Amitriptyline and imipramine inhibit the release of acetylcholine from parasympathetic nerve terminals in the rat iris

The electrically stimulated release of [3H]acetylcholine from the parasympathetic nerve terminals of the rat iris in vitro is increased in a dose-dependent manner by scopolamine but is decreased by the tricyclic antidepressants amitriptyline and imipramine. The increased release in the presence of scopolamine seems to be due to the blockade of a presynaptic muscarinic autoreceptor that, in the drug-free state, inhibits the release of acetylcholine. However, at drug concentrations that should have comparable antimuscarinic potency, the antidepressants inhibit the release of acetylcholine. This suggests that the anticholinergic side effects of the antidepressants may be due to the reduced release of acetylcholine from parasympathetic nerve terminals as well as a possible direct postsynaptic muscarinic receptor blocking action. Whatever the mechanism of this action, the antidepressants do not have the same effect as scopolamine at the presynaptic muscarinic autoreceptor in the rat iris.     

Scopolamine administration modulates muscarinic, nicotinic and NMDA receptor systems

Studies on the effect of scopolamine on memory are abundant but so far only regulation of the muscarinic receptor (M1) has been reported. We hypothesized that levels of other cholinergic brain receptors as the nicotinic receptors and the N-methyl-D-aspartate (NMDA) receptor, known to be involved in memory formation, would be modified by scopolamine administration.C57BL/6J mice were used for the experiments and divided into four groups. Two groups were given scopolamine 1 mg/kg i.p. (the first group was trained and the second group untrained) in the multiple T-maze (MTM), a paradigm for evaluation of spatial memory. Likewise, vehicle-treated mice were trained or untrained thus serving as controls. Hippocampal levels of M1, nicotinic receptor alpha 4 (Nic4) and 7 (Nic7) and subunit NR1containing complexes were determined by immunoblotting on blue native gel electrophoresis.Vehicle-treated trained mice learned the task and showed memory retrieval on day 8, while scopolamine-treatment led to significant impairment of performance in the MTM. At the day of retrieval, hippocampal levels for M1, Nic7 and NR1 were higher in the scopolamine treated groups than in vehicle-treated groups.The concerted action, i.e. the pattern of four brain receptor complexes regulated by the anticholinergic compound scopolamine, is shown. Insight into probable action mechanisms of scopolamine at the brain receptor complex level in the hippocampus is provided. Scopolamine treatment is a standard approach to test cognitive enhancers and other psychoactive compounds in pharmacological studies and therefore knowledge on mechanisms is of pivotal interest.     

The effects of muscarinic receptor blockers on the turnover rate of acetylcholine in various regions of the rat brain.

The actions of antimuscarinic agents (benztropine, trihexyphenidyl, and scopolamine) on the dynamics of acetylcholine (ACh) in central cholinergic neurons were examined in various rat brain areas. It was found that the pattern of changes in ACh turnover (TRACh) elicited by these drugs exhibited marked regional variations. After administration of the anticholinergic drugs, the TRACh in hippocampus and thalamus was increased, in cortex it was decreased, and in striatum it was unchanged. ACh concentration in the cortex and striatum was decreased while in hippocampus and thalamus ACh levels were unaltered. Further analysis of the cholinergic septo-hippocampal pathway using lesions of the fimbria-fornix and local drug injections into the septum argue against an in vivo action of these drugs on presynaptic or cell body muscarinic autoreceptors. Moreover, the data suggest that muscarinic receptor blockers cause an increased TRACh only in those areas where a feedback loop is operative, possibly by inhibiting a neuronal feedback loop involving at least one noncholinergic interneuron.     

Anticonvulsant effects of phencynonate hydrochloride and other anticholinergic drugs in soman poisoning: neurochemical mechanisms

Previous studies have paid little attention to the anticonvulsant effect of anticholinergic drugs that act on both muscarinic (M) and nicotinic (N) receptors during soman-induced seizures. Therefore, with the establishment of a soman-induced seizures model in rats, this study evaluated the efficacy in preventing soman-induced convulsions of two antagonists of both the M and N receptors, phencynonate hydrochloride (PCH) and penehyclidine hydrochloride (8018), which were synthesized by our institute, and of other anticholinergic drugs, and investigated the mechanisms of their antiseizures responses. Male rats, previously prepared with electrodes to record electroencephalographic (EEG) activity, were pretreated with the oxime HI-6 (125 mg kg-1, i.p.) 30 min before they were administered soman (180 microg kg-1, s.c.). All animals developed seizures subsequent to this treatment. Different drugs were given at different times (5, 20 and 40 min after seizures onset) and their anticonvulsant effects were monitored and compared using the two variables, i.e. the dose that could totally control the ongoing seizures, as well as the speed of seizures control. The anticonvulsant effects of atropine, scopolamine and 8018 decreased with the progression of the seizures, and they eventually lost their anticonvulsant activity when the seizures had progressed for 40 min. In contrast, PCH showed good anticonvulsant effectiveness at 5 and 20 min, and especially at 40 min after seizures onset. Of the anticholinergic drugs tested, atropine, scopolamine, and 8018 showed no obvious protection against pentylenetetrazol (PTZ)-induced convulsions or N-methyl-D-aspartate (NMDA)-induced lethality in mice. However, PCH antagonized the PTZ-induced convulsions in a dose-dependant manner with an ED50 of 10.8 mg kg-1, i.p. (range of 7.1-15.2 mg kg-1) and partly blocked the lethal effects of NMDA in mice. PCH also dose-dependently inhibited NMDA-induced injury in rat primary hippocampal neuronal cultures, suggesting a possible neuroprotective action in vivo. In conclusion, our study suggests that the mechanisms of PCH action against soman-induced seizures might differ from those of the M receptor antagonists atropine and scopolamine, and that of the antagonist of both the M and N receptors, 8018. The pharmacological profile of PCH might include anticholinergic and anti-NMDA properties. Compared with the currently recommended anticonvulsant drug diazepam, with known NMDA receptor antagonists such as MK-801 and with conventional anticholinergics such as scopolamine and atropine, the potent anticonvulsant effects of PCH during the entire initial 40 min period of soman poisoning, and its fewer adverse effects, all suggest that PCH might serve as a new type of anticonvulsant for the treatment of seizures induced by soman.     

Molecular mechanism of doxorubicin-induced anticholinergic effect in guinea-pig atria

The molecular mechanisms of anticholinergic actions of doxorubicin were examined by electrophysiological methods in atria and myocytes isolated from guinea-pig heart. A direct anticholinergic action of doxorubicin was confirmed with antagonistic action on carbachol-induced negative inotropic effect in atria. Both carbachol and adenosine produced shortening of action potential duration in atria measured by a microelectrode method. Doxorubicin (10-100 microM) inhibited the carbachol-induced action potential shortening in a concentration-dependent manner. However, doxorubicin did not antagonize the shortening elicited by adenosine. The whole-cell voltage clamp technique was performed to induce the muscarinic acetylcholine-receptor-operated K+ current (IK.ACh) in atrial myocytes loaded with GTP or GTPgammaS, a nonhydrolysable analogue of GTP. Doxorubicin (1-100 microM) suppressed carbachol-induced IK.ACh in a concentration-dependent manner (IC50 = 5.6 microM). In contrast, doxorubicin (10 and 100 microM) suppressed neither adenosine-induced IK.ACh nor GTPgammaS-induced IK.ACh. These results indicate that doxorubicin produces a direct anticholinergic effect through the muscarinic receptors in atrial myocytes.     

Nefiracetam (DM-9384) Preserves Hippocampal Neural Cell Adhesion Molecule-Mediated Memory Consolidation Processes During Scopolamine Disruption of Passive Avoidance Training in the Rat

Abstract: Scopolamine (0.15 mg/kg), a muscarinic antagonist, when administered during training or at a discrete 6-h posttraining time point, is demonstrated to inhibit the recall of a step-down passive avoidance response when tested at 24 and 48 h after task acquisition. Nefiracetam (3 mg/ kg), a piracetam-related nootropic, when given with scopolamine during training tended to improve task recall, and this effect was more pronounced when given at the 6-h posttraining time. Co-administration of nefiracetam with scopolamine was not necessary to achieve the antiamnesic action, as nefiracetam given during training significantly improved the memory deficits produced by scopolamine at the 6-h posttraining time. The paradigm-specific increase in hippocampal neural cell adhesion molecule sialylation, which is observed during consolidation of a passive avoidance response, was attenuated by the presence of scopolamine during training and at the 6-h posttraining time, and this effect was reversed by co-administration of nefiracetam, albeit in a paradigm-independent manner. These results suggest nefiracetam exerts a neurotrophic action that protects memory consolidation from drug inter- ventive insults.     

The role of muscarinic cholinoreceptors in the retrieval of the instrumental food conditioned reflex in cats

It was demonstrated in cat experiments that impairment of the retrieval of appetitive instrumental conditioning observed after systemic administration of antagonists of muscarinic central cholinoreceptors scopolamine (a nonselective M1 antagonist) and trihexyphenidyl (relatively selective M1 antagonist) could be connected with central and peripheral side effects of these antagonists. It was established that in case of the absence of side effects (low doses of trihexyphenidyl, 1 mg/kg) the blockade of M1-cholinoreceptors led either to selective fall-off of the motor instrumental reaction with in the presence of contextual behavior and other conditioned reactions or the appearance of errors that seemingly was indicative of the disturbance of triggering and realization of the motor program as the most important component of conditioning performance. The systemic injection of trihexyphenidyl (10 mg/kg), scopolamine (0.03 and 0.06 mg/kg) and nonselective peripheral antagonist methylscopolamine (0.03 mg/kg) led to changes in the general functional state (disorders in the emotional and motivational sphere), the expression of which depended on the individual sensitivity to anticholinergic drugs. The disturbance of CR retrieval observed in parallel with side reactions was characterized by a complete cessation of conditioning and apparently was not associated with memory deterioration.     

On the long acting gastric antisecretory activity of LM 24056 A non H2 antagonist

LM 24056, a phenothiazine derivative with no central effects, can be classified as a non anti H₂ antisecretory agent with a long duration of action. Its activity was demonstrated orally at low dose in pentagastrin stimulated Shay rat and in Heidenhain pouch in dog against gastrin, pentagastrin, carbachol and test meal. LM 24056 was inactive in histamine induced secretion. LM 24056 possesses very weak affinity to muscarinic receptors in vitro and in vivo. It has negligible anticholinergic properties in rats and mice at the peripheral level but no effect at the central level. The long lasting antisecretory action of LM 24056 may be supported by the persistent presence in plasma of a desmethyl metabolite at higher concentrations tham that of LM 24056 at any time. Contrary to LM 24056 sulfoxide and LM 24056 sulfone, desmethyl LM 24056 is a more potent antisecretory drug than LM 24056. Desmethyl LM 24056 possesses more marked affinity to peripheral muscarinic receptor than LM 24056. As the administration of therapeutic doses of LM 24056 was not followed by anticholinergic side-effects, it may be suggested that LM 24056 activity is related to a “prodrug like effect”. Finally the activity of LM 24056 may be related to LM 24056 itself and/or a desmethyl metabolite.     

Overexpression of <Emphasis Type="Italic">odc</Emphasis> (ornithine decarboxylase) in <Emphasis Type="Italic">Datura innoxia</Emphasis> enhances the yield of scopolamine

Scopolamine is widely used for its anticholinergic properties. Because of higher physiological activity and less side effects the world demand of scopolamine is estimated to be ten times greater than other anticholinergic agents, hyoscyamine and atropine. Since natural production is limited, alternatives are required to boost the production. We report the introduction of mouse odc gene of polyamine biosynthesis pathway which is also the primary pathway of tropane alkaloids in Datura innoxia. Polyamines, mainly putrescine, serve as the common metabolite for tropane alkaloids and nicotine. We have overexpressed odc gene to modulate the metabolic flux downstream and eventually achieved higher accumulation of scopolamine in transgenic plants. Among six independent transformed lines one line (O10) produced scopolamine (0.258 μg/g dry weight) almost six times higher than that produced by control plants (0.042 μg/g DW). To our knowledge, this is the first report of odc overexpression in D. innoxia leading to higher scopolamine yield.     

Neuromodulatory Propensity of Bacopa monniera Against Scopolamine-Induced Cytotoxicity in PC12 Cells Via Down-Regulation of AChE and Up-Regulation of BDNF and Muscarnic-1 Receptor Expression

Scopolamine is a competitive antagonist of muscarinic acetylcholine receptors, and thus classified as an anti-muscarinic and anti-cholinergic drug. PC12 cell lines possess muscarinic receptors and mimic the neuronal cells. These cells were treated with different concentrations of scopolamine for 24 h and were protected from the cellular damage by pretreatment with Bacopa monniera extract (BME). In current study, we have explored the molecular mechanism of neuromodulatory and antioxidant propensity of (BME) to attenuate scopolamine-induced cytotoxicity using PC12 cells. Our results elucidate that pretreatment of PC12 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by 3 μg/ml scopolamine to 54.83 and 30.30 % as evidenced by MTT and lactate dehydrogenase assays respectively. BME (100 μg/ml) ameliorated scopolamine effect by down-regulating acetylcholine esterase and up-regulating brain-derived neurotropic factor and muscarinic muscarinic-1 receptor expression. BME pretreated cells also showed significant protection against scopolamine-induced toxicity by restoring the levels of antioxidant enzymes and lipid peroxidation. This result indicates that the scopolamine-induced cytotoxicity and neuromodulatory changes were restored with the pretreatment of BME.     

The effect of a synthetic anticholinergic agent, N-methyl hyoscine methyl sulfate, on the exocrine pancreatic secretion of the alert dog]

The inhibitory action of N-methyl hyoscine methyl sulphate (N-methyl scopolamine, an anticholinergic drug) has been studied on the exocrine pancreatic secretion stimulated by secretin + caerulein on the conscious dog provided with Thomas cannulae. The dose-response curve shows an "all or nothing" effect on protein secretion since 0.38 microgram/kg. The inhibition of water and bicarbonate secretion is only observed from 12 micrograms/kg. The inhibitory effect of this drug was much greater than that obtained with similar molar quantities of atropine and no central effect has been observed.     

Effects of anticholinergic and cholinesterase blocking drugs on appetitive behavior under different deprivation conditions

The effects of scopolamine HBr (0.125?1.0 mg/kg) methscopolamine bromide (0.125?1.0 mg/kg), physostigmine sulphate (0.05?0.4 mg/kg), and neostigmine bromide (0.025?0.2 mg/kg) were studied under four different states of deprivation. The dependent measures were differentiated into two appetitive behaviors: lever pressing for food reward and for water reward. The dosages of both anticholinergic agents were effective in reducing appetitive behavior for food reward. The central acting anticholinergic (scopolamine) alone suppressed appetitive behavior for water reward. The effect of cholinesterase blockade was significantly effected by the locus of action with physostigmine suppressing both forms of appetitive behavior while the effect of peripheral cholinesterase blockade on appetitive behavior was specific to suppressing lever pressing for food reward. There were significant interactions of these agents with the deprivation conditions which were particularly evident in the effects of the less extensive acting drugs.     

Neurotensin concentrations in rat striatum and nucleus accumbens: Further studies of their regulation

The possible influence of cholinergic and dopaminergic mechanisms on neurotensin-containing neurones was examined at two different levels; nucleus accumbens and striatum in the rat brain. The acute treatment with the anticholinergic drugs atropine and scopolamine increased neurotensin concentrations in the striatum and, in the former case, also in the nucleus accumbens. Subchronic administration of atropine resulted in tolerance to its neurotensin-elevating action within the accumbens, but not within the striatum. Combined treatment with submaximal doses of haloperidol and atropine resulted in increases in neurotensin content which were greater than those seen with either agent alone. This was true regardless of whether the drugs were administered acutely or subchronically. This observation demonstrated that the tolerance phenomena occurring after subchronic elozapine and fluperlapine were not attributable to their anticholinergic activity. The control of striatal and accumbal neurotensin content by antidopaminergic and anticholinergic drugs seemed to be quite specific: drugs with actions on noradrenergic, serotoninergic, GABA-ergic and opiate systems did not influence the neurotensin content in these two structures. Preliminary studies on the effects of haloperidol on neurotensin release from striatal slices in vitro and that of cycloheximide on haloperidol's effect in vivo, suggest a possible inhibitory action of dopamine receptor blockade on neurotensin release.     

Modulation of Second Messengers in the Nervous System of Larval Manduca sexta by Muscarinic Receptors

Abstract: Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta . Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M co-eluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [³H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.     

Tolerance to an anticholinergic agent is paralleled by increased binding to muscarinic receptors in rat brain and increased behavioral response to a centrally active cholinomimetic

Pretreatment of rats with agents with strong antimuscarinic activity in the CNS (scopolamine, benztropine, trihexyphenidyl, amitriptyline, and thioridazine) but not their inactive congeners (desipramine, fluphenazine, or haloperidol) led to significant increases in the maximum apparent density of binding sites for 3H-QNB in cerebral cortical or striatal membranes. The dopamine agonist bromocriptine induced a similar effect that was blocked by haloperidol in striatum. None of these treatments altered the apparent affinity of the test ligand. Tolerance to the behavioral activating action of scopolamine developed over two weeks of daily treatment. This change was paralleled by an increase in 3H-QNB binding in cerebral cortex which was dependent on the dose and duration of treatment with scopolamine and persisted for a week following two weeks of treatment. Scopolamine pretreatment led to a significant increase in basal, spontaneous motor activity in the rat, but also to a marked increase in the motor-inhibitory actions of the centrally active muscarinic agonist pilocarpine. These results add to the impression that decreased availability of ACh agonists can significantly increase the availability and functional activity of central muscarinic ACh receptors to reflect "disuse supersensitivity."     

The effect of estrogen treatment on scopolamine inhibition of lordosis.

Previous evidence indicates that the cholinergic muscarinic antagonist, scopolamine, inhibits lordosis in female rats. In the experiments reported here, the effects of various doses and repeated administrations of estrogen on the scopolamine inhibition of lordosis were examined. In the first experiment, intraperitoneal injections of scopolamine (1 mg/rat) completely inhibited lordosis in ovariectomized rats primed with low doses of estradiol benzoate (0.25 or 0.5 micrograms for 3 days) and progesterone (500 micrograms). However, scopolamine was significantly less effective in inhibiting lordosis in females primed with a higher dose of estradiol benzoate (25 micrograms for 3 days) and progesterone (500 micrograms). When hormone priming was repeated on subsequent weeks, scopolamine continued to inhibit lordosis in females that received 0.25 micrograms estradiol benzoate but was less effective in females primed with 0.5 micrograms. Scopolamine failed to inhibit lordosis in females treated with 25 micrograms estradiol benzoate on these later tests. In the second experiment, various doses of scopolamine (1, 2, or 4 mg/rat) were administered intraperitoneally to females primed with the highest dose of estradiol benzoate (25 micrograms) and progesterone (500 micrograms). Lordosis was inhibited equally by all scopolamine doses during the first week. As in the first experiment, scopolamine failed to inhibit lordosis at all doses on subsequent weeks of testing. These results indicate that the ability of scopolamine to inhibit lordosis is reduced by increasing the dose or the number of estrogen exposures. Because higher doses of scopolamine failed to restore its inhibitory effect on lordosis an upregulation of muscarinic receptors by estrogen cannot account for the reduced effectiveness of scopolamine.     

The effect of sodium channel activators on muscarinic receptors of neuroblastoma cells

Incubation of neuroblastoma NIE 115 cells with veratrine leads to an apparent reduction in the number of muscarinic acetylcholine receptors assayed by [3H]scopolamine methyl chloride binding. No true down-regulation of the receptors occurs but a component of veratrine with muscarinic receptor affinity, which is not veratridine, enters the intracellular water space during the incubation period and competes with [3H]scopolamine methyl chloride for the muscarinic binding sites in subsequent ligand binding assays unless it is carefully washed away. Treatment of cells with the agonist carbamoylcholine does, however, lead to a true downregulation of muscarinic receptors.     

Muscarinic antagonists attenuate dizocilpine-induced hypermotility in mice.

Pretreatment of mice with the muscarinic receptor antagonists scopolamine and atropine attenuated the hypermotility (but not the depression of rearing) induced by a low dose of dizocilpine maleate [(+)-MK-801; 0.1 mg/kg, i.p.], a non-competitive NMDA antagonist. In contrast, the muscarinic blockers failed to affect hypermotility induced by equieffective doses of phencyclidine (1 mg/kg, i.p.) or d-amphetamine (2 mg/kg, i.p.). These results suggest differences between the mechanism of behavioral activation produced by dizocilpine and phencyclidine, and demonstrate the potential of muscarinic blockade for diminishing the behavioral toxicity of NMDA antagonists.     

Effects of Scopolamine and Melatonin Cotreatment on Cognition,Neuronal Damage,and Neurogenesis in the Mouse Dentate Gyrus

It has been demonstrated that melatonin plays important roles in memory improvement and promotes neurogenesis in experimental animals. We examined effects of melatonin on cognitive deficits, neuronal damage, cell proliferation, neuroblast differentiation and neuronal maturation in the mouse dentate gyrus after cotreatment of scopolamine (anticholinergic agent) and melatonin. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected for 2 and/or 4 weeks to 8-week-old mice. Scopolamine treatment induced significant cognitive deficits 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly improved spatial learning and short-term memory impairments. Two and 4 weeks after scopolamine treatment, neurons were not damaged/dead in the dentate gyrus, in addition, no neuronal damage/death was shown after cotreatment of scopolamine and melatonin. Ki67 (a marker for cell proliferation)- and doublecortin (a marker for neuroblast differentiation)-positive cells were significantly decreased in the dentate gyrus 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly increased Ki67- and doublecortin-positive cells compared with scopolamine-treated group. However, double immunofluorescence for NeuN/BrdU, which indicates newly-generated mature neurons, did not show double-labeled cells (adult neurogenesis) in the dentate gyrus 2 and 4 weeks after cotreatment of scopolamine and melatonin. Our results suggest that melatonin treatment recovers scopolamine-induced spatial learning and short-term memory impairments and restores or increases scopolamine-induced decrease of cell proliferation and neuroblast differentiation, but does not lead to adult neurogenesis (maturation of neurons) in the mouse dentate gyrus following scopolamine treatment.     

Ondansetron amelioration of scopolamine induced cognitive deficits in three-panel runway apparatus in rats

Effect of ondansetron (5-HT3-receptor antagonist) was studied on the working memory deficits induced by scopolamine, a muscarinic receptor antagonist in rats using a three-panel runway apparatus. Varying doses of scopolamine (0.1-0.56mg/kg, ip) were administered alone or in combination with ondansetron (0.01-1.0 mg/kg, ip) and memory errors and latency period of the session were recorded on a three-panel runway apparatus. Treatment with scopolamine (0.56 mg/kg) produced working memory deficits in rats. Treatment with ondansetron (1.0 mg/kg) significantly reduced the scopolamine-induced working memory deficits.