M1 Muscarinic Receptor Antagonist Research Articles

Improving the Tolerability of Anticholinergic Agents in the Treatment of Overactive Bladder

Pharmacological treatment for overactive bladder has centred around the interruption of the detrusor activity that is central to urge and incontinence symptoms. The majority of patients with this disorder are treated with antimuscarinic agents. These drugs have been demonstrated to improve urgency, frequency of micturition and urge incontinence, all of which are primary symptoms of overactive bladder; however, they are also commonly associated with anticholinergic adverse effects, most notably dry mouth. Attempts to increase tolerability have included the development of advanced formulations that regulate release of the active ingredient and the development of pharmacological agents that target the desired bladder receptors more specifically and accurately. Although all agents provide good efficacy, tolerability is greatly affected by the formulation used to deliver the active pharmacological agent, as well as the specificity of the targeted receptors. Clinical trials involving a transdermal formulation of oxybutynin have shown that this delivery method may be associated with a lower incidence of anticholinergic adverse events compared with both the immediate-release and the extended-release oral formulations of traditional agents, as well as the most recently approved agents - trospium chloride, solifenacin and darifenacin. Much is still being learned about the function and specificity of muscarinic receptors, which will support the development of agents with sustained efficacy and enhanced tolerability compared with the available formulations to date. These include the S-isomer of oxybutynin, as well as selective muscarinic M2 receptor antagonists.

Role of hippocampal M2 muscarinic receptors in the estrogen-induced enhancement of working memory

We have previously demonstrated that acetylcholine, acting at M2 muscarinic receptors, mediates the estradiol-induced increase in hippocampal N-methyl- d-aspartate receptor binding and the associated enhancement in working memory. The goal of present experiment was to investigate the role of hippocampal M2 receptors in the behavioral aspects of these effects. Ovariectomized rats were trained to locate a hidden escape platform on a matching-to-place version of the water maze in which the platform was moved to a new location for each session of four daily trials. Following 18 days of training, rats were randomly assigned to receive one of the following treatments: 1) injections of oil vehicle delivered 72 and 48 h before testing and continuous delivery of vehicle into the dorsal hippocampus via bilateral cannulae implants connected to osmotic minipumps; 2) injections of estradiol benzoate (EB) delivered 72 and 48 h before testing and continuous delivery of vehicle into the hippocampus; 3) injections of EB delivered 72 and 48 h before testing and continuous delivery of the M2 muscarinic receptor antagonist, AFDX 116, into the hippocampus; and 4) injections of EB delivered 72 and 48 h before testing and continuous delivery of AFDX 116 into a control site in the cortex. Chronic administration of AFDX 116 into the hippocampus, but not the cortex, significantly attenuated an estrogen-induced enhancement in performance on a working memory task in the water maze as indicated by increased latency and increased path length to locate an escape platform during a test trial when a 90 min delay was imposed between the first and second trials. These results indicate that acetylcholine acts at M2 muscarinic receptors located in the hippocampus to mediate the positive effects exerted by estrogen on working memory.

Darifenacin: a novel M3 muscarinic selective receptor antagonist for the treatment of overactive bladder

Darifenacin is a novel M3 muscarinic selective receptor antagonist for once-daily treatment of overactive bladder (OAB), a highly prevalent, chronic and debilitating disease defined by urinary urgency with or without urge incontinence, usually with increased frequency of micturition and nocturia. In vitro, darifenacin is a potent and specific muscarinic receptor antagonist with ≤ 59-fold higher selectivity for muscarinic M3 receptors relative to other muscarinic receptor subtypes. This profile may, therefore, confer clinical efficacy in the treatment of OAB, with a lower propensity for adverse effects and safety issues related to blockade of other muscarinic receptor subtypes. Indeed, consistent with its low relative affinity for M1 and M2 receptors, no effects on cognitive function and heart-rate variability, respectively, have been observed with darifenacin. Subsequent large-scale clinical trials have confirmed that darifenacin (at doses of 7.5 and 15 mg/day) results in central nervous system and cardiac adverse events comparable to placebo, and provides early and meaningful improvement across a range of OAB symptoms including incontinence episodes, urgency and urinary frequency. On the basis of such findings, darifenacin would appear to meet the current need for an effective OAB pharmacotherapy that is efficacious, well-tolerated and, more importantly, minimises the risk of safety-related adverse effects.

Determination of SCH 211803 by nanoelectrospray infusion mass spectrometry: evaluation of matrix effect and comparison with liquid chromatography–tandem mass spectrometry

A high throughput assay for SCH 211803, an M2 muscarinic receptor antagonist in human plasma using nanoelectrospray infusion tandem mass spectrometry is described. Sample processing consisted of protein precipitation followed by solid phase extraction using octadecasilyl resin-filled pipette tips on a liquid handling robotic system. The sample extracts were infused directly to the mass spectrometer using a nanoelectrospray interface in a silicon chip format. SCH 211803 was quantified in plasma over the concentration range of 1–1000 ng/mL. In comparison with a liquid chromatography–tandem mass spectrometry assay, the nanoelectrospray method has comparable accuracy, precision and limit of quantitation, with a nine-fold improvement in sample throughput. Using the nanoelectrospray assay, ion suppression was evaluated and found to be 15%. This represented a four-fold reduction in matrix suppression when compared to a conventional electrospray source operating in the flow injection analysis mode at a flow rate common for LC–MS/MS analysis.

Distinct properties of carbachol- and DHPG-induced network oscillations in hippocampal slices

The aim of this study was to compare and contrast the properties of gamma oscillations induced by activation of muscarinic acetylcholine or metabotropic glutamate receptors in the CA3 region of rat hippocampal slices. Both carbachol and the group I metabotropic glutamate receptor agonist, ( RS)-3,5-dihydroxyphenylglycine (DHPG), induced network oscillations in the gamma-frequency range (30–100 Hz). The M1 muscarinic receptor antagonist, pirenzepine, blocked carbachol-, but enhanced DHPG-induced oscillations, whereas LY 341495, an antagonist at metabotropic glutamate receptors, abolished DHPG-, but left carbachol-induced oscillations unchanged. There were significant differences in the peak frequency, maximal power, and spectral width of the two oscillations. Pharmacological experiments showed that both types of oscillation depend on fast excitatory and inhibitory synaptic transmission. Interestingly, activation of neurokinin-1 receptors by substance P fragment or enhancement of inhibitory synaptic currents by the benzodiazepine ligand, zolpidem, boosted DHPG-, but reduced the power of carbachol-induced oscillations. These results suggest that, although carbachol and DHPG might activate similar conductances in individual pyramidal cells, the oscillations they induce in slices involve different network mechanisms, most likely by recruiting distinct types of GABAergic interneuron.

Effects of anticholinergic drugs selective for muscarinic receptor subtypes on prepulse inhibition in mice

The effects of anticholinergic drugs selective for muscarinic receptor subtypes on prepulse inhibition of acoustic startle response were determined in mice. The prepulse inhibition is associated with sensorimotor information processing in the brain. The anticholinergic agent scopolamine (0.3 mg/kg, s.c.) significantly attenuated prepulse inhibition, while the drug (1–10 mg/kg, s.c.) had no effects on startle amplitude as an indicator of startle response. The muscarinic M1 receptor antagonist pirenzepine (0.1–10 μg/mouse, i.c.v.) and the muscarinic M2 receptor antagonist AF-DX116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (0.1–10 μg/mouse, i.c.v.) had no effects on prepulse inhibition or startle amplitude. The muscarinic M3 receptor antagonist 4-DAMP (1,1-dimethyl-4-diphenylacetoxy-piperidinium iodide) (30 μg/mouse, i.c.v.) and the muscarinic M4 receptor antagonist tropicamide (0.1 μg/mouse, i.c.v.) significantly attenuated prepulse inhibition, while tropicamide (0.01 μg/mouse, i.c.v.) but not 4-DAMP (10 and 30 μg/mouse, i.c.v.) produced a significant increase in startle amplitude. These results suggest that the blockade of muscarinic M3 and M4 receptors leads to the disruption of prepulse inhibition.

Regulation of rat mesencephalic GABAergic neurones through muscarinic receptors.

Central dopamine neurones are involved in regulating cognitive and motor processes. Most of these neurones are located in the ventral mesencephalon where they receive abundant intrinsic and extrinsic GABAergic input. Cholinergic neurones, originating from mesopontine nuclei, project profusely in the mesencephalon where they preferentially synapse onto local GABAergic neurones. The physiological role of this cholinergic innervation of GABAergic neurones remains to be determined, but these observations raise the hypothesis that ACh may regulate dopamine neurones indirectly through GABAergic interneurones. Using a mesencephalic primary culture model, we studied the impact of cholinergic agonists on mesencephalic GABAergic neurones. ACh increased the frequency of spontaneous IPSCs (151 +/- 49%). Selective activation of muscarinic receptors increased the firing rate of isolated GABAergic neurones by 67 +/- 13%. The enhancement in firing rate was Ca(2+) dependent since inclusion of BAPTA in the pipette blocked it, actually revealing a decrease in firing rate accompanied by membrane hyperpolarization. This inhibitory action was prevented by tertiapin, a blocker of GIRK-type K(+) channels. In addition to its excitatory somatodendritic effect, activation of muscarinic receptors also acted presynaptically, inhibiting the amplitude of unitary GABAergic synaptic currents. Both the enhancement in spontaneous IPSC frequency and presynaptic inhibition were abolished by 4-DAMP (100 nm), a preferential M3 muscarinic receptor antagonist. The presence of M3-like receptors on mesencephalic GABAergic neurones was confirmed by immunocytochemistry. Taken together, these results demonstrate that mesencephalic GABAergic neurones can be regulated directly through muscarinic receptors. Our findings provide new data that should be helpful in better understanding the influence of local GABAergic neurones during cholinergic activation of mesencephalic circuits.

Influence of gender on K+-induced cerebral vasodilatation.

It is not known whether cerebral vasoprotective mechanisms in females include increased function of arterial K+ channels. We hypothesized that vasodilator responses mediated by activation of inwardly rectifying K+ (K(IR)) channels are greater in cerebral arteries of female versus male rats and that this is due to the effects of estrogen. Changes in basilar artery diameter were measured with a cranial window preparation in anesthetized Sprague-Dawley rats. K+ (5 and 10 mmol/L) caused greater vasodilatation in females (percent maximum, 21+/-3% and 58+/-7%, respectively) versus males (11+/-1% and 37+/-4%, respectively; P<0.05). In contrast, vasodilator responses to aprikalim (1 and 3 micromol/L) or acetylcholine (ACh, 1 and 10 micromol/L) did not differ between the genders. The selective K(IR) channel inhibitor barium ion (30 micromol/L) decreased basilar artery diameter in males but not females (-7+/-1% versus -2+/-1%, P<0.05) and selectively inhibited K+-induced vasodilatation by approximately 50% in both groups. Ovariectomy of female rats resulted in smaller vasodilator effects of K+, and chronic treatment of these rats with 17beta-estradiol (0.01 mg/kg per day for 7 days) normalized K+-induced vasodilatation. Furthermore, the selective M2 muscarinic ACh receptor antagonist methoctramine (1 micromol/L) increased responses to K+ in males to levels equivalent to responses in females but had no effect on responses to K+ in females. K+ is a more powerful vasodilator in the female versus male cerebral circulation. This difference is estrogen dependent and could be due to a lack of M2 muscarinic ACh receptor-induced inhibition of K(IR) channel activation by K+ in female cerebral arteries.

Muscarinic M2 Receptor Stimulation of Cav1.2b Requires Phosphatidylinositol 3-Kinase, Protein Kinase C, and c-Src

This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 micromol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 micromol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 micromol/L), by the c-Src inhibitor PP2 (10 micromol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gbetagamma. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 micromol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.

Darifenacin

Darifenacin is a selective muscarinic M3-receptor antagonist that has been evaluated in clinical trials in patients with overactive bladder syndrome (OAB) using a controlled-release formulation. In multicentre, randomised, double-blind trials in patients with OAB, darifenacin 7.5 or 15 mg once daily for 12 weeks significantly reduced the frequency of urinary incontinence, frequency of micturition and frequency and severity of urgency versus placebo. A significant difference from placebo was apparent 2 weeks after starting treatment. At a dosage of 30 mg once daily, darifenacin significantly prolonged warning time compared with placebo. Darifenacin 15 mg once daily for 2 weeks was as effective as oxybutynin 5 mg three times daily at reducing the frequency of urinary incontinence and frequency and severity of urgency in patients with OAB. Darifenacin was generally well tolerated in clinical trials. The most common adverse events were dry mouth and constipation. CNS tolerability appeared to be similar to that of placebo. Darifenacin had no adverse effect on cognitive function in healthy elderly volunteers.

Profile of spinal and supra-spinal antinociception of (−)-linalool

We previously reported that administration of (−)-linalool, the naturally occurring enantiomer in essential oils, induced a significant reduction in carrageenin-induced oedema and in acetic acid-induced writhing. The latter effect was completely antagonised by the muscarinic receptor antagonist atropine and by the opioid receptor antagonist naloxone. To further characterise the antinociceptive profile of (−)-linalool, we studied its effect in the hot plate and the formalin in tests. In addition, to determine the possible involvement of the cholinergic, opioidergic and dopaminergic systems, we tested the effects of atropine, pirenzepine, a muscarinic M1 receptor antagonist, naloxone, sulpiride, a dopamine D2 receptor antagonist and ( R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine (SCH-23390), a dopamine D1 receptor antagonist on (−)-linalool-induced antinociception. Moreover, since K + channels seem to play an important role in the mechanisms of pain modulation, we examined the effect of glibenclamide, an ATP-sensitive K + channel inhibitor on (−)-linalool-induced antinociception. The administration of (−)-linalool (100 and 150 mg/kg, s.c.) increased the reaction time in the hot-plate test. Moreover, (−)-linalool (50 and 100 mg/kg) produced a significant reduction in the early acute phase of the formalin model, but not in the late tonic phase. The highest dose (150 mg/kg) caused a significant antinociceptive effect on both phases. The antinociceptive effects of (−)-linalool were decreased by pre-treatment with atropine, naloxone, sulpiride and glibenclamide but not by pirenzepine and SCH-23390. These results are in agreement with the demonstrated pharmacological properties of linalool, mainly its cholinergic, local anaesthetic activity and its ability to block NMDA receptors. Furthermore, a key role seems to be played by K + channels, whose opening might be the consequence of a stimulation of muscarinic M2, opioid or dopamine D2 receptors.

Long-term effects of BIBN-99, a selective muscarinic M2 receptor antagonist, on improving spatial memory performance in aged cognitively impaired rats

Aged Long–Evans rats were screened for spatial memory deficits using the Morris water maze task. Rats found to have impaired performance on the task (aged-impaired, AI) were then treated with a selective muscarinic M2 receptor antagonist, 5,11-dihydro-8-chloro-11-[[4-[3-[(2,2-dimethyl-1-oxopentyl)ethylamino]propyl]-1-piperidinyl]acetyl]-6 H-pyrido[2,3- b][1,4]benzodiazepin-6-one (BIBN-99; 0.5 mg/kg, s.c.), for 3 successive days while receiving additional water maze training. BIBN-99 significantly improved performance in the task during the 3 days of drug treatment. Treatment was then ceased for the remainder of the study and rats were tested again in the water maze on days 10, 17, and 24. Compared to vehicle-treated rats, enhanced performance was observed in the AI rats that had previously been treated with BIBN-99. These results indicate that BIBN-99 enhances spatial learning in AI animals and that enhanced (or long-term) memory persists in the absence of the drug. In a second experiment, a 2-month delay was imposed in between the original water maze screening and the drug treatment regime. Again, BIBN-99 significantly improved performance in AI rats. This latter study suggests that reference memory does not decay, even in an AI animal that had displayed poor learning following original water maze screening. Together, these studies help provide further insight into possible mechanism(s) of reference memory and its potential clinical usefulness.

Muscarinic M(3) receptor antagonists with (2R)-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxyphenylacetamide Structures. Part 2.

Optimization of the amine part of our original muscarinic M(3) receptor antagonist 1 was performed to identify M(3) receptor antagonists that are superior to 1. Compounds carrying a variety of diamine moieties without hydrophobic substituent on the nitrogen atom were screened against the binding affinity for the M(3) receptor and the selectivity for M(3) over the M(1) and M(2) receptors. This process led to a 4-aminopiperidinamide (2l) with a K(i) value of 5.1 nM and with a selectivity of the M(3) receptor that was 46-fold greater than that of the M(2) receptor. Further derivatization of 2l by inserting a spacer group or by incorporating alkyl group(s) into the amine part resulted in the identification of an 4-(aminoethyl)piperidinamide 2l-b with a K(i) value of 3.7 nM for the M(3) receptor and a selectivity for the M(3) receptor that was 170-fold greater than that of the M(2) receptor.

Treatment of dementia with neurotransmission modulation

The prevalence of dementia is growing in developed countries where elderly patients are increasing in numbers. Neurotransmission modulation is one approach to the treatment of dementia. Cholinergic precursors, anticholinesterases, nicotine receptor agonists and muscarinic M2 receptor antagonists are agents that enhance cholinergic neurotransmission and that depend on having some intact cholinergic innervation to be effective in the treatment of dementia. The cholinergic precursor choline alfoscerate may be emerging as a potential useful drug in the treatment of dementia, with few adverse effects. Of the anticholinesterases, donepezil, in addition to having a similar efficacy to tacrine in mild-to-moderate Alzheimer’s disease (AD), appears to have major advantages; its use is associated with lower drop-out rates in clinical trials, a lower incidence of cholinergic-like side effects and no liver toxicity. Rivastigmine is efficacious in the treatment in dementia with Lewy bodies, a condition in which the other anticholinesterases have not been tested extensively to date. Galantamine is an anticholinesterase and also acts as an allosteric potentiating modulator at nicotinic receptors to increase the release of acetylcholine. Pooled data from clinical trials of patients with mild-to-moderate AD suggest that the benefits and safety profile of galantamine are similar to those of the anticholinesterases. Selective nicotine receptor agonists are being developed that enhance cognitive performance without influencing autonomic and skeletal muscle function, but these have not yet entered clinical trial for dementia. Unlike the cholinergic enhancers, the M1 receptor agonists do not depend upon intact cholinergic nerves but on intact M1 receptors for their action, which are mainly preserved in AD and dementia with Lewy bodies. The M1 receptor-selective agonists developed to date have shown limited efficacy in clinical trials and have a high incidence of side effects. A major recent advancement in the treatment of dementia is memantine, a non-competitive antagonist at NMDA receptors. Memantine is beneficial in the treatment of severe and moderate-to-severe AD and may also be of some benefit in the treatment of mild-to-moderate vascular dementia. Drugs that modulate 5-HT, somatostatin and noradrenergic neurotransmission are also being considered for the treatment of dementia.

The intracellular pathway of the acetylcholine-induced contraction in cat detrusor muscle cells.

1. The present study was aimed to investigate intracellular pathways involved in acetylcholine (ACh)-induced contraction in cat detrusor muscle cells 2. Contraction was expressed as per cent shortening of length of individually isolated smooth muscle cells obtained by enzymatic digestion. Dispersed intact and permeabilized cells were prepared for the treatment of drugs and antibody to enzymes, respectively. Using Western blot, we confirmed the presence of related proteins. 3. The maximal contraction to ACh was generated at 10(-11) M. This response was preferentially antagonized by M3 muscarinic receptor antagonist rho-fluoro-hexahydrosiladifenidol (rhoF-HSD) but not by the M1 antagonist pirenzepine and the M2 muscarinic receptor antagonist methoctramine. We identified G-proteins (Gq/11), (Gs), (G0), (Gi1), (Gi2) and (Gi3) in the bladder detrusor muscle. ACh-induced contraction was selectively inhibited by (Gq/11) antibody but not to other G subunit. 4. The phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor neomycin reduced ACh-induced contraction. However, the inhibitors of the phospholipase D, the phospholipase A2 and protein kinase C did not attenuate the ACh-induced contraction. ACh-induced contraction was inhibited by antibody to PLC-beta1 but not PLC-beta3 and PLC-gamma. Thapsigargin or strontium, which depletes or blocks intracellular calcium release, inhibited ACh-induced contraction. Inositol 1,4,5-triphosphate IP3 receptor inhibitor heparin reduced ACh-induced contraction. 5. These results suggest that in cat detrusor muscle contraction induced by ACh is mediated via M3 muscarinic receptor-dependent activation of Gq/11 and PLC-beta1 and IP3-dependent Ca(2+) release.

Role of the M2 muscarinic receptor pathway in lidocaine-induced potentiation of the relaxant response to atrial natriuretic peptide in bovine tracheal smooth muscle.

We earlier reported that lidocaine augments the relaxation and accumulation of guanosine 3',5'-cyclic monophosphate produced by atrial natriuretic peptide (ANP) in bovine tracheal smooth muscle contracted with methacholine. However, the mechanism of that augmentation remains to be elucidated. In this study, we examined the role of muscarinic receptor-mediated signalling in the potentiation of ANP-induced relaxation by lidocaine. Lidocaine (100 micro M) augmented the relaxant responses to ANP in methacholine (0.3 microM)-contracted bovine tracheal smooth muscle but had no effect on the relaxant effects of ANP in preparations contracted with 100 micro M histamine. Treatment of tracheal preparations with methoctramine (0.03 microM), an M2 muscarinic receptor antagonist, enhanced ANP-induced relaxation and this treatment abolished the synergistic action of lidocaine on ANP. In radioligand-binding experiments, lidocaine concentration dependently displaced the specific binding of [3H]- N-methyl scopolamine to cloned human M2 and M3 muscarinic receptors expressed in Chinese hamster ovary cells. These results suggest that lidocaine acts as an M2 muscarinic receptor antagonist, thereby potentiating the relaxant responses to ANP in the bovine tracheal smooth muscle contracted with muscarinic receptor agonists.

Orally active and selective benzylidene ketal M2 muscarinic receptor antagonists for the treatment of Alzheimer's disease

AbstractNumerous studies have implicated the cholinergic system as a target for Alzheimer's disease (AD) therapy. The currently marketed drugs for treatment of AD are acetylcholinesterase inhibitors, which increase levels of the neurotransmitter acetylcholine (ACh) by blocking its metabolic degradation. However, this mechanism results in a non‐selective enhancement of cholinergic activity in peripheral organs as well as brain, which could lead to detrimental side effects. Cholinergic enhancement in brain can also be achieved by blocking presynaptic release‐regulating autoreceptors. M2 muscarinic receptor activation inhibits ACh release in brain regions that are critical for learning and memory processes. Thus, M2 antagonists may prove beneficial for relief of cognitive deficits in AD. In this review, early discoveries of M2 muscarinic antagonists based on dibenzodiazepinones, natural products, piperazines, and piperidines are described. More specifically, the development of a benzylidene ketal class of M2 muscarinic antagonists is reviewed. Characterization and optimization of these agents have led to the identification of compounds with high M2 receptor affinity and subtype selectivity. These M2 antagonists also facilitate ACh release and enhance cognition in preclinical experimental paradigms. While clinical efficacy of these compounds has not been evaluated, M2 antagonists represent a promising treatment for AD. Drug Dev. Res. 56:310–320, 2002. © 2002 Wiley‐Liss, Inc.

Intrastriatal infusions of methoctramine improve memory in cognitively impaired aged rats

Alterations of striatal cholinergic markers may correlate with cognitive impairments in aged rats. M2 muscarinic receptors were found to be presynaptic inhibitory autoreceptors on striatal cholinergic interneurons. The effect of bilateral intrastriatal infusions of the M2 muscarinic receptor antagonist methoctramine was assessed, in cognitively impaired aged (24–26 months) Long-Evans female rats, on memory performances in a water maze. Compared with vehicle infusions, methoctramine injected bilaterally (1 μg/side) in the dorsolateral striatum, significantly improved procedural memory performance while having no effect on spatial working memory. Our results suggest that, in cognitively impaired aged rats, the blockade of M2 muscarinic receptors in the dorsolateral striatum improves procedural memory probably by enhancing the release of acetylcholine.

Muscarinic Receptors Control Postprandial Release of Glucagon-Like Peptide-1: In Vivo and in Vitro Studies in Rats

Plasma levels of glucagon-like peptide-1 (GLP-1) rise rapidly after nutrient ingestion through an indirect mechanism triggered from the proximal intestine and involving the vagus nerve that stimulates the L cell in the distal gut. The role of muscarinic receptors in this pathway was thus investigated using the anesthetized rat and fetal rat intestinal cells (FRIC) in culture. GLP-1 secretion from the distal gut increased 5-fold after 3 ml corn oil were placed into the proximal duodenum (P < 0.001). Atropine (a nonspecific muscarinic receptor antagonist) completely inhibited fat-induced GLP-1 secretion in vivo (P < 0.01). Pirenzepine (an M1 muscarinic receptor antagonist) also inhibited fat-induced GLP-1 secretion in vivo, by 91 +/- 6% (P < 0.01). Gallamine (an M2 muscarinic receptor antagonist) and 4-diphenylacetoxy-N-methylpiperidine (an M3 muscarinic receptor antagonist) had no effect. Incubating FRIC cultures with bethanechol (a muscarinic receptor agonist) stimulated GLP-1 secretion to 200 +/- 22% of control (P < 0.01). Pirenzepine and gallamine significantly inhibited bethanechol-stimulated GLP-1 secretion, by 96 +/- 12% and 98 +/- 8%, respectively (P < 0.01). Unexpectedly, 4-diphenylacetoxy-N-methylpiperidine stimulated GLP-1 secretion by FRIC cells, to 324 +/- 52% of the control value (P < 0.01). Double immunofluorescent staining using GLP-1 and M1, M2, and M3 muscarinic receptor antibodies showed expression of the three subtypes of muscarinic receptors by the L cells in rat ileal sections and FRIC cultures. These results demonstrate the role of M1 muscarinic receptors expressed by L cells in the control of postprandial secretion of GLP-1. M2 muscarinic receptors also seem to play a role in controlling GLP-1 secretion by fetal, but not adult, L cells.

Pharmacological regulation of the neuronal control of airway mucus secretion.

The dominant neural control of human airway mucus secretion is cholinergic. There is no adrenergic control and sensory-efferent control is equivocal. Recent advances have identified several mechanisms that inhibit neurogenic mucus secretion. Muscarinic M3 receptor antagonists and tachykinin NK1 receptor antagonists inhibit neurogenic secretion. Muscarinic M2 receptors, nitric oxide and vasoactive intestinal peptide are inhibitory and regulate the magnitude of neurogenic secretion. The opening of large-conductance calcium-activated potassium channels is a common endogenous inhibitory mechanism and may represent the best therapeutic target. None of these inhibitory options are currently being targeted specifically for therapy of airway hypersecretion.