N-methylscopolamine Research Articles

Challenges in reproducing the standard lithium Pilocarpine model of temporal lobe epilepsy in wistar rats: troubleshooting strategies

Introduction: Animal model of temporal lobe epilepsy (TLE) provide an essential platform to study the disease pathophysiology, and disease modifying management protocols. Lithium –pilocarpine model is one of the most favored model. Many practical difficulties have been encountered while creating this model in this study as well as by other authors. We have systematically attempted to compile all issues and their possible solutions in establishment of Li-Pi model of status epilepticus in wistar rats, to provide a dependable protocol with improved seizure induction rates and lower mortality rates. Materials and Methods: Young small size male wistar rats (100-150gm) were injected with lithium chloride (LiCl) (127 mg/kg, i.p.), followed by methyl scopolamine (1mg/kg, i.p.) after 18-22 hrs. Precisely after 30 minutes, dose of pilocarpine (i.p.) was administered. Post seizures, diazepam (10 mg/kg) was injected to reduce seizures severity and to increase survival rate and rats were fed glucose (10% of body weight) through oral gavage. Model creation was validated by histological examination of hippocampus. The rats which did not develop seizures, were reutilized for the model generation after drug wash-out period of 48 hours. Results: Initial model creation was unsuccessfully attempted in 75 rats. Standard described protocol of three incremental doses of pilocarpine (10mg/kg, maximum of 30mg/kg) did not elicit epilepsy. Loading dose of 30mg/kg pilocarpine caused stage 1 epilepsy in 60% rats; second dose of pilocarpine (30mg/kg), caused stage 2 epilepsy in 70% rats. Successful TLE model creation was seen in 33 out of 57 rats, using proposed modification in which 2 incremental doses of pilocarpine (30mg/kg each) were followed by 3 doses of 10mg/kg each; 6 rats died. Conclusions: The study provides a detailed procedure for lithium pilocarpine model creation and various pitfalls encountered. Modified pilocarpine dosage led to improved seizure induction rates (58%) and lower mortality rates (10%). Keywords: Epilepsy animal model; Status epilepticus; lithium-pilocarpine; Animal mortality

Novel Method Development and Validation for UV–Visible Spectrophotometric Analysis of Methscopolamine Bromide

Novel Method Development and Validation for UV–Visible Spectrophotometric Analysis of Methscopolamine Bromide

Development and Optimization of Methscopolamine Bromide Gastroretentive Floating Tablets Using 32 Factorial Design.

The aim of this study was to formulate methscopolamine floating drug delivery system to increase its gastro retention for further enhancement of absorption and overall bioavailability. Direct compression method was used to formulate floating drug delivery system of methscopolamine bromide.: Different amount of HPMC, PVP K25, and MCC were used for preparation of tablets. The prepared tablets were evaluated for thickness, hardness, weight variation, floating lag time, swelling index and in-vitro drug release. All the formulations showed less than 10% of weight variation. The hardness and thickness of all the formulations were within the range of 3.7-4.2 kg/cm2 and 3.63-3.83 mm respectively. Floating lag time for all the formulations was reported in seconds. The degree of swelling was reported in range of 82.10-85.83%. In vitro release was carried out for 24 h. The maximum release was shown by F1 (93.947%) while the minimum release was observed for F4 (90.420%). The best formulation was optimized on the basis of percentage cumulative drug release, floating lag time and swelling index. F1 found to be the best formulation. Further on analyzing the drug release mechanism, F1 found to exhibit korsmeyer peppas model of drug release. Floating gastroretentive tablet of methscopolamine bromide was successfully developed using direct compression method with potential to enhance the drug absorption and effective treatment of peptic ulcer.

Peripheral effects of vagus nerve stimulation on anxiety and extinction of conditioned fear in rats.

Vagus nerve stimulation (VNS) enhances extinction of conditioned fear in rats. Previous findings support the hypothesis that VNS effects on extinction are due to enhanced consolidation of extinction memories through promotion of plasticity in extinction-related brain pathways however, alternative explanations are plausible. According to one hypothesis, VNS may produce a hedonic effect and enhance extinction through counter-conditioning. According to another hypothesis, VNS reduces anxiety during exposure and this weakens the association of conditioned stimuli with aversive conditioned responses. The present set of experiments (1) used conditioned place preference (CPP) to identify potential rewarding effects associated with VNS and (2) examined the peripheral effects of VNS on anxiety and extinction enhancement. Male Sprague–Dawley rats were surgically implanted with cuff electrodes around the vagus nerve and subjected to a CPP task in which VNS and sham stimulation were each paired with one of two distinct contexts over the course of 5 d. Following this procedure, rats did not show a place preference, suggesting that VNS is not rewarding or aversive. The role of the peripheral parasympathetic system in the anxiolytic effect of VNS on the elevated plus maze was examined by blocking peripheral muscarinic receptors with intraperitoneal administration of methyl scopolamine prior to VNS. Methyl scopolamine blocked the VNS-induced reduction in anxiety but did not interfere with VNS enhancement of extinction of conditioned fear, indicating that the anxiety-reducing effect of VNS is not necessary for the extinction enhancement.

Anti-cholinergics mecamylamine and scopolamine alleviate motion sickness-induced gastrointestinal symptoms through both peripheral and central actions

Enhanced cholinergic activity contributes to the production of complex autonomic manifestations of motion sickness (MS). However, whether anti-cholinergics exert their anti-MS effects through central or peripheral actions remained unclarified. In the present study, we investigated the effects of mecamylamine (MEC) and scopolamine (SCOP) on rotation-induced gastrointestinal symptoms (conditioned gaping and defecation), locomotion disturbances (hypoactivity and impaired balance performance), hypothermia as well as Fos expression in vestibulo-autonomic regions in rats. We also observed the effects of hexamethonium (HEX) and methyl scopolamine (MSCP) on those MS behavioral responses. The efficacy of all these drugs on rotation-induced emesis and other MS symptoms in cats was also examined. We found that intragastric administration of MEC and SCOP inhibited rotation-induced gaping and defecation in rats, but only MEC showed a dose-dependent manner. MEC aggravated rotation-induced balance disorder and failed to attenuate rotation-induced hypothermia as the SCOP did. MEC was more effective for inhibiting Fos expression in the caudal vestibular nucleus and nucleus of solitary tract than SCOP. Intraperitoneal injection of HEX and MSCP also significantly alleviated rotation-induced gastrointestinal symptoms, and showed benefit to balance performance in rats. In cats, MEC, SCOP and HEX had prophylactic effects against rotation-induced emesis and salivation, and deceased non-retching/vomiting symptoms, but MSCP only attenuated emesis. It suggested that MEC and SCOP might alleviate gastrointestinal symptoms of MS via inhibiting peripheral autonomic nervous system and central vestibulo-autonomic pathways. The nicotinic acetylcholine receptor inhibitors like MEC might be new candidates against gastrointestinal symptoms induced by MS or other vestibular disorders.

Does Thermosalient Effect Have to Concur with a Polymorphic Phase Transition? The Case of Methscopolamine Bromide

In this paper, we report for the first time an observed thermosalient effect that is not accompanied with a phase transition. Our experiments found that methscolopamine bromide—a compound chemically very similar to another thermosalient material, oxitropium bromide—exhibited crystal jumps during heating in the temperature range of 323–340 K. The same behavior was observed during cooling at a slightly lower temperature range of 313–303 K. Unlike other thermosalient solids reported so far, no phase transition was observed in this system. However, similar to other thermosalient materials, methscolopamine showed unusually large and anisotropic thermal expansion coefficients. This indicates that the thermosalient effect in this compound is caused by a different mechanism compared to all other reported materials, where it is governed by sharp and rapid phase transition. By contrast, thermosalient effect seems to be a continuous process in methscolopamine bromide.

Characterization of methanthelinium binding and function at human M1-M5 muscarinic acetylcholine receptors.

Firstly, it was determined whether methanthelinium bromide (MB) binds to human M1-M5 (hM1-hM5) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [3H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100μM of MB ranging from none in hM3 to 4.6-fold in hM2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [3H]NMS (0.2 and 2.0nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logKI (± S.E.) hM3 = 8.71 ± 0.15, hM1 = 8.68 ± 0.14, hM5 = 8.58 ± 0.07, hM2 = 8.27 ± 0.07 to hM4 = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logKB: hM1 = 9.53 ± 0.05, hM4 = 9.33 ± 0.05, hM5 = 8.80 ± 0.05, hM2 = 8,79± 0.06, to hM3 = 8.43 ± 0.04. In conclusion, MB, below 1μM, binds competitively and non-selectively (except for the difference between hM3 vs. hM4) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM1 and hM4.

Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant

Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

Verapamil Blocks Scopolamine Enhancement Effect on Memory Consolidation in Passive Avoidance Task in Rats.

Our recent data have indicated that scopolamine, a non-selective muscarinic receptor antagonist, improves memory consolidation, in a passive avoidance task, tested in rats. It has been found that verapamil, a phenylalkylamine class of the L-type voltage-dependent calcium channel antagonist, inhibits [3H] N-methyl scopolamine binding to M1 muscarinic receptors. However, there are no data about the effect of verapamil on memory consolidation in the passive avoidance task, in rats. The purpose of the present study was to examine the effects of verapamil (0.5, 1.0, 2.5, 5.0, 10, or 20 mg/kg i.p.) as well as the interaction between scopolamine and verapamil on memory consolidation in the step-through passive avoidance task, in Wistar rats. Our results showed that verapamil (1.0 and 2.5 mg/kg) administered immediately after the acquisition task significantly increased the latency of the passive avoidance response, on the 48 h retested trial, improving memory consolidation. On the other hand, verapamil in a dose of 5 mg/kg, that per se does not affect memory consolidation, significantly reversed the memory consolidation improvement induced by scopolamine (1 mg/kg, i.p., administered immediately after verapamil treatment) but did not change the passive avoidance response in rats treated by an ineffective dose of scopolamine (30 mg/kg). In conclusion, the present data suggest that (1) the post-training administration of verapamil, dose-dependently, improves the passive avoidance response; (2) verapamil, in ineffective dose, abolished the improvement of memory consolidation effect of scopolamine; and (3) exists interaction between cholinergic muscarinic receptors and calcium homeostasis-related mechanisms in the consolidation of emotional memory.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Muscarinic acetylcholine receptor binding affinities of pethidine analogs.

A series of pethidine analogs were synthesized and their affinities for the [(3)H]N-methyl-scopolamine (NMS) binding site on muscarinic acetylcholine receptors (mAChRs) were determined using M1, M3 or M5 human mAChRs expressed by Chinese hamster ovary (CHO) cell membranes. Compound 6b showed the highest binding affinities at M1, M3 and M5 mAChRs (Ki=0.67, 0.37, and 0.38 μM, respectively).

Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors.

Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M(2)-selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M(2) and M(3) receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein-fused receptors and an Alexa-555-conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor. We confirm the hypothesis that methoctramine high-affinity binding to the M(2) receptors involves simultaneous interaction with both the orthosteric binding site and the allosteric binding site located between the second and third extracellular loops. Methoctramine can bind solely with low affinity to the allosteric binding site on the extracellular domain of NMS-occupied M(2) receptors by interacting primarily with glutamate 175 in the second extracellular loop. In this mode, methoctramine physically prevents dissociation of NMS from the orthosteric binding site. Our results also demonstrate that lysine 523 in the third extracellular loop of the M(3) receptors forms a hydrogen bond with glutamate 219 of the second extracellular loop that hinders methoctramine binding to the allosteric site at this receptor subtype. Impaired interaction with the allosteric binding site manifests as low-affinity binding of methoctramine at the M(3) receptor.

Long-Term Activation upon Brief Exposure to Xanomleline Is Unique to M1 and M4 Subtypes of Muscarinic Acetylcholine Receptors

Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

Role of the third intracellular loop in the subtype-specific internalization and recycling of muscarinic M2 and M4 receptors.

Muscarinic M2, M4, and M2-M4 chimera receptors were transiently expressed in HEK-293 tsA201 cells, and agonist-dependent internalization of these receptors and recycling of internalized receptors were examined by measuring the amount of cell-surface receptors as [3H]N-methylscopolamine (NMS) binding activity. Coexpression of a dominant negative form of dynamin (DN-dynamin,dynamin K44A) greatly reduced the agonist-dependent internalization of M4 receptors but not of M2 receptors, as was reported by Vögler et al. (J Biol Chem 273, 12155-12160, 1998).The agonist-dependent internalization of M2/M4-i3/M2 chimera receptors (M2 receptors with the i3 loop replaced by that of M4 receptors) was greatly reduced by co-expression of DN-dynamin as was the case for M4 receptors, whereas the agonist-dependent internalization of M4/M2-i3/M4 chimera receptors was hardly affected by co-expression of DN-dynamin as was the case for M2 receptors.Internalized M2/M4-i3/M2 receptors as well as internalized M4 receptors were shown to be recycled back to the cell surface after removal of agonists, whereas no recycling was observed for M4/M2-i3/M4 receptors as well as M2 receptors. These results indicate that the i3 loops of M2 and M4 receptors take a major role in their agonist-dependent internalization and recycling.

Comparison of subcellular distribution and functions between exogenous and endogenous M1 muscarinic acetylcholine receptors

AimsRecombinant systems have been used for evaluating the properties of G-protein-coupled receptors (GPCRs) on the assumption of cell surface expression. However, many GPCRs, including muscarinic acetylcholine receptors (mAChRs), have also been reported to be distributed in intracellular organelles in native tissues and cell lines. In this study, we compared the pharmacological profiles of exogenously and endogenously expressed M1-mAChRs, and evaluated the functional properties of these receptors. Main methodsRecombinant M1-mAChRs were expressed exogenously in Chinese hamster ovary cells (CHO-M1 cells) and compared with endogenously expressed M1-mAChRs in N1E-115 neuroblastoma cells. The pharmacological and functional profiles were evaluated using cell-permeable antagonists (1-quinuclidinyl-benzilate (QNB), pirenzepine and atropine) and cell-impermeable antagonists (N-methylscopolamine (NMS) or MT-7). Key findingsM1-mAChRs were seen at the cell surface and intracellular sites in both cell lines. Under whole cell conditions, intracellular M1-mAChRs were mainly recognized by cell-permeable ligands, but scarcely by cell-impermeable ligands (at less than 100nM). In CHO-M1 cells, M1-mAChR activation by carbachol resulted in Ca2+ mobilization, ERK1/2 phosphorylation and a reduction in thymidine incorporation, all of which were completely inhibited by MT-7, indicating the involvement of surface M1-mAChRs. In N1E-115 cells, Ca2+ mobilization occurred through surface M1-mAChRs, whereas ERK1/2 phosphorylation and acceleration of thymidine incorporation were mediated through intracellular M1-mAChRs. SignificanceExogenous and endogenous M1-mAChRs are present at both the cell surface and the intracellular organelles, and the pharmacological properties of geographically distinct M1-mAChRs are different, and may depend on cell background and/or exogenous or endogenous origin.

Sudden unexpected death in a mouse model of Dravet syndrome

Sudden unexpected death in epilepsy (SUDEP) is the most common cause of death in intractable epilepsies, but physiological mechanisms that lead to SUDEP are unknown. Dravet syndrome (DS) is an infantile-onset intractable epilepsy caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes brain type-I voltage-gated sodium channel NaV1.1. We studied the mechanism of premature death in Scn1a heterozygous KO mice and conditional brain- and cardiac-specific KOs. Video monitoring demonstrated that SUDEP occurred immediately following generalized tonic-clonic seizures. A history of multiple seizures was a strong risk factor for SUDEP. Combined video-electroencephalography-electrocardiography revealed suppressed interictal resting heart-rate variability and episodes of ictal bradycardia associated with the tonic phases of generalized tonic-clonic seizures. Prolonged atropine-sensitive ictal bradycardia preceded SUDEP. Similar studies in conditional KO mice demonstrated that brain, but not cardiac, KO of Scn1a produced cardiac and SUDEP phenotypes similar to those found in DS mice. Atropine or N-methyl scopolamine treatment reduced the incidence of ictal bradycardia and SUDEP in DS mice. These findings suggest that SUDEP is caused by apparent parasympathetic hyperactivity immediately following tonic-clonic seizures in DS mice, which leads to lethal bradycardia and electrical dysfunction of the ventricle. These results have important implications for prevention of SUDEP in DS patients.

M1 is a major subtype of muscarinic acetylcholine receptors on mouse colonic epithelial cells

Muscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified. We characterized the pharmacological profile of mAChRs on mouse colonic crypts, employing [(3)H]-N-methyl scopolamine chloride as a radioligand and several subtype-selective chemicals, and the functional aspect by measuring short-circuit current (I sc) in Ussing chambers and by evaluating MAP kinase phosphorylation in mouse colonic mucosal sheets. The mAChRs were detected on the crypts (K d=163.2±32.3pM, B max=47.3±2.6fmol/mg of total cell protein). Muscarinic toxin 7 (MT-7, M1 subtype selective) gave a displacement curve with high affinity, but there was a part insensitive to MT-7 (18.8±0.4% of the total specific binding). The MT-7-insensitive component was displaced completely by darifenacin (M3 selective) with high affinity. ACh induced an increase in I sc, which was significantly enhanced by MT-7 but was completely inhibited by darifenacin or atropine. Colitis induction resulted in a significant decrease in the density of mAChRs, which occurred mainly in the MT-7-sensitive component (M1 subtype). Immunological experiments exhibited a reduction of M1 but not of M3 signal after colitis induction. Muscarinic stimulation induced an increase in MAP kinase phosphorylation, which was completely suppressed by MT-7 and was attenuated by inflammation, in mouse colonic epithelium. These results suggest that mAChRs in mouse colonic epithelial cells consist of two subtypes, M1 (80%) and M3 (20%). The major M1 subtype was likely to regulate epithelial chloride secretion negatively and was susceptible to inflammation and may be relevant to inflammatory gut dysfunction.

Treatment with endotracheal therapeutics after sarin microinstillation inhalation exposure increases blood cholinesterase levels in guinea pigs

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the blood and tissues of animals that are treated with a number of endotracheally aerosolized therapeutics for protection against inhalation toxicity to sarin. Therapeutics included, aerosolized atropine methyl bromide (AMB), scopolamine or combination of AMB with salbutamol, sphingosine 1-phosphate, keratinocyte growth factor, adenosine A1 receptor antisense oligonucleotide (EPI2010), 2,3-diacetyloxybenzoic acid (2,3 DABA), oxycyte, and survanta. Guinea pigs exposed to 677.4 mg/m3 or 846.5 mg/m3 (1.2 LCt50) sarin for 4 min using a microinstillation inhalation exposure technique and treated 1 min later with the aerosolized therapeutics. Treatment with all therapeutics significantly increased the survival rate with no convulsions throughout the 24 h study period. Blood AChE activity determined using acetylthiocholine as substrate showed 20% activity remaining in sarin-exposed animals compare to controls. In aerosolized AMB and scopolamine-treated animals the remaining AChE activity was significantly higher (45–60%) compared to sarin-exposed animals (p < 0.05). Similarly, treatment with all the combination therapeutics resulted in significant increase in blood AChE activity in comparison to sarin-exposed animals although the increases varied between treatments (p < 0.05). BChE activity was increased after treatment with aerosolized therapeutics but was lesser in magnitude compared to AChE activity changes. Various tissues showed elevated AChE activity after therapeutic treatment of sarin-exposed animals. Increased AChE and BChE activities in animals treated with nasal therapeutics suggest that enhanced breathing and reduced respiratory toxicity/lung injury possibly contribute to rapid normalization of chemical warfare nerve agent inhibited cholinesterases.

EFFECT OF AN ANTI-ACETYLCHOLINE DRUG, METHSCOPOLAMINE BROMIDE, ON ULCER FORMATION AND GASTRIC MUCUS.

Abstract A 30-hr period of restraint in rats was followed by the appearance of ulcers in the gastric corpus coincidental with a marked reduction of gastric juice volume, acid and also of hexosamine which was used as an estimation of mucus content. Methscopolamine (Pamine), an anti-acetylcholine drug, prevented ulcer formation, reduced further volume and acid output but produced a 3–4 fold increase in hexosamine concentration. Tissue (corpus and antrum) hexosamine was moderately reduced by restraint. In the corpus, this was counteracted by methscopolamine but antrum hexosamine was not influenced by this drug. The anti-ulcer property of methscopolamine may be due not only to its effect on acid secretion but also to the rise in gastric mucus concentration that it produced.