested that acetylcholine (ACh), the major vagal nerve neurotransmitter, protected against cardiomyocyte injury by activating AMP-activated protein kinase (AMPK). This study investigated the role of ER stress in endothelial cells during H/R and explored the beneficial effects of ACh. Our results showed that H/R triggered ER stress and apoptosis in endothelial cells, evidenced by the elevation of glucose-regulated protein 78, cleaved caspase-12 and C/EBP homologous protein expression. ACh significantly decreased ER stress and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling positive cells and restored ER ultrastructural changes induced by H/R, possibly via protein kinase-like ER kinase and inositol-requiring kinase 1 pathways. Additionally, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3 AChR) inhibitor, abolished ACh-mediated increase in AMPK phosphorylation during H/R. Furthermore, M3 AChR or AMPK siRNA abrogated the ACh-elicited the attenuation of ER stress in endothelial cells, indicating that the salutary effects of ACh were likely mediated by M3 AChR-AMPK signaling. Overall, ACh activated AMPK through M3 AChR, thereby inhibited H/R-induced ER stress and apoptosis in endothelial cells. We have suggested for the first time that AMPK may function as an essential intermediate step between M3 AChR stimulation and inhibition of ER stress-associated apoptotic pathway during H/R, which may help to develop novel therapeutic approaches targeting ER stress to prevent or alleviate ischemia/reperfusion injury.

M2muscarinic receptors inhibit cell proliferation and migration in urothelial bladder cancer cells.

The role of muscarinic receptors in several diseases including cancer has recently emerged. To evaluate the hypothesis that muscarinic acetylcholine receptors may play a role in bladder cancer as well as in other tumor types, we investigated their expression in bladder tumor specimens. All examined samples expressed the M1, M2 and M3 receptor subtypes. We also found that the level of M2 transcripts, but not those of M1 or M3, significantly increased with the tumor histologic grade. In view of these results, we proceeded to investigate whether the M2 agonist Arecaidine had any effect on in vitro cell growth and migration of T24 cells, a bladder tumor cell line expressing the muscarinic receptors, including the M2 subtype. We observed that Arecaidine significantly reduced T24 and 5637 cell proliferation and migration in a concentration dependent manner. The silencing of M2 receptor by siRNA in T24 and 5637 cell lines showed the inability of Arecaidine (100 µM) to inhibit cell proliferation after 48 hours, whereas the use of M1 and M3 antagonists in T24 appeared not to counteract the Arecaidine effect, suggesting that the inhibition of cell proliferation was directly dependent on M2 receptor activation. These data suggest that M2 muscarinic receptors may play a relevant role in bladder cancer and represent a new attractive therapeutic target.

The use of a modified [3H]4-DAMP radioligand binding assay with increased selectivity for muscarinic M3 receptor shows that cortical CHRM3 levels are not altered in mood disorders.

[(3)H]4-DAMP is a radioligand that has been used to quantify levels of the muscarinic receptor CHRM3 protein in situ. However, in addition to high affinity binding to CHRM3, [(3)H]4-DAMP binds with low affinity to CHRM1 confounding the potential to discriminate between changes in these two muscarinic receptors. We have developed a [(3)H]4-DAMP binding assay, optimised for measuring CHRM3 protein levels in the cortex, with minimal selectivity towards CHRM1. The selectivity of our assay towards CHRM3 was confirmed using recombinant receptor-expressing, cell lysate preparations. [(3)H]4-DAMP binding levels were similar between wildtype and CHRM1 knockout mice, confirming that the amount of [(3)H]4-DAMP binding to CHRM1 was negligible. We used this assay to measure CHRM3 protein levels in the frontal pole, obtained post-mortem from subjects with bipolar disorder (n = 15), major depressive disorder (n = 15) and matched controls (n = 20) and showed that [(3)H]4-DAMP binding was not altered in either bipolar disorder or major depressive disorder. Western blotting confirmed that CHRM3 protein levels were unchanged in these subjects.

Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: a target molecule for lung cancer therapy.

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.

Proline rich-oligopeptides: diverse mechanisms for antihypertensive action.

Bradykinin-potentiating peptides from Bothrops jararaca (Bj) discovered in the early 1960s, were the first natural inhibitors of the angiotensin-converting enzyme (ACE). These peptides belong to a large family of snake venom proline-rich oligopeptides (PROs). One of these peptides, Bj-PRO-9a, was essential for defining ACE as effective drug target and development of captopril, an active site-directed inhibitor of ACE used worldwide for the treatment of human arterial hypertension. Recent experimental evidences demonstrated that cardiovascular effects exerted by different Bj-PROs are due to distinct mechanisms besides of ACE inhibition. In the present work, we have investigated the cardiovascular actions of four Bj-PROs, namely Bj-PRO-9a, -11e, -12b and -13a. Bj-PRO-9a acts upon ACE and BK activities to promote blood pressure reduction. Although the others Bj-PROs are also able to inhibit the ACE activity and to potentiate the BK effects, our results indicate that antihypertensive effect evoked by them involve new mechanisms. Bj-PRO-11e and Bj-PRO-12b involves induction of [Ca(2+)]i transients by so far unknown receptor proteins. Moreover, we have suggested argininosuccinate synthetase and M3 muscarinic receptor as targets for cardiovascular effects elicited by Bj-PRO-13a. In summary, the herein reported results provide evidence that Bj-PRO-mediated effects are not restricted to ACE inhibition or potentiation of BK-induced effects and suggest different actions for each peptide for promoting arterial pressure reduction. The present study reveals the complexity of the effects exerted by Bj-PROs for cardiovascular control, opening avenues for the better understanding of blood pressure regulation and for the development of novel therapeutic approaches.

The inhibitory effects of antimuscarinic autoantibodies in the sera of primary Sjogren syndrome patients on the gastrointestinal motility.

Impairment of gastrointestinal tract (GI) function, including delayed gastric emptying and colonic dysmotility, are common features of primary Sjögren's syndrome (SS). However, the pathogenesis remains largely unknown. The aim of the current study was to investigate the role of functional autoantibodies to the muscarinic receptor in mediating GI dysfunction associated with primary SS. The effect of SS or normal immunoglobulin G (IgG) on smooth muscle (SM) motility was assessed by comparing the amplitude of carbachol (CCh) or electrical field stimulation (EFS) - induced muscle contraction before and after IgG application. Muscarinic receptor type 3 (M3R) played a dominant role in both colon and gastric SM contraction, while M2R was partly involved in gastric smooth muscle contraction. Preincubation for 1h of the colon and gastric SM strips with 1mg/ml purified IgG from the sera of four primary SS patients (SS IgG) significantly inhibited carbachol-induced smooth muscle contraction (CISC) over a range of CCh concentrations, whereas IgG from healthy controls had little effect. Incubation of the colon SM strips with SS IgG also inhibited EFS-induced colon muscle contraction, which was mimicked by the M3R-selective blocker, 4-DAMP. SR1403330, an NK1 antagonist, had little effect on EFS-mediated colonic SM contraction. The results suggest that autoantibodies isolated from primary SS patients' sera inhibit muscarinic receptor-mediated cholinergic neurotransmission in mouse colon and stomach, which may provide clues for explaining the GI dysfunction seen in patients with primary SS.

Activation of muscarinic M3 receptors inhibits large-conductance voltage- and Ca2+-activated K+ channels in rat urinary bladder smooth muscle cells.

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 µM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 µM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 µM), but not muscarinic M2 receptors with methoctramine (1 µM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 µM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca(2+) for BK channel activation, carbachol (1 µM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca(2+). In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM.

Autoradiographic Distribution of Muscarinic Acetylcholine Receptor Subtypes in Rat Cochlear Nucleus.

The cochlear nucleus (CN) receives cholinergic inputs primarily from centrifugal pathways. There is evidence that the effects of these cholinergic inputs may be mediated mainly by muscarinic acetylcholine receptors. We used 1-[N-methyl-3H]scopolamine (NMS) to study muscarinic receptor binding in the rat CN autoradiographically. To determine which muscarinic receptor subtypes participate in the binding, we included competition assays using the unlabeled subtype-preferential ligands pirenzepine, AF-DX 116, 4-DAMP, HHSiD, and tropicamide to compete with [3H]NMS for binding. Our results suggest that NMS binding density in the CN is about a tenth of that in the facial nucleus. Inside the CN, the highest binding was found in granular regions, followed in order by the dorsal CN (DCN) fusiform soma layer, the DCN molecular layer, the DCN deep layer, the anteroventral CN (AVCN) and posteroventral CN (PVCN). Binding in the interstitial nucleus (auditory nerve root) was similar to background. The results of the competition assays suggest that the M2 receptor subtype predominates in VCN, M4 in the DCN fusiform soma layer, and both subtypes in DCN molecular and deep layers. M4 and M3 subtypes predominated in the granular region of AVCN, while M1 and M2 were more prominent in the granular region of PVCN. The results show similarities to those obtained with pharmacological and immunohistochemical methods, but also some discrepancies. The different distributions of the different muscarinic receptor subtypes suggest that the effects of cholinergic inputs may differ among CN subregions, in agreement with in vivo pharmacological results. Overall, the centrifugal cholinergic influences on information processing in the CN may especially involve M2 and M4 receptors.