Action Of ACh Research Articles

Basal forebrain cholinergic activity is necessary for upward firing rate homeostasis in the rodent visual cortex.

Bidirectional homeostatic plasticity allows neurons and circuits to maintain stable firing in the face of developmental or learning-induced perturbations. In the primary visual cortex (V1), upward firing rate homeostasis (FRH) only occurs during active wake (AW) and downward during sleep, but how this behavioral state-dependent gating is accomplished is unknown. Here, we focus on how AW enables upward FRH in V1 of juvenile Long Evans rats. A major difference between quiet wake (QW), when upward FRH is absent, and AW, when it is present, is increased cholinergic (ACh) tone, and the main cholinergic projections to V1 arise from the horizontal diagonal band of the basal forebrain (HDB ACh). We therefore chemogenetically inhibited HDB ACh neurons while inducing upward homeostatic compensation using direct activity-suppression in V1. We found that synaptic scaling up and intrinsic homeostatic plasticity, two important cellular mediators of upward FRH, were both impaired when HDB ACh neurons were inhibited. Most strikingly, HDB ACh inhibition flipped the sign of intrinsic plasticity so that it became anti-homeostatic, and this effect was phenocopied by knockdown of the M1 ACh receptor in V1, indicating that this modulation of intrinsic plasticity is the result of direct actions of ACh within V1. Finally, we found that upward FRH induced by visual deprivation was completely prevented by HDB ACh inhibition. Together, our results show that HDB ACh modulation is a key enabler of upward homeostatic plasticity and FRH, and more broadly suggest that neuromodulatory inputs can segregate upward and downward homeostatic plasticity into distinct behavioral states.

Donepezil inhibits neuromuscular junctional acetylcholinesterase and enhances synaptic transmission and function in isolated skeletal muscle.

Donepezil, a piperidine inhibitor of acetylcholinesterase (AChE) prescribed for treatment of Alzheimer's disease, has adverse neuromuscular effects in humans, including requirement for higher concentrations of non-depolarising neuromuscular blockers during surgery. Here, we examined the effects of donepezil on synaptic transmission at neuromuscular junctions (NMJs) in isolated nerve-muscle preparations from mice. We measured effects of therapeutic concentrations of donepezil (10 nM to 1 μM) on AChE enzymic activity, muscle force responses to repetitive stimulation, and spontaneous and evoked endplate potentials (EPPs) recorded intracellularly from flexor digitorum brevis muscles from CD01 or C57BlWldS mice. Donepezil inhibited muscle AChE with an approximate IC50 of 30 nM. Tetanic stimulation in sub-micromolar concentrations of donepezil prolonged post-tetanic muscle contractions. Preliminary Fluo4-imaging indicated an association of these contractions with an increase and slow decay of intracellular Ca2+ transients at motor endplates. Donepezil prolonged spontaneous miniature EPP (MEPP) decay time constants by about 65% and extended evoked EPP duration almost threefold. The mean frequency of spontaneous MEPPs was unaffected but the incidence of 'giant' MEPPs (gMEPPs), some exceeding 10 mV in amplitude, was increased. Neither mean MEPP amplitude (excluding gMEPPs), mean EPP amplitude, quantal content or synaptic depression during repetitive stimulation were significantly altered by concentrations of donepezil up to 1 μM. Adverse neuromuscular signs associated with donepezil therapy, including relative insensitivity to neuromuscular blockers, are probably due to inhibition of AChE at NMJs, prolonging the action of ACh on postsynaptic nicotinic acetylcholine receptors but without substantively impairing evoked ACh release.

Colocalization of M1 muscarinic cholinergic receptors and NMDA receptors in dendrites and spines of pyramidal neurons of the mouse basolateral amygdala: An ultrastructural analysis

The cholinergic innervation of the neocortex, hippocampus, and basolateral amygdala is critical for higher cognitive functions, including attention and memory. One action of ACh in the hippocampus is the potentiation of NMDA receptor (NMDAR) currents in pyramidal neurons (PNs) by M1 muscarinic receptors (M1Rs). The increase in these currents enhances long-term potentiation (LTP), an important mechanism for memory formation. Ultrastructural observations in the hippocampus revealed that M1Rs and NMDARs were colocalized in hippocampal PN dendrites, consistent with electrophysiological studies demonstrating M1R-NMDAR interactions. Similar to the hippocampus, activation of M1Rs have been shown to be critical for mnemonic functions in the anterior basolateral nucleus of the amygdala (BLa). In the present study dual-labeling immunoelectron microscopy was used to determine if there was colocalization of M1Rs and NMDARs in neurons of the mouse BLa. We found extensive colocalization of these receptors in dendrites and spines of BLa PNs, and most of the M1Rs were membrane-associated where they could be activated by released acetylcholine. These results suggest that M1Rs in BLa PNs could be targeted by M1R positive allosteric modulators (PAMs), resulting in amelioration of memory impairments in neuropsychiatric disorders, such as Alzheimer’s disease, by potentiating NMDAR currents in the amygdala.

M1 receptors interacting with NMDAR enhance delay-related neuronal firing and improve working memory performance.

The recurrent excitatory circuits in dlPFC underlying working memory are known to require activation of glutamatergic NMDA receptors (NMDAR). The neurons in these circuits also rely on acetylcholine to maintain persistent activity, with evidence for actions at both nicotinic α7 receptors and muscarinic M1 receptors (M1R). It is known that nicotinic α7 receptors interact with NMDAR in these circuits, but the interactions between M1R and NMDAR on dlPFC neuronal activity are unknown. Here, we investigated whether M1Rs contribute to the permissive effects of ACh in dlPFC circuitry underlying working memory via interactions with NMDA receptors. We tested interactions between M1Rs and NMDARs in vivo on single neuron activity in rhesus macaques performing a working memory task, as well as on working memory behavior in rodents following infusion of M1R and NMDAR compounds into mPFC. We report that M1R antagonists block the enhancing effects of NMDA application, consistent with M1R permissive actions. Conversely, M1R positive allosteric modulators prevented the detrimental effects of NMDAR blockade in single neurons in dlPFC and on working memory performance in rodents. These data support an interaction between M1R and NMDARs in working memory circuitry in both primates and rats, and suggest M1Rs contribute to the permissive actions of ACh in primate dlPFC. These results are consistent with recent data suggesting that M1R agonists may be helpful in the treatment of schizophrenia, a cognitive disorder associated with NMDAR dysfunction.

Second M3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium.

The bronchodilator tiotropium binds not only to its main binding site on the M3 muscarinic receptor but also to an allosteric site. Here, we have investigated the functional relevance of this allosteric binding and the potential contribution of this behaviour to interactions with long-acting β-adrenoceptor agonists, as combination therapy with anticholinergic agents and β-adrenoceptor agonists improves lung function in chronic obstructive pulmonary disease. ACh, tiotropium, and atropine binding to M3 receptors were modelled using molecular dynamics simulations. Contractions of bovine and human tracheal smooth muscle strips were studied. Molecular dynamics simulation revealed extracellular vestibule binding of tiotropium, and not atropine, to M3 receptors as a secondary low affinity binding site, preventing ACh entry into the orthosteric binding pocket. This resulted in a low (allosteric binding) and high (orthosteric binding) functional affinity of tiotropium in protecting against methacholine-induced contractions of airway smooth muscle, which was not observed for atropine and glycopyrrolate. Moreover, antagonism by tiotropium was insurmountable in nature. This behaviour facilitated functional interactions of tiotropium with the β-agonist olodaterol, which synergistically enhanced bronchoprotective effects of tiotropium. This was not seen for glycopyrrolate and olodaterol or indacaterol but was mimicked by the interaction of tiotropium and forskolin, indicating no direct β-adrenoceptor-M3 receptor crosstalk in this effect. We propose that tiotropium has two binding sites at the M3 receptor that prevent ACh action, which, together with slow dissociation kinetics, may contribute to insurmountable antagonism and enhanced functional interactions with β-adrenoceptor agonists.

A crosstalk between muscarinic and CRF2 receptors regulates cellular adhesion properties of human colon cancer cells

Patients with inflammatory bowel disease often suffer from chronic and relapsing intestinal inflammation that favor the development of colitis associated cancer. An alteration of the epithelial intestinal barrier function observed in IBD is supposed to be a consequence of stress. It has been proposed that corticotrophin-releasing factor receptor (CRF2), one of the two receptors of CRF, the principal neuromediator of stress, acts on cholinergic nerves to induce stress-mediated epithelial barrier dysfunction. Non-neuronal acetylcholine (Ach) and muscarinic receptors (mAchR) also contribute to alterations of epithelial cell functions. In this study, we investigated the mechanisms through which stress and Ach modulate epithelial cell adhesive properties. We show that Ach-induced activation of mAchR in HT-29 cells results in cell dissociation together with changes in cell-matrix contacts, which correlates with the acquisition of invasive potential consistent with a matrix metalloproteinase (MMP) mode of invasion. These processes result from mAchR subsequent stimulation of the cascade of src/Erk and FAK activation. Ach-induced secretion of laminin 332 leads to α3β1 integrin activation and RhoA-dependent reorganization of the actin cytoskeleton. We show that Ach-mediated effects on cell adhesion are blocked by astressin 2b, a CRF2 antagonist, suggesting that Ach action depends partly on CRF2 signaling. This is reinforced by the fact that Ach-mediated activation of mAchR stimulates both the synthesis and the release of CRF2 ligands in HT-29 cells (effects blocked by atropine). In summary, our data provides evidence for a novel intracellular circuit involving mAchR acting on CRF2-signaling that could mediate colonic mucosal barrier dysfunction and exacerbate mucosal inflammation.

Nicotine promotes rooting in leaf explants of in vitro raised seedlings of tomato, Lycopersicon esculentum Miller var. Pusa Ruby

Nicotine promotes rooting in leaf explants of tomato (Lycopersicon esculentum Miller var. Pusa Ruby). Nicotine at 10−9 to 10−3M concentrations was added to the MS basal medium. The optimum response (three-fold increase in rooting) was obtained at 10−7M nicotine-enriched MS medium. At the same level i.e. 10−7M Nicotine induced dramatic increase (11-fold) in the number of secondary roots per root. We have shown earlier that exogenous acetylcholine induces a similar response in tomato leaves. Since nicotine is an agonist of one of the two acetylcholine receptors in animals, its ability to simulate ACh action in a plant system suggests the presence of the same molecular mechanism operative in both, animal and plant cells.

MiR-132 inhibits lipopolysaccharide-induced inflammation in alveolar macrophages by the cholinergic anti-inflammatory pathway

ABSTRACTObjective: Although microRNA-132 (miR-132) has been shown to be involved in the inflammatory regulation, its role in sepsis-induced lung injury is unknown. We hypothesized that miR-132 attenuated lipopolysaccharide (LPS)-induced inflammation of alveolar macrophages by targeting acetylcholinesterase (AChE) and enhancing the acetylcholine (ACh)-mediated cholinergic anti-inflammatory response. Methods: The LPS-treated rat alveolar macrophage cell line NR8383 was used as the inflammatory model. To assess the effect of miR-132, alveolar macrophages were transfected with miR-132 mimic or inhibitor. Results: We found that miR-132 was upregulated in LPS-stimulated alveolar macrophages. Induction of AChE mRNA showed an inverse pattern with respect to AChE protein and activity, suggesting posttranscriptional regulation of AChE. Utilizing miR-132 mimic transfection, we found that overexpression of miR-132 enhanced the ACh-mediated cholinergic anti-inflammatory reaction by targeting AChE mRNA in LPS-treated alveolar macrophages. Blockage of miR-132 using miR-132 inhibitor reversed the Ach action upon LPS-induced release of inflammatory mediators and reduction in AchE protein/activity. Moreover, in the presence of ACh, upregulation of miR-132 suppressed LPS-induced nuclear translocation of NF-κB and production of STAT3 and phosphorylated STAT3, while downregulation of miR-132 enhanced the nuclear translocation of NF-κB. Conclusion: We propose that miR-132 functions as a negative regulator of the inflammatory response in alveolar macrophages by potentiating the cholinergic anti-inflammatory pathway, and represents a potential therapeutic leverage point in modulating inflammatory responses.

Dose-limiting inhibition of acetylcholinesterase by ladostigil results from the rapid formation and fast hydrolysis of the drug–enzyme complex formed by its major metabolite, R-MCPAI

Ladostigil is a pseudo reversible inhibitor of acetylcholinesterase (AChE) that differs from other carbamates in that the maximal enzyme inhibition obtainable does not exceed 50–55%. This could explain the low incidence of cholinergic adverse effects induced by ladostigil in rats and human subjects. The major metabolite, R-MCPAI is believed to be responsible for AChE inhibition by ladostigil in vivo. Therefore we determined whether the ceiling in AChE inhibition resulted from a limit in the metabolism of ladostigil to R-MCPAI by liver microsomal enzymes, or from the kinetics of enzyme inhibition by R-MCPAI. Ladostigil reduces TNF-α in lipopolysaccharide-activated microglia. In vivo, it may also reduce pro-inflammatory cytokines by inhibiting AChE and increasing the action of ACh on macrophages and splenic lymphocytes. We also assessed the contribution of AChE inhibition in the spleen of LPS-injected mice to the anti-inflammatory effect of ladostigil. As in other species, AChE inhibition by ladostigil in spleen, brain and plasma did not exceed 50–55%. Since levels of R-MCPAI increased with increasing doses of ladostigil we concluded that there was no dose or rate limitation of metabolism. The kinetics of enzyme inhibition by R-MCPAI are characterized by a rapid formation of the drug–enzyme complex and fast hydrolysis which limits the attainable degree of AChE inhibition. Ladostigil and its metabolites (1–100nM) decreased TNF-α in lipopolysaccharide-activated macrophages. Ladostigil (5 and 10mg/kg) also reduced TNF-α in the spleen after injection of lipopolysaccharide in mice. However, AChE inhibition contributed to the anti-inflammatory effect only at a dose of 10mg/kg.

The impact of hydroquinone on acetylcholine esterase and certain human carbonic anhydrase isoenzymes (hCA I, II, IX, and XII)

Carbonic anhydrases (CAs) are widespread and the most studied members of a great family of metalloenzymes in higher vertebrates including humans. CAs were investigated for their inhibition of all of the catalytically active mammalian isozymes of the Zn2+-containing CA, (CA, EC 4.2.1.1). On the other hand, acetylcholinesterase (AChE. EC 3.1.1.7), a serine protease, is responsible for ACh hydrolysis and plays a fundamental role in impulse transmission by terminating the action of the neurotransmitter ACh at the cholinergic synapses and neuromuscular junction. In the present study, the inhibition effect of the hydroquinone (benzene-1,4-diol) on AChE activity was evaluated and effectively inhibited AChE with Ki of 1.22 nM. Also, hydroquinone strongly inhibited some human cytosolic CA isoenzymes (hCA I and II) and tumour-associated transmembrane isoforms (hCA IX, and XII), with Kis in the range between micromolar (415.81 μM) and nanomolar (706.79 nM). The best inhibition was observed in cytosolic CA II.

PT339 The Ang 1-7 mediated vascular and coronary reactivity in diabetic endothelial dysfunction

Introduction: Ang 1-7 appeares as an intrigant peptide regulating AT1 receptor activity and effects. Objectives: The in vitro evaluation of rat aorta and coronary system response on action of the different neuroendocrine factors, inclusively Ang 1-7, in diabetes induced endothelial dysfunction. Methods: Diabetes induced endothelial dysfunction (DED) was reproduced by streptozotocin administration in rats. Using the models of isolated aorta ring and isovolumic heart perfusion the vascular response (relaxant or constricting) and coronary flux were determined on action of acetylcholine (Ach), bradykinin, Ang 1-7, Ang II (inclusively in condition of mas receptor blockade by A779), hydrogen peroxide, norepinephrine and endothelin-1. Results: The Ach action up on phenylephrine induced aorta ring contraction manifested in DED by significant vasorelaxation reduction by up to 36% vs control pattern. Remarkably, Ang 1-7 (10-10 M) has induced an aortic ring relaxation similarly to control level, achieving a range of 11,4-26,5%. The Ang II induced constrictor plateau is significantly elevated in DED by 51,9%. However, it decreases by 32,8% when a premedication by Ang 1-7 is performed, and, contrarily, raises by 36,4% when mas receptors are blocked by A779. Norepinephrine and endothelin-1 have induced in DED an aorta contraction increasing by 34-42%. The analogous events were fixed in heart. Thus, functional coronary reserve is reduced in DED on Ach and bradykinin action by 38 and 31%, respectively. Nevertheless, coronary reserve is not affected during Ang 1-7 action compared to control index: 8,84 vs 9,86% (p>0,05). Coronary flux on Ang II action is decreased more pronounced by 46%, but the decline is limited by almost 50% in condition of Ang 1-7 administration. The mas receptor blocking led to Ang II induced coronary flow diminution more by 52%. Interestingly, the hyperpolarization induced coronary dilation level related to hydrogen peroxide action is not impaired in DED. Conclusion: 1. Vasorelaxation and coronary dilation effects of Ang 1-7 are not impaired in diabetes induced endothelial dysfunction, event that may be emphasized as compensator pattern considering the fact of affected cholinergic vascular response. 2. Ang 1-7 significantly reduces Ang II induced vasoand coronary constricting effects, but mas receptor blocking, contrarily, leads to their augmentation. Disclosure of Interest: None Declared

PT340 Age-related impairment in androgen stimulation of vascular endothelial growth factor-mediated angiogenesis

Introduction: Ang 1-7 appeares as an intrigant peptide regulating AT1 receptor activity and effects. Objectives: The in vitro evaluation of rat aorta and coronary system response on action of the different neuroendocrine factors, inclusively Ang 1-7, in diabetes induced endothelial dysfunction. Methods: Diabetes induced endothelial dysfunction (DED) was reproduced by streptozotocin administration in rats. Using the models of isolated aorta ring and isovolumic heart perfusion the vascular response (relaxant or constricting) and coronary flux were determined on action of acetylcholine (Ach), bradykinin, Ang 1-7, Ang II (inclusively in condition of mas receptor blockade by A779), hydrogen peroxide, norepinephrine and endothelin-1. Results: The Ach action up on phenylephrine induced aorta ring contraction manifested in DED by significant vasorelaxation reduction by up to 36% vs control pattern. Remarkably, Ang 1-7 (10-10 M) has induced an aortic ring relaxation similarly to control level, achieving a range of 11,4-26,5%. The Ang II induced constrictor plateau is significantly elevated in DED by 51,9%. However, it decreases by 32,8% when a premedication by Ang 1-7 is performed, and, contrarily, raises by 36,4% when mas receptors are blocked by A779. Norepinephrine and endothelin-1 have induced in DED an aorta contraction increasing by 34-42%. The analogous events were fixed in heart. Thus, functional coronary reserve is reduced in DED on Ach and bradykinin action by 38 and 31%, respectively. Nevertheless, coronary reserve is not affected during Ang 1-7 action compared to control index: 8,84 vs 9,86% (p>0,05). Coronary flux on Ang II action is decreased more pronounced by 46%, but the decline is limited by almost 50% in condition of Ang 1-7 administration. The mas receptor blocking led to Ang II induced coronary flow diminution more by 52%. Interestingly, the hyperpolarization induced coronary dilation level related to hydrogen peroxide action is not impaired in DED. Conclusion: 1. Vasorelaxation and coronary dilation effects of Ang 1-7 are not impaired in diabetes induced endothelial dysfunction, event that may be emphasized as compensator pattern considering the fact of affected cholinergic vascular response. 2. Ang 1-7 significantly reduces Ang II induced vasoand coronary constricting effects, but mas receptor blocking, contrarily, leads to their augmentation. Disclosure of Interest: None Declared

A novel kinetic model for polysaccharide dissolution during atmospheric acetic acid pretreatment of sugarcane bagasse

Acetic acid (AcH) pretreatment of sugarcane bagasse with the catalysis of sulfuric acid (SA) could greatly enhance the enzymatic digestibility of cellulose. However, polysaccharide dissolution happened inevitably during the pretreatment. It was found that the simplest model, which assumes that the total polysaccharides were reactive to be dissolved, could not well describe the kinetic behavior of polysaccharide dissolution. A novel pseudo-homogenous kinetic model was thus developed by introducing a parameter termed as "potential dissolution degree" (δ(d)) based on the multilayered structure of cell wall. It was found that solid xylan and glucan dissolutions were a first-order reaction with respect to the dissolvable fraction. Due to the delignification action of AcH, polysaccharide dissolutions were enhanced in AcH media compared with those in aqueous system. Acetylizations of cellulose and sugars were also observed, and AcH concentration showed a significant influence on the degree of acetylization.

Role of endothelium, acetylocholine and calcium ions in Bay K8644- and KCl-induced contraction

The aim of this study was to establish the involvement of acetylcholine (Ach) and calcium ions in modulating contractions induced by Bay K8644 (an agonist of calcium channels located in the cell membrane) and KCl (at depolarizing concentrations), and also to examine the importance of the vascular endothelium in the activity of Bay K8644. The study was performed on perfused Wistar rat tail arteries. Contraction induced by Bay K8644 with the participation of intracellular (in calcium‑free physiological salt solution, FPSS) and extracellular (in physiological salt solution, PSS, following the emptying of the cellular Ca2+ stores) pools of Ca2+ and the addition of nitro-L-arginine (L‑NNA; nitric oxide synthase inhibitor) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; an inhibitor of soluble guanylyl cyclase) was studied. In addition, the effect of Ach on the contraction response was analyzed and the results were compared with the depolarizing action of KCl. The effects of 8Br‑cGMP on the artery contraction induced by Bay K8644 prior to and following removal of the endothelium were compared. Bay K8644 and KCl in PSS induced vascular contraction, which was reduced with the addition of Ach. The spasmolytic Ach action did not occur in the presence of L‑NNA and ODQ. 8Br‑cGMP reduced the contraction of arterial walls (with and without endothelium) induced by Bay K8644. The increase in vascular tone induced by Bay K8644 and KCl was independent of the intracellular calcium ion pool. The relaxant effect of Ach on the responses stimulated by Bay K8644 and KCl indicated the participation of nitric oxide in modulating the reactivity of the arteries to the factors examined, resulting in an influx of Ca2+ into the cell.

Targeting Neuronal Nicotinic Receptors in Cancer: New Ligands and Potential Side-Effects

In the nervous system, the neuronal nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory postsynaptic potentials as well as slower paracrine actions of ACh. They are also widely expressed in non-nervous tissue, including the neoplastic, which is intriguing as smoking is an established risk factor for cancer. Moreover, recent evidence attributes to the gene cluster coding for the α3/α5/β4 nAChR subunits a role in both development of lung cancer and nicotine addiction. Many cellular effects of nicotine and the tobacco-derived carcinogenic N-nitrosamines are probably caused by nAChR activation, which regulates cell proliferation, migration, apoptosis and neoangiogenesis. Nonetheless, the precise nAChR roles in tumors are difficult to determine because cancer cells express a wide variety of nicotinic subunits, whose function is unclear. Patented compounds which selectively target nAChRs subtypes are increasingly available and will hopefully allow better understanding of the physiology of these channels in specific cell types, as well as suggest novel diagnostic and therapeutic approaches. At the present state, however, thorough functional studies of these compounds are still limited and whether they act as agonists, antagonists or partial agonists is often unclear. Such a blurred distinction between activators and inhibitors makes detailed studies in expression systems sorely needed for both physiological understanding and outlining the possible side-effects.

Targeting Neuronal Nicotinic Receptors in Cancer: New Ligands and Potential Side-Effects

In the nervous system, the neuronal nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory postsynaptic potentials as well as slower paracrine actions of ACh. They are also widely expressed in non-nervous tissue, including the neoplastic, which is intriguing as smoking is an established risk factor for cancer. Moreover, recent evidence attributes to the gene cluster coding for the 3/5/4 nAChR subunits a role in both development of lung cancer and nicotine addiction. Many cellular effects of nicotine and the tobacco-derived carcinogenic N-nitrosamines are probably caused by nAChR activation, which regulates cell proliferation, migration, apoptosis and neoangiogenesis. Nonetheless, the precise nAChR roles in tumors are difficult to determine because cancer cells express a wide variety of nicotinic subunits, whose function is unclear. Patented compounds which selectively target nAChRs subtypes are increasingly available and will hopefully allow better understanding of the physiology of these channels in specific cell types, as well as suggest novel diagnostic and therapeutic approaches. At the present state, however, thorough functional studies of these compounds are still limited and whether they act as agonists, antagonists or partial agonists is often unclear. Such a blurred distinction between activators and inhibitors makes detailed studies in expression systems sorely needed for both physiological understanding and outlining the possible side-effects.

Non‐neuronal release of ACh plays a key role in secretory response to luminal propionate in rat colon

Colonic chloride secretion is induced by chemical stimuli via the enteric nervous reflex. We have previously demonstrated that propionate stimulates chloride secretion via sensory and cholinergic systems of the mucosa in rat distal colon. In this study, we demonstrate non-neuronal release of ACh in the secretory response to propionate using an Ussing chamber. Mucosa preparations from the colon, not including the myenteric and submucosal plexuses, were used. Luminal addition of propionate and serosal addition of ACh caused biphasic changes in short-circuit current (Isc). TTX (1 μm) had no effects, while atropine (10 μm) significantly inhibited the Isc response to propionate and abolished that to ACh. In response to luminal propionate stimulation, ACh was released into the serosal fluid. A linear relationship was observed between the maximal increase in Isc and the amounts of ACh released 5 min after propionate stimulation. This ACh release induced by propionate was not affected by atropine and bumetanide, although both drugs significantly reduced the Isc responses to propionate. Luminal addition of 3-chloropropionate, an inactive analogue of propionate, abolished both ACh release and Isc response produced by propionate. RT-PCR analysis indicated that isolated crypt cells from the distal colon expressed an enzyme of ACh synthesis (ChAT) and transporters of organic cation (OCTs), but not neuronal CHT1 and VAChT. The isolated crypt cells contained comparable amounts of ACh to the residual muscle tissues including nerve plexuses. In conclusion, the non-neuronal release of ACh from colonocytes coupled with propionate stimulation plays a key role in chloride secretion, via the paracrine action of ACh on muscarinic receptors of colonocytes.

Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion

Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion.

The role of cholinergic system in neuronal plasticity: focus on visual cortex and muscarinic receptors.

This review is focused on the basal forebrain (BFB) cholinergic system, cholinergic receptors and cholinoceptive target areas such as the neocortex, all of which are intimately involved in high cognitive functions and synaptic plasticity. The neurons of the BfB synthe-size acetylcholine (ACh) whose action is mediated by two subclasses of receptors, namely nicotinic and muscarinic receptors. using the visual system as a model, the aim here is to integrate and discuss the current knowledge on anatomy, ontogeny and function of the BfB cholinergic system. This signaling system represents the anatomo-functional basis of ach action on neuronal network, neuronal plasticity and cognitive functions. cholinergic system role on higher brain functions has received increasing attention since the first observation of a. alzheimer (1907) reporting dramatic changes of the BfB cholinergic neuro-anatomy in one of the most devastating neurodegenerative diseases of adult brain, i.e. alzheimer’s disease (ad). in addition to this observation, later work demonstrated its participation in deep re-arrangements of brain connectivity such as the regulation of neuronal plasticity during maturation of cortical sensory maps, in adult and aged brain.

Pathology‐specific effects of the IKur/Ito/IK,ACh blocker AVE0118 on ion channels in human chronic atrial fibrillation

This study was designed to establish the pathology-specific inhibitory effects of the IKur/Ito/IK,ACh blocker AVE0118 on atrium-selective channels and its corresponding effects on action potential shape and effective refractory period in patients with chronic AF (cAF). Outward K+-currents of right atrial myocytes and action potentials of atrial trabeculae were measured with whole-cell voltage clamp and microelectrode techniques, respectively. Outward currents were dissected by curve fitting. Four components of outward K+-currents and AF-specific alterations in their properties were identified. Ito was smaller in cAF than in SR, and AVE0118 (10 microM) apparently accelerated its inactivation in both groups without reducing its amplitude. Amplitudes of rapidly and slowly inactivating components of IKur were lower in cAF than in SR. The former was abolished by AVE0118 in both groups, the latter was partially blocked in SR, but not in cAF, even though its inactivation was apparently accelerated in cAF. The large non-inactivating current component was similar in magnitude in both groups, but decreased by AVE0118 only in SR. AVE0118 strongly suppressed AF-related constitutively active IK,ACh and prolonged atrial action potential and effective refractory period exclusively in cAF. In atrial myocytes of cAF patients, we detected reduced function of distinct IKur components that possessed decreased component-specific sensitivity to AVE0118 most likely as a consequence of AF-induced electrical remodelling. Inhibition of profibrillatory constitutively active IK,ACh may lead to pathology-specific efficacy of AVE0118 that is likely to contribute to its ability to convert AF into SR.