Hymenaea rubriflora Ducke stem bark extract has vasorelaxant and contractile inhibition capacity

Abstract We investigated the vasodilatory effects of Hymenaea rubriflora Ducke stem bark extract (HRHAc). Vascular reactivity of the aortic rings of Wistar rats was tested by in vitro cumulative doses (0.1 - 729 µg/mL). Rats (n=5) were treated with 25 (G25), 50 (G50) and 100 (G100) mg/ kg of HR-HAc or saline (control group - CG) for four weeks. An in vitro assay resulted in dose-dependent relaxation of the aortic rings with functional endothelium, which was inhibited in the presence of L-NAME. Rings of the treated animals increased acetylcholine relaxing potency at all doses, with a greater effect on G50 (pD2 = 7.8±0.1, Emax = 95.6±1.1) and a decreased contractile potency to phenylephrine in G25 (pD2 = 6.9±0.06, Emax = 61.5±6.0%) and G50 (pD2= 6.6±0.06, Emax = 71.0±8.5%) when compared to the CG in the presence and absence of endothelium (pD2= 6.4± 0.1, 6.4±0.1 and 6.9±0.1, respectively). Cumulative doses of nitroprusside resulted in increased relaxing potency in all treated groups and maintained Emax at 100%. It is concluded that HR-HAc has vasorelaxant capacity and inhibitory vascular contraction activity applied either directly to aortic rings or after treatment with in vivo supplementation, which places this extract as a potential nutraceutical or pharmacological agent for treating diseases associated with vascular dysfunction.

Ameliorative potential of phloridzin in type 2 diabetes-induced memory deficits in rats.

Diabetes associated oxidative stress and impaired cholinergic neurotransmission causes cognitive deficits. Although phloridzin shows antioxidant- and insulin sensitizing-activities, its ameliorative potential in diabetes-induced memory dysfunction remains unexplored. In the present study, type 2 diabetes (T2D) was induced by streptozotocin (35 mg/kg, intraperitoneal) in rats on ad libitum high-fat diet. Diabetic animals were treated orally with phloridzin (10 and 20 mg/kg) for four weeks. Memory functions were evaluated by passive avoidance test (PAT) and novel object recognition (NOR) test. Brains of rats were subjected to biochemical analysis of glutathione (GSH), brain-derived neurotrophic factor (BDNF), malonaldehyde (MDA) and acetylcholinesterase (AChE). Role of cholinergic system in the effects of phloridzin was evaluated by scopolamine pre-treatment in behavioral studies. While diabetic rats showed a significant decrease in step through latency in PAT, and exploration time and discrimination index in NOR test; a substantial increase in all parameters was observed following phloridzin treatment. Phloridzin reversed abnormal levels of GSH, BDNF, MDA and AChE in the brain of diabetic animals. Moreover, in silico molecular docking study revealed that phloridzin acts as a potent agonist at M1 receptor as compared to acetylcholine. Viewed collectively, reversal of T2D-induced memory impairment by phloridzin might be attributed to upregulation of neurotrophic factors, reduced oxidative stress and increased cholinergic signaling in the brain. Therefore, phloridzin may be a promising molecule in the management of cognitive impairment comorbid with T2D.

Stable desensitization of α7 nicotinic acetylcholine receptors by NS6740 requires interaction with S36 in the orthosteric agonist binding site.

NS6740 is an α7 nicotinic acetylcholine receptor-selective partial agonist with low efficacy for channel activation, capable of promoting the stable conversion of the receptors to nonconducting (desensitized) states that can be reactivated with the application of positive allosteric modulators (PAMs). In spite of its low efficacy for channel activation, NS6740 is an effective activator of the cholinergic anti-inflammatory pathway. We observed that the concentration-response relationships for channel activation, both when applied alone and when co-applied with the PAM PNU-120596 are inverted-U shaped with inhibitory/desensitizing activities dominant at high concentrations. We evaluated the potential importance of recently identified binding sites for allosteric activators and tested the hypotheses that the stable desensitization produced by NS6740 may be due to binding to these sites. Our experiments were guided by molecular modeling of NS6740 binding to both the allosteric and orthosteric activation sites on the receptor. Our results indicate that with α7C190A mutants, which have compromised orthosteric activation sites, NS6740 may work at the allosteric activation sites to promote transient PAM-dependent currents but not the stable desensitization seen with wild-type α7 receptors. Modeling NS6740 in the orthosteric binding sites identified S36 as an important residue for NS6740 binding and predicted that an S36V mutation would limit NS6740 activity. The efficacy of NS6740 for α7S36V receptors was reduced to zero, and applications of the compound to α7S36V receptors failed to induce the desensitization observed with wild-type receptors. The results indicate that the unique properties of NS6740 are due primarily to binding at the sites for orthosteric agonists.

ICH3, a selective alpha7 nicotinic acetylcholine receptor agonist, modulates adipocyte inflammation associated with obesity.

PURPOSE: The alpha7 nicotinic acetylcholine receptor (α7nAChR), involved in the modulation of inflammation and insulin sensitivity, is downregulated in white adipose tissue (WAT) of obese patients. This study aims to test the ability of a selective synthetic α7nAChR agonist, the spirocyclic Δ2-isoxazoline derivative (R)-(-)-ICH3 (ICH3), to counteract acute inflammation and obesity-associated modifications in WAT. METHODS: We employed the LPS-septic shock murine model, human primary adipocytes and diet-induced obese (DIO) mice. Inflammatory factor expression was assessed by ELISA and quantitative real-time PCR. Flow cytometry was employed to define WAT inflammatory infiltrate. Insulin signaling was monitored by quantification of AKT phosphorylation. RESULTS: In the septic shock model, ICH3 revealed antipyretic action and reduced the surge of circulating cytokines. In vitro, ICH3 stimulation (10 µM) preserved viability of human adipocytes, decreased IL-6 mRNA (P < 0.05) and blunted LPS-induced peak of TNFα (P < 0.05) and IL-6 (P < 0.01). Chronic administration of ICH3 to DIO mice was associated with lower numbers of CD8+ T cells (P < 0.05) and to changed WAT expression of inflammatory factors (Hp, P < 0.05; CD301/MGL1, P < 0.01; Arg-1, P < 0.05). As compared to untreated, ICH3 DIO mice exhibited improved insulin signaling in the skeletal muscle (P < 0.01) mirrored by an improved response to glucose load (ipGTT: P < 0.05 at 120 min). CONCLUSIONS: We proved that ICH3 is an anti-inflammatory drug, able to reduce inflammatory cytokines in human adipocytes and to blunt the effects of obesity on WAT inflammatory profile, on glucose tolerance and on tissue insulin sensitivity.

Synthesis of Novel Nicotinic Ligands with Multimodal Action: Targeting Acetylcholine α4ß2, Dopamine and Serotonin Transporters.

Nicotinic acetylcholine receptors (nAChRs), serotonin transporters (SERT) and dopamine transporters (DAT) represent targets for the development of novel nicotinic derivatives acting as multiligands associated with different health conditions, such as depressive, anxiety and addiction disorders. In the present work, a series of functionalized esters structurally related to acetylcholine and nicotine were synthesized and pharmacologically assayed with respect to these targets. The synthesized compounds were studied in radioligand binding assays at α4ß2 nAChR, h-SERT and h-DAT. SERT experiments showed not radioligand [3H]-paroxetine displacement, but rather an increase in the radioligand binding percentage at the central binding site was observed. Compound 20 showed Ki values of 1.008 ± 0.230 µM for h-DAT and 0.031 ± 0.006 µM for α4ß2 nAChR, and [3H]-paroxetine binding of 191.50% in h-SERT displacement studies, being the only compound displaying triple affinity. Compound 21 displayed Ki values of 0.113 ± 0.037 µM for α4ß2 nAChR and 0.075 ± 0.009 µM for h-DAT acting as a dual ligand. Molecular docking studies on homology models of α4ß2 nAChR, h-DAT and h-SERT suggested potential interactions among the compounds and agonist binding site at the α4/ß2 subunit interfaces of α4ß2 nAChR, central binding site of h-DAT and allosteric modulator effect in h-SERT.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo. Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (ß cooperativity). Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

Possible mechanism of Vitis vinifera L. flavones on neurotransmitters, synaptic transmission and related learning and memory in Alzheimer model rats.

BACKGROUND: This study explored the possible mechanism of flavones from Vitis vinifera L. (VTF) on neurotransmitters, synaptic transmission and related learning and memory in rats with Alzheimer disease (AD). METHODS: The researchers injected amyloid-ß(25-35) into the hippocampus to establish AD model rats. The Sprague-Dawley (SD) rats were divided into a control group, a donepezil group, an AD model group, a VTF low-dose group, a VTF medium-dose group and a VTF high-dose group. The researchers detected the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) according to kit instructions. The protein expression of brain-derived neurotrophic factor (BDNF), synaptotagmin-1 (SYT1) and cyclic adenosine monophosphate response element binding protein (CREB) in the rats' hippocampi was detected by immunohistochemistry and Western blot, and the gene expression of cAMP-regulated enhancer (CRE) was detected by real-time quantitative polymerase chain reaction (PCR). RESULTS: VTF may enhance the protein expression of p-CREB, BDNF and SYT1 in rat hippocampi, depending on dose. The messenger RNA (mRNA) level of CREB was significantly higher in the VTF high-dose group than in the model group, which was consistent with the results of Western blotting. VTF may reduce the activity of AChE and increase that of ChAT in rat hippocampi. Finally, VTF effectively improved the learning and memory abilities of AD rats. CONCLUSIONS: VTF can promote synaptic plasticity and indirectly affect the expression of cholinergic neurotransmitters, which may be one mechanism of VTF protection in AD rats.

Endogenous acetylcholine regulates neuronal and astrocytic vascular endothelial growth factor expression levels via different acetylcholine receptor mechanisms.

Vascular endothelial growth factor (VEGF), a signaling molecule involved in angiogenesis, plays an important role in neuroprotection and neurogenesis. In the present study, we aimed to elucidate the mechanisms underlying endogenous acetylcholine (ACh)-induced VEGF expression in neurons and astrocytes, and identify the neuronal cells contributing to its expression in the medial septal area, a nuclear origin of cholinergic neurons mainly projecting to the hippocampus. The mRNA expression and secretion of VEGF were measured by RT-PCR and ELISA using mouse primary cultured cortical neurons and astrocytes. VEGF expression in the medial septal area was assessed by RT-PCR and immunostaining using mice treated with tacrine [9-amino-1,2,3,4-tetrahydro-acridine HCl (THA); 2.5 mg/kg, i.p.] once daily for 7 days. The THA treatment increased VEGF mRNA expression in neurons in a manner that was reversed by mecamylamine, a nicotinic ACh receptor (AChR) antagonist, whereas in mouse primary cultured astrocytes, carbachol, but not THA dose-dependently increased VEGF mRNA expression and secretion in a manner that was inhibited by scopolamine, a muscarinic AChR inhibitor. In in vivo studies, the administration of THA significantly increased the expression of VEGF in medial septal cholinergic neurons and the effects of THA were significantly blocked by mecamylamine. THA also significantly increased the expression levels of a phosphorylated form of VEGF receptor 2 (p-VEGFR2), an activated form of VEGFR2. The present results suggest that endogenous ACh plays an up-regulatory role for VEGF expression in neurons and astrocytes via different mechanisms. Moreover, endogenous ACh-induced increases in VEGF levels appear to activate VEGFR2 on medial septal cholinergic neurons via an autocrine mechanism.

Interaction between noradrenergic and cholinergic signaling in amygdala regulates anxiety- and depression-related behaviors in mice.

Medications that target the noradrenergic system are important therapeutics for depression and anxiety disorders. More recently, clinical studies have shown that the α2-noradrenergic receptor (α2AR) agonist guanfacine can decrease stress-induced smoking relapse during acute abstinence, suggesting that targeting the noradrenergic system may aid in smoking cessation through effects on stress pathways in the brain. Acetylcholine (ACh), like the nicotine in tobacco, acts at nicotinic acetylcholine receptors (nAChRs) to regulate behaviors related to anxiety and depression. We therefore investigated interactions between guanfacine and ACh signaling in tests of anxiolytic and antidepressant efficacy in female and male C57BL/6J mice, focusing on the amygdala as a potential site of noradrenergic/cholinergic interaction. The antidepressant-like effects of guanfacine were blocked by shRNA-mediated knockdown of α2AR in amygdala. Knockdown of the high-affinity ß2 nAChR subunit in amygdala also prevented antidepressant-like effects of guanfacine, suggesting that these behavioral effects require ACh signaling through ß2-containing nAChRs in this brain area. Ablation of NE terminals prevented the anxiolytic- and antidepressant-like effects of the nicotinic partial agonist cytisine, whereas administration of the cholinesterase antagonist physostigmine induced a depression-like phenotype that was not altered by knocking down α2AR in the amygdala. These studies suggest that ACh and NE have opposing actions on behaviors related to anxiety and depression and that cholinergic signaling through ß2-containing nAChRs and noradrenergic signaling through α2a receptors in neurons of the amygdala are critical for regulation of these behaviors.

Monosodium glutamate impairs the contraction of uterine visceral smooth muscle ex vivo of rat through augmentation of acetylcholine and nitric oxide signaling pathways.

The aim of the study was to examine the toxic effects of Monosodium glutamate (MSG), an extensively used food additive, on the contraction of uterine visceral smooth muscle (UVSM) in rat and to elucidate the probable neurocrine mechanism involved in it. MSG produced significant potentiation of the force and inhibition of frequency of uterus recorded ex vivo in chronic MSG exposure and in single dose acute experiments. MSG also produced significant potentiation of force of acetylcholine induced contraction and no alterations in atropine induced contraction of uterus. Further, MSG produced significant increase in force and frequency of contraction of neostigmine incubated uterus. We have found significant potentiation of the post pause force of contraction of uterus when MSG was applied in adrenaline incubated uterus. MSG also produced significant decrease in frequency of contraction of sodium nitroprusside incubated uterus; increase in frequency of N-ω-Nitro-l-Arginine Methyl Ester incubated uterus and no significant changes in frequency of contraction of methylene blue incubated uterus. These results indicate that MSG potentiates the force of contraction of UVSM predominantly by augmenting the activity of cholinergic intrinsic efferents and inhibits the frequency of contraction probably by augmenting the activity of nitrergic efferents. In conclusion, MSG potentiates the force and inhibits the frequency of contraction of UVSM, and the MSG induced effect is probably mediated through the augmentation of acetylcholine and nitric oxide signaling pathways.

Rutin hydrate ameliorates cadmium chloride-induced spatial memory loss and neural apoptosis in rats by enhancing levels of acetylcholine, inhibiting JNK and ERK1/2 activation and activating mTOR signalling.

This study aimed at studying the potential neuroprotective effect of Rutin hydrate (RH) alone or in conjugation with α-tocopherol against cadmium chloride (CdCl2)-induced neurotoxicity and cognitive impairment in rats and to investigate the mechanisms of action. Rats intoxicated with CdCl2 were treated with the vehicle, RH, α-tocopherol or combined treatment were examined, and compared to control rats received vehicle or individual doses of either drug. Data confirmed that RH improves spatial memory function by increasing acetylcholine availability, boosting endogenous antioxidant potential, activating cell survival and inhibiting apoptotic pathways, an effect that is more effective when RH was conjugated with α-tocopherol. Mechanism of RH action includes activation of PP2A mediated inhibiting of ERK1/2 and JNK apoptotic pathways and inhibition of PTEN mediated activation of mTOR survival pathway. In conclusion, RH affords a potent neuroprotection against CdCl2-induced brain damage and memory dysfunction and co-administration of α-tocopherol enhances its activity.

Cholinergic Targets in Lung Cancer.

Lung cancers express an autocrine cholinergic loop in which secreted acetylcholine can stimulate tumor growth through both nicotinic and muscarinic receptors. Because activation of mAChR and nAChR stimulates growth; tumor growth can be stimulated by both locally synthesized acetylcholine as well as acetylcholine from distal sources and from nicotine in the high percentage of lung cancer patients who are smokers. The stimulation of lung cancer growth by cholinergic agonists offers many potential new targets for lung cancer therapy. Cholinergic signaling can be targeted at the level of choline transport; acetylcholine synthesis, secretion and degradation; and nicotinic and muscarinic receptors. In addition, the newly describe family of ly-6 allosteric modulators of nicotinic signaling such as lynx1 and lynx2 offers yet another new approach to novel lung cancer therapeutics. Each of these targets has their potential advantages and disadvantages for the development of new lung cancer therapies which are discussed in this review.

Synaptic muscarinic response types in hippocampal CA1 interneurons depend on different levels of presynaptic activity and different muscarinic receptor subtypes.

Depolarizing, hyperpolarizing and biphasic muscarinic responses have been described in hippocampal inhibitory interneurons, but the receptor subtypes and activity patterns required to synaptically activate muscarinic responses in interneurons have not been completely characterized. Using optogenetics combined with whole cell patch clamp recordings in acute slices, we measured muscarinic responses produced by endogenously released acetylcholine (ACh) from cholinergic medial septum/diagonal bands of Broca inputs in hippocampal CA1. We found that depolarizing responses required more cholinergic terminal stimulation than hyperpolarizing ones. Furthermore, elevating extracellular ACh with the acetylcholinesterase inhibitor physostigmine had a larger effect on depolarizing versus hyperpolarizing responses. Another subpopulation of interneurons responded biphasically, and periodic release of ACh entrained some of these interneurons to rhythmically burst. M4 receptors mediated hyperpolarizing responses by activating inwardly rectifying K(+) channels, whereas the depolarizing responses were inhibited by the nonselective muscarinic antagonist atropine but were unaffected by M1, M4 or M5 receptor modulators. In addition, activation of M4 receptors significantly altered biphasic interneuron firing patterns. Anatomically, interneuron soma location appeared predictive of muscarinic response types but response types did not correlate with interneuron morphological subclasses. Together these observations suggest that the hippocampal CA1 interneuron network will be differentially affected by cholinergic input activity levels. Low levels of cholinergic activity will preferentially suppress some interneurons via hyperpolarization and increased activity will recruit other interneurons to depolarize, possibly because of elevated extracellular ACh concentrations. These data provide important information for understanding how cholinergic therapies will affect hippocampal network function in the treatment of some neurodegenerative diseases.

Use of an α3ß4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties.

Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3ß4 binding sites and other subunits, including ß4, ß2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 ß4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and ß4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and ß4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3ß4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for ß4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.

Micromechanical measurement of AChBP binding for label-free drug discovery.

A potential binding assay based on binding-driven micromechanical motion is described. Acetylcholine binding protein (AChBP) was used to modify a microcantilever. The modified microcantilever was found to bend on application of the naturally occurring agonist (acetylcholine) or the antagonist (nicotine and d-tubocurarine). Control experiments show that microcantilevers modified without AChBP do not respond to acetylcholine, nicotine, and d-tubocurarine. K(d) values obtained for acetylcholine, nicotine, and d-tubocurarine are similar to those obtained from radio-ligand binding assays. These results suggest that the microcantilever system has potential for use in label free, drug screening applications.

Central activation of the cholinergic anti-inflammatory pathway reduces surgical inflammation in experimental post-operative ileus.

BACKGROUND AND PURPOSE: Electrical stimulation of the vagus nerve reduces intestinal inflammation following mechanical handling, thereby shortening post-operative ileus in mice. Previous studies in a sepsis model showed that this cholinergic anti-inflammatory pathway can be activated pharmacologically by central administration of semapimod, an inhibitor of p38 mitogen-activated protein kinase. We therefore evaluated the effect of intracerebroventricular (i.c.v.) semapimod on intestinal inflammation and post-operative ileus in mice. EXPERIMENTAL APPROACH: Mice underwent a laparotomy or intestinal manipulation 1 h after i.c.v. pre-treatment with semapimod (1 µg·kg(-1) ) or saline. Drugs were administered through a cannula placed in the left lateral ventricle 1 week prior to experimentation. Twenty-four hours after surgery, gastric emptying was measured using scintigraphy, and the degree of intestinal inflammation was assessed. Finally, activation of brain regions was assessed using quantitative immunohistochemistry for c-fos. KEY RESULTS: Intestinal manipulation induced inflammation of the manipulated intestine and significantly delayed gastric emptying, 24 h after surgery in saline-treated animals. Semapimod significantly reduced this inflammation and improved gastric emptying. Vagotomy enhanced the inflammatory response induced by intestinal manipulation and abolished the anti-inflammatory effect of semapimod. Semapimod but not saline induced a significant increase in c-fos expression in the paraventricular nucleus, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve. CONCLUSIONS AND IMPLICATIONS: Our findings show that i.c.v. semapimod reduces manipulation-induced intestinal inflammation and prevented post-operative ileus. This anti-inflammatory effect depends on central activation of the vagus nerve.

Possible role of cholinesterase inhibitors on memory consolidation following hypobaric hypoxia of rats.

High altitude (HA) generates a deleterious effect known as hypobaric hypoxia (HBH). This causes severe physiological and psychological changes such as acute mountain sickness (AMS) and cognitive functions in terms of learning and memory. The present study has evaluated the effect of cholinesterase inhibitors on memory consolidation following HBH. Adult male Sprague Dawley rats (80-90 days old) with an average body weight of 250 ± 25 g were used. Rats were assessed memory consolidation by using Morris water maze (MWM) for 8 days. After assessment of memory consolidation, rats were then exposed to HBH in stimulated chamber for 7 days at 6,100 m. After exposure to HBH, the memory consolidation of rats has been assessed in MWM. The results showed that there was memory consolidation impairment in HBH-exposed rats as compared to normoxic rats in terms of time spent in quaradents, rings, and counters. The rats which have been treated with physostigmine (PHY) and galantamine (GAL) showed better time spent in quaradents, rings, and counters as compared with hypoxic rats. In conclusion, the cholinesterase inhibitors could ameliorate the impairment of memory consolidation following HBH.

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms.

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.

Memogain is a galantamine pro-drug having dramatically reduced adverse effects and enhanced efficacy.

Memogain (Gln-1062) is an inactive pro-drug of galantamine, the latter being a plant alkaloid approved for the treatment of mild to moderate Alzheimer's disease. Memogain has more than 15-fold higher bioavailability in the brain than the same doses of galantamine. In the brain, Memogain is enzymatically cleaved to galantamine, thereby regaining its pharmacological activity as a cholinergic enhancer. In animal models of drug-induced amnesia, Memogain produced several fold larger cognitive improvement than the same doses of galantamine, without exhibiting any significant levels of gastrointestinal side effects that are typical for the unmodified drug and other inhibitors of cholinesterases, such as donepezil and rivastigmin. In the ferret, dramatically reduced emetic and behavioral responses were observed when Memogain was administered instead of galantamine. Based on these and other preclinical data, Memogain may represent an advantageous drug treatment for Alzheimer's disease, combining much lesser gastrointestinal side effects and considerably higher potency in enhancing cognition, as compared to presently available drugs.