Cholinergic Receptor Subtypes Research Articles

Oxantel is an N-type (methyridine and nicotine) agonist not an L-type (levamisole and pyrantel) agonist: classification of cholinergic anthelmintics in Ascaris

Three pharmacological subtypes of cholinergic receptors have been distinguished in Ascaris suum using a muscle contraction assay and classical pharmacological techniques. The receptor subtypes are: a B-subtype (sensitive to bephenium); an L-subtype (sensitive to levamisole and pyrantel); and an N-subtype (sensitive to nicotine and methyridine). Oxantel is a cholinergic anthelmintic that was first introduced for the treatment of whipworm, Trichuris, infections in children. Here, we compare the subtype selectivity of oxantel with thenium and other cholinergic anthelmintics. We used the A. suum assay to derive p A 2 values for the agonists: oxantel, thenium, bephenium, levamisole, pyrantel, nicotine and methyridine with the antagonists: paraherquamide, 2-desoxyparaherquamide and methyllycaconitine. p A 2 values, rather than p K B values, were determined for all agonists when it was found that Schild slopes for some agonists were significantly less than 1.0. The p A 2 of oxantel was 6.58±0.25 for paraherquamide; 5.39±0.28 for 2-desoxyparaherquamide; 7.01±0.19 for methyllycaconitine. Comparison of p A 2 values using cluster analysis showed that oxantel was grouped with nicotine and methyridine, the N-subtype agonists. Thenium had p A 2s of 7.84±0.41 for paraherquamide; 5.52±0.50 for 2-desoxyparaherquamide; 6.33±0.19 for methyllycaconitine. Cluster analysis placed thenium between the L-subtype agonists and the B-subtype agonist. The therapeutic significance of classification of cholinergic anthelmintics is discussed. Combination of oxantel and pyrantel would have therapeutic advantages, covering N- and L-subtypes, and so increasing spectrum of action and reducing the potential for development of resistance. Our results predict that oxantel may remain effective in some nematode isolates that have become levamisole- and pyrantel-resistant.

Effects of myo-Inositol Versus Fluoxetine and Imipramine Pretreatments on Serotonin 5HT2Aand Muscarinic Acetylcholine Receptors in Human Neuroblastoma Cells

myo-Inositol (mI) is a key metabolic precursor to the phospoinositide (PI) metabolic pathway as a key component of central G-protein coupled receptor signaling systems, including several subtypes of adrenergic, cholinergic, serotonergic and metabotropic glutamatergic receptors. High dose mI has also been shown to be clinically effective in the treatment of obsessive-compulsive disorder, as well as panic and depression, although its mechanism of action remains elusive. The current study aimed to investigate the possible modulatory role of mI versus fluoxetine or imipramine pretreatments on serotonin-2A receptor (5HT2A-R) and muscarinic acetylcholine receptor (mAChR) function and binding in in vitro systems. After pretreating human neuroblastoma cells with different concentrations of mI, fluoxetine, or imipramine, receptor function was measured by second messenger [3H]-IPx accumulation and [35S]-GTPgammaS binding to G alpha(q) protein. Total [3H]-mI uptake into cells was measured, as well as specific receptor binding to determine receptor binding after the pretreatments. Results suggest that mI reduces 5HT2A-R function at the receptor-G protein level. While fluoxetine also reduced 5HT2A-R function, but to a lesser degree, imipramine increased 5HT2A-R function, which may explain why mI seems to be effective exclusively in selective serotonin reuptake inhibitor-sensitive disorders. In addition mI, and at high concentrations fluoxetine and imipramine, also reduces mAChR function. Furthermore the results suggest that the attenuating effect of mI on mAChRs is partially dependent on the PI metabolic pathway. The data provide novel information on understanding the mechanism of action of mI in depression and related anxiety disorders and added to the evidence suggesting a role for the cholinergic system in the pathophysiology of depression.

Oral Poster 20: Regional Profile of Adrenergic, Cholinergic and Serotonergic Receptor Subtypes in the Human Female Urethra

Oral Poster Presentations: AUGS-SGS Abstracts: 2004 Joint Scientific Meeting The American Urogynecologic Society and The Society of Gynecologic Surgeons

Age-related changes of muscarinic cholinergic receptor subtypes in the striatum of Fisher 344 rats

Striatum expresses a cholinergic system involved in the regulation of its activity and changes in striatal cholinergic receptors may be related to cognitive impairment. This study has investigated muscarinic cholinergic M1–M5 receptor subtype expression in striatum of Fischer 344 rats aged 6 (young), 15 (adult) and 22 months (senescent) to assess the contribution of different muscarinic cholinergic receptor subtypes in age-related changes of striatal cholinergic neurotransmission. Western blot analysis revealed the expression of the M1–M5 muscarinic receptor subtytpes in the striatum of rats of the three age groups investigated. Both radioligand binding assay and light microscope autoradiography showed in young rats a M4>M1>M2>M3>M5 rank order of receptor density. With the exception of M1 receptor, the density of which is similar in the dorsal (motor) and ventral (limbic) striatum, other receptor subtypes were more abundant in ventral than in dorsal striatum. M1 receptor expression was unchanged between young and adult rats and decreased in senescent animals both in dorsal and ventral striatum. In dorsal striatum M2 and M5 receptor expression did not show age-related changes, whereas in ventral striatum it was slightly decreased in adult rats compared to young or senescent cohorts. M3 receptor expression did not show age-related modifications, whereas a progressive age-related decrease of M4 receptor was found, both in dorsal and ventral striatum. These data indicate a heterogeneous response to age of different muscarinic receptor subtypes. Striatal cholinergic markers are thought to correlate with cognitive impairment in aged rats. In view of this, the identification of age-related changes of striatal muscarinic receptor subtypes may contribute to develop cholinergic strategies to counter cholinergic neurotransmission changes occurring with aging.

Localization of the m5 muscarinic cholinergic receptor in rat circle of Willis and pial arteries

The expression and microanatomical localization of the muscarinic cholinergic m5 receptor subtype was investigated in rat circle of Willis and pial arteries by in situ hybridization, immunoblotting and immunohistochemistry. In situ hybridization histochemistry revealed a strong signal in the endothelium of circle of Willis and pial arteries and a moderate signal in the tunica media of the same arteries, within smooth muscle. Exposure of membranes of arteries to anti-m5 receptor protein antibodies caused the development of a band of approximately 81 kDa. Immunohistochemistry revealed the accumulation of m5 receptor protein immunoreactivity primarily within endothelium of circle of Willis and cerebral arteries and to a lesser extent in the tunica media, within smooth muscle. Medium (external diameter 200–100 μm) and small-sized (external diameter smaller than 100 μm) pial arteries displayed a significantly higher immune staining than large-sized pial arteries or circle of Willis arteries. The above data that are consistent with recent functional studies reporting cholinergic dilation of cerebral blood vessels mediated via a m5 receptor, have shown that both endothelial and muscular components of cerebral arteries synthesize and express a muscarinic m5 receptor. In view of the peculiar localization in cerebral vessels, handling of the muscarinic m5 receptor may be considered as an approach in the treatment of cerebrovascular disease.

Contribution of acetylcholine to visual cortex plasticity

Acetylcholine is involved in a variety of brain functions. In the visual cortex, the pattern of cholinergic innervation varies considerably across different mammalian species and across different cortical layers within the same species. The physiological effects of acetylcholine in the visual cortex display complex responses, which are likely due to cholinergic receptor subtype composition in cytoplasm membrane as well as interaction with other transmitter systems within the local neural circuitry. The functional role of acetylcholine in visual cortex is believed to improve the signal-to-noise ratio of cortical neurons during visual information processing. Available evidence suggests that acetylcholine is also involved in experience-dependent visual cortex plasticity. At the level of synaptic transmission, activation of muscarinic receptors has been shown to play a permissive role in visual cortex plasticity. Among the muscarinic receptor subtypes, the M 1 receptor seems to make a predominant contribution towards modifications of neural circuitry. The signal transduction cascade of the cholinergic pathway may act synergistically with that of the NMDA receptor pathway, whose activation is a prerequisite for cortical plasticity.

Role of central cholinergic receptor sub-types in spatial working memory: a five-arm maze task in mice provides evidence for a functional role of nicotinic receptors in mediating trace access processes

A delayed-matching spatial working memory protocol in a 5-arm maze was used to test the hypothesis of differential roles for central nicotinic and muscarinic cholinergic receptors in mediating task performance. In experiment 1, using a within subjects-repeated design, groups of C57Bl/6 mice, previously trained to criterion with a 4 h retention interval separating presentation and test phases, received i.p. injections of either saline, scopolamine (0.8 mg/kg), mecamylamine (8.0 mg/kg), or the combination of scopolamine and mecamylamine before re-testing. Injections were given either, a) 15 min pre-presentation or, b) 30 s, c) 15 min, d) 3 h 45 min post-presentation in order to differentially affect the acquisition, trace maintenance and recall phases. Significant decreases in correct responses were observed for each drug treatment but the effects were a function of the time of treatment. Results of condition d), (i.e.15 min before retention test) confirm previous reports of severe disruption by each antagonist and their combination on retention. However, conditions a–c) show a constant disruption by scopolamine, increasing disruption by mecamylamine, whereas the combined treatment was without effect. Although the data show that central nicotinic and muscarinic antagonists both modulate working memory performance, they indicate first, that scopolamine-induced “amnesia” results, not from selective post-synaptic M1 muscarinic blockade but from indirect over-activation of nicotinic receptors. Second, the observation of high levels of retention although nicotinic and muscarinic receptors had undergone combined blockade during a large part of the retention interval is incompatible with the concept that test-induced activation of central cholinergic neurones mediates memory trace maintenance. Finally, taken with data from experiment 2, using a short (20 min) treatment-to-test interval, we conclude that central nicotinic receptors play a key role in attentional processes enabling working memory trace access during retrieval.

Bidirectional modulation of visual plasticity by cholinergic receptor subtypes in the frog optic tectum.

Cholinergic input to the optic tectum is necessary for visual map maintenance. To understand why, we examined the effects of activation of the different cholinergic receptor subtypes in tectal brain slices and determined whether the retinotectal map was affected by manipulations of their activity in vivo. Both alpha-bungarotoxin sensitive and insensitive nicotinic receptor agonists increased spontaneous postsynaptic currents (sPSCs) in a subpopulation of patch-clamped tectal cells; application of subtype selective receptor antagonists reduced nicotine-induced increases in sPSCs. Activation of alpha-bungarotoxin insensitive nicotinic receptors also induced substantial inward current in some cells. Muscarinic receptor mediated outward current responses were blocked by the M2-like muscarinic receptor antagonists himbacine or AF-DX 384 and mimicked by application of the M2-like agonist oxotremorine. A less frequently observed muscarinic response involving a change in sPSC frequency appeared to be mediated by M1-like muscarinic receptors. In separate experiments, pharmacological manipulation of cholinergic receptor subtype activation led to changes in the activity-dependent visual map created in the tectum by retinal ganglion cell terminals. Chronic exposure of the tectum to either alpha-bungarotoxin insensitive, alpha-bungarotoxin sensitive or M1-like receptor antagonists resulted in map disruption. However, treatment with the M2-like receptor antagonist, AF-DX 384, compressed the map. We conclude that nicotinic or M1-like muscarinic receptors control input to tectal cells while alpha-bungarotoxin insensitive nicotinic receptors and M2-like muscarinic receptors change tectal cell responses to that input. Blockade of the different cholinergic receptor subtypes can have opposing effects on map topography that are consistent with expected effects on tectal cell activity levels.

Postsynaptic muscarinic M1 receptors activate prefrontal cortical EEG of C57BL/6J mouse.

Recent pharmacological studies exploring the functional roles of muscarinic cholinergic receptor (mAChR) subtypes in prefrontal cortex of C57BL/6J (B6) mouse have provided evidence for a presynaptic M2 autoreceptor. The B6 mouse was chosen for these studies because it is a genetically well-characterized model that also provides the genomic background for many genetically modified mice. In addition to increasing ACh release, one functional consequence of pharmacologically blocking the cortical M2 autoreceptor is activation of the contralateral prefrontal cortical EEG. To date, the mechanisms through which M2 autoreceptor antagonism causes cortical EEG activation have not been investigated. The present study tested the hypothesis that, in the B6 mouse, prefrontal cortical ACh activates the contralateral prefrontal EEG via postsynaptic M1 receptors. This hypothesis was tested in 15 mice using in vivo microdialysis delivery of muscarinic antagonists with simultaneous quantification of ACh release, number of 7- to 14-Hz EEG spindles, and fast Fourier transformation analysis of prefrontal EEG. Dialysis delivery of the nonsubtype selective muscarinic antagonist scopolamine (10 nM) significantly (P = 0.01) increased ACh release. Quantitative EEG analysis showed that scopolamine did not alter contralateral prefrontal cortical EEG. To differentiate mAChR subtypes mediating pre- versus postsynaptic responses, additional experiments used muscarinic antagonists with different affinities for the five mAChR subtypes. Microdialysis delivery of 3 nM AF-DX 116, a muscarinic antagonist with relatively high affinity for the M2 and M4 subtypes, significantly (P < 0.01) increased prefrontal cortical ACh release and activated EEG in the contralateral prefrontal cortex. EEG activation was characterized by a significant decrease in number of 7- to 14-Hz EEG spindles (P < 0.0001) and power (Vrms) of EEG slow waves (P < 0.05). Microdialysis delivery of 3 nM AF-DX 116 plus 3 nM pirenzepine, a relatively selective M1 and M4 muscarinic antagonist, also significantly (P < 0.01) increased ACh release but did not decrease the number of EEG spindles and did not change EEG slow waves. The differential EEG and ACh responses to dialysis delivery of the muscarinic antagonists support the conclusion that, in B6 mouse, postsynaptic muscarinic receptors of the M1 subtype are a primary site by which ACh activates the EEG.

Immunochemical and immunocytochemical characterization of cholinergic markers in human peripheral blood lymphocytes

Cholinergic markers and the expression of M 2–M 5 muscarinic cholinergic receptor subtypes were investigated in human peripheral blood lymphocytes by Western blot analysis and immunocytochemistry. The totality of peripheral blood lymphocytes express acetylcholine (ACh) immunoreactivity, choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular ACh transporter (VAChT) and M 2–M 5 muscarinic cholinergic receptor protein immunoreactivity. Western blot analysis performed independently on T and B lymphocytes using anti-ChAT and anti-AChE antibodies revealed labelling of single bands of approximately 68–70 and 70 kDa, respectively, whereas VAChT was bound to two bands of approximately 80 and 45 kDa. The pattern of immunoblotting was similar in membranes of lymphocytes and striatum, used as a reference brain tissue. Western blot analysis using anti M 2–M 5 receptor antibodies revealed labelling of single bands of approximately 55, 85–90, 50 and 81 kDa, respectively. Confocal laser immunofluorescence showed the localization of ACh and VAChT immunoreactivity in punctiform areas likely corresponding to cytoplasmic vesicles. ChAT and AChE were diffused to the cytoplasm and plasma membrane. Muscarinic receptor immunoreactivity was located in lymphocyte plasma membrane. Although the role of lymphocyte cholinergic system is still unclear, the demonstration of cholinergic markers in T and B human blood lymphocytes supports the view that a cholinergic systems may contribute to the regulation of immune function. The characterization of these cholinergic markers may also contribute to define if their evaluation can be used for assessing the status of brain cholinergic system.

Response of Rats to Low Levels of Sarin

The purpose of this study was to determine whether exposure to levels of sarin causing no overt clinical signs would cause more subtle, adverse health effects that persisted after the exposure ended. Inhalation exposures of male Fischer 344 rats to 0, 0.2, or 0.4 mg/m 3 of sarin for 1 h/day for 1, 5, or 10 days under normal (25°C) and heat-stressed (32°C) conditions were completed and observations were made at 1 day and 1 month after the exposures. The sarin exposures had no observed effects on body weight, respiration rate, and minute volume during exposure nor in body temperature and activity during the 30-day recovery period. There was no evidence of cellular changes in brain determined by routine histopathology nor of any increase in apoptosis. Brain mRNA for interleukin (IL)-1β, tumor necrosis factor-α, and IL-6 was increased in a dose-dependent manner. Autoradiographic studies demonstrated that M1 cholinergic receptor site densities were unchanged at 1 day after repeated exposures with or without heat stress. At 30 days, there was a decrease in M1 receptors in the olfactory tubercle (with and without heat), and, with heat stress, M1 sites also decreased in a dose-dependent manner in the frontal cortex, anterior olfactory nucleus, and hippocampus. M3 receptor sites were not affected by sarin exposure alone. In the presence of heat stress, there was an upregulation in binding site densities in the frontal cortex, olfactory tubercle, anterior nucleus, and striatum immediately after exposure, and these effects persisted at 30 days. Although red blood cell acetylcholinesterase (AChE) was not greatly inhibited by the 1-day exposure, there were 30 and 60% inhibitions after repeated exposures at the low and high doses, respectively. Histochemical staining for AChE demonstrated that sarin exposure alone reduced AChE in the cerebral cortex, striatum, and olfactory bulb. Sarin exposure under heat stress reduced AChE staining in the hippocampus, an area important for memory function. Thus, repeated exposures under heat-stress conditions, to levels of sarin that would not be noticed clinically, resulted in delayed development of brain alterations in cholinergic receptor subtypes that may be associated with memory loss and cognitive dysfunction.

Serum Response Factor Activation by Muscarinic Receptors via RhoA: NOVEL PATHWAY SPECIFIC TO M1 SUBTYPE INVOLVING CALMODULIN, CALCINEURIN, AND Pyk2

The muscarinic cholinergic receptor (mAChR) subtypes share high sequence similarity except in their third intracellular loop and COOH terminus, domains thought to be involved in signal transduction. Subtypes M1, M3, and M5 couple mainly through Galpha(q/11), and M2 and M4 couple mainly through Galpha(i/o). Whether subtypes within each of these two groups differ in their signaling pathways remains to be resolved. This study focused on nuclear signaling pathways leading to activation of the transcription factor, serum response factor (SRF). Genes encoding M1, M2, and M3 were co-expressed in Jurkat T lymphocytes with a reporter gene driven by a mutant serum response element, SRE.L, which responds to SRF activation. We show that only M1 mAChR activated SRF through a pathway involving the small GTPase RhoA, with no response observed for M2 and M3. Transfection of GTPase-deficient Galpha subunits (GalphaQL; constitutively active form) demonstrated that SRF was activated by Galpha(13)QL but only marginally by Galpha(q)QL and Galpha(12)QL in Jurkat cells. Yet transfection of regulator of G protein-signaling protein, RGS2 and RGS4, which inhibit Galpha(q/11) activity, indicated that Galpha(q/11) and Ca(2+) mobilization were required for SRF activation by M1. Calmodulin inhibitors suppressed the M1 and the Galpha(13)QL pathways, acting both upstream and downstream of RhoA. However, calcineurin inhibitors and the tyrosine kinase inhibitor genistein selectively suppressed SRF activation by M1, but not by Galpha(13)QL, indicating the presence of separate pathways. The calmodulin-dependent tyrosine kinase Pyk2 was also activated by M1 but not M3, and Pyk2 appears also to play a role in M1-SRF activation, as judged by experiments with two dominant-negative Pyk2 mutants. These results reveal a novel calmodulin-dependent RhoA-SRF signaling pathway unique to the M1 mAChR subtype.

Differential gene expression of cholinergic muscarinic receptor subtypes in male and female normal human urinary bladder

Objectives To study the mRNA expression of each muscarinic receptor subtype in bladder areas involved in micturition, such as the bladder dome, neck, and trigone. Our study focused on the analysis of the gene expression of muscarinic receptors in the human male and female urinary bladder. Other than the well-known role of bladder parasympathetic innervation, an extensive study of the muscarinic receptor mRNA distribution in male and female urinary bladder is still lacking. Methods The study was carried out on 5 female (age 56 ± 10 years) and 5 male (age 70 ± 9 years) patients. The patients selected for this study did not have any lower urinary tract symptoms, as determined by International Prostate Symptom Score questionnaire. The mRNAs encoding muscarinic receptor subtypes were assessed by reverse transcription-polymerase chain reaction, followed by Southern blot analysis. Results Using a molecular approach, we demonstrated the presence of all muscarinic receptor subtypes in the different urinary bladder areas involved in micturition; in particular, our data indicated that mRNAs encoding muscarinic receptors are largely expressed in all examined bladder areas, both in men and women, although with some remarkable differences and a peculiar distribution. Conclusions Our results indicate that the pharmacology of the human bladder may be more complex than previously recognized. Furthermore, the choice to study each biopsy as a single sample and not use a pool of tissues allowed us to point out the individual variability between subjects and sex-related differences in the expression profile of muscarinic receptor subtype mRNAs.

Paraherquamide and 2-deoxy-paraherquamide distinguish cholinergic receptor subtypes in Ascaris muscle.

Paraherquamide is a novel natural anthelmintic product with a mode of action that is incompletely characterized. Nicotine and cholinergic-anthelmintic agonists of different chemical classes were used to produce contraction in Ascaris muscle strips. Paraherquamide and a semisynthetic derivative, 2-deoxy-paraherquamide, antagonized these responses. Analysis of the actions of the antagonists was made using the simple competitive model and nonlinear regression to estimate the pK(B) values of the antagonists. The analysis was tested using Clark plots. The pK(B) values for paraherquamide were: nicotine, 5.86 +/- 0.14; levamisole, 6.61 +/- 0.19; pyrantel, 6.50 +/- 0.11; and bephenium, 6.75 +/- 0.15. The pK(B) of nicotine was significantly different from the pK(B) values for levamisole, pyrantel, and bephenium, showing that paraherquamide can distinguish a subtype of cholinergic receptors sensitive to nicotine and a subtype of cholinergic receptors sensitive to levamisole, pyrantel, and bephenium. The pK(B) values for 2-deoxy-paraherquamide were: levamisole, 5.31 +/- 0.13; pyrantel, 5.63 +/- 0.10; and bephenium, 6.07 +/- 0.13. The Clark plots of the antagonism illustrated the degree of fit to the competitive model for 2-deoxy-paraherquamide. 2-Deoxy-paraherquamide selectively antagonized the effects of bephenium; the pK(B) values of levamisole and pyrantel were significantly different from the pK(B) of bephenium. Paraherquamide and 2-deoxy-paraherquamide are selective competitive cholinergic antagonists that distinguish subtypes of cholinergic receptor in Ascaris muscle corresponding to nicotine-, levamisole-, and bephenium-sensitive receptors.

Expression of peripheral blood lymphocyte muscarinic cholinergic receptor subtypes in airway hyperresponsiveness

The expression of muscarinic cholinergic receptor subtypes was investigated in peripheral blood lymphocytes (PBL) of bronchial asthma patients by a combined kinetic and equilibrium labeling technique for radioligand binding assay of muscarinic cholinergic receptor subtypes and by receptor immunochemistry and immunocytochemistry. An increased expression of M2 and to a lesser extent of M5 receptors and no changes of M4 receptor were observed in PBL of asthmatics compared to control individuals. The increase was related to bronchial hyperresponsiveness detected by methacholine challenge test. Analysis of M3 receptor expression revealed biphasic changes, with a decreased receptor density in patients with normal, mild and moderate responses to methacholine test and a recovery to levels similar to those found in healthy individuals in severe responders to methacholine test. The demonstration of a different expression of lymphocyte muscarinic receptors in asthma suggests that cholinergic system may participate to a molecular framework influencing immune functions in asthma.

Luminescence in ophiuroids (Echinodermata) does not share a common nervous control in all species

Study of the control mechanisms of light emission in invertebrates shows the involvement of several neurotransmitters. In ophiuroids, only one species (Amphipholis squamata) has so far been characterized for luminescence control, which seems to be cholinergic, with an influence of several excitatory and inhibitory neuromodulators (amino acids, catecholamines, neuropeptides S1 and S2, purines). The aim of this work is to investigate the nature of control mechanisms of light emission in three luminous ophiuroid species, A. filiformis, O. aranea and O. californica, in order to see whether or not they share common mechanisms. Luminescence induced by general depolarisation of tissues using KCl (200 mmol l(-1)) shows different patterns, according to species. Only A. filiformis emits light in response to acetylcholine. In this species, the involvement of both muscarinic and nicotinic receptors is proposed, since atropine and tubocurarine (at 10(-3) mol l(-1)) inhibited 99 % and 71 %, respectively, of the light emitted. Study of the subtypes of cholinergic receptors involved in photogenesis revealed that several subtypes of muscarinic receptors might be involved. It was also clearly shown that ophiuroids did not share a common mechanism of nervous control of luminescence in all species.

Muscarinic cholinergic receptor subtypes in the hippocampus of aged rats

In spite of the suggestion of impaired muscarinic function in adult-onset cognitive disorders, data on the expression of muscarinic receptors in the hippocampus as a function of age are inconsistent. One reason may be that the majority of investigations were unable to differentiate the five brain muscarinic receptors subtypes. In this study, using a protocol based on a combination of both kinetic and equilibrium binding approaches, we have assessed the expression and the density of M1–M5 muscarinic cholinergic receptors in the hippocampus of Fisher 344 rats aged 6, 15 and 22 months. An age-related decrease of the density of M1 receptor was found in pyramidal neurons of the CA1 subfield. In this area, other subtypes of muscarinic receptors were unchanged with the exception of a loss of M2 receptor in the radial layer. In the CA3 subfield, receptor changes involved M2, M3 and M5 subtypes, whereas in the dentate gyrus, the main changes affected M1 and M2 receptors of the granular layer and M2 and M3 receptors of the molecular layer. The above findings indicate that analysis of age-related changes of different muscarinic cholinergic receptors might represent a useful contribution to identifying the basis of cholinergic neurotransmission impairment in adult-onset cognitive dysfunction.

Activation of muscarinic M 1 receptors by acetylcholine to increase glucose uptake into cultured C 2C 12 cells

Regulation of glucose metabolism by cholinergic nervous activation has been demonstrated. In an attempt to evaluate the role of cholinergic receptor subtype in this regulation of glucose metabolism, we employed cultured myoblast C 2C 12 cells to investigate the glucose uptake in the present study. Acetylcholine (ACh) enhanced the uptake of radioactive glucose into C 2C 12 cells at the concentration range of 0.001 to 1.0 μmol/l. This effect was suppressed by the muscarinic antagonist atropine. Effect of ACh on muscarinic receptors was further supported by the blockade of scopolamine, another classical antagonist. Thus, activation of muscarinic receptors to enhance the radioactive glucose uptake into C 2C 12 cells can be considered. Moreover, pirenzepine, the antagonist of muscarinic M 1 receptors, competitively antagonized the action of ACh in C 2C 12 cells. However, methoctramine at concentration sufficient to inhibit the muscarinic M 2 receptors failed to produce similar effect. Similarly, 4-DAMP at effective concentration to block muscarinic M 3 receptors lacked the influence. An activation of muscarinic M 1 receptors seems responsible for the action of ACh in C 2C 12 cells. Pharmacological inhibition of phospholipase C by U73312 resulted in a concentration-dependent decrease in ACh-stimulated uptake of radioactive glucose into C 2C 12 cells. However, treatment with U73343, the inactive congener, failed to block the action of ACh. Moreover, both chelerythrine and GF 109203X diminished the action of ACh at concentrations sufficient to inhibit protein kinase C. Therefore, the obtained data suggest that increase of the glucose uptake evoked by ACh is mainly due to the activation of muscarinic M 1 receptors in cultured myoblast C 2C 12 cells.

Defining the neuromolecular action of myo-inositol : Application to obsessive–compulsive disorder

Dietary inositol is incorporated into neuronal cell membranes as inositol phospholipids where it serves as a key metabolic precursor in G protein-coupled receptors. In the brain, several subtypes of adrenergic, cholinergic, serotonergic and metabotropic glutamatergic receptors are coupled to the hydrolysis of phosphoinositides (PI) with myo-inositol (MI) crucial to the resynthesis of PI and the maintenance and effectiveness of signalling. Despite a mode of action that remains illusive, MI has demonstrated therapeutic efficacy in obsessive–compulsive disorder (OCD), putative OCD-spectrum disorders, as well as panic and depression. Behavioural and biochemical studies indicate that this efficacy does not involve simply the replenishing of the membrane PI pool. In addition to its precursory role in cell signalling, inositol lipids alter receptor sensitivity, can direct membrane trafficking events, and have been found to modulate an increasing array of signalling proteins. These effects may afford MI an ability to modulate the interaction between neurotransmitters, drugs, receptors and signalling proteins. This paper reviews the neuromolecular and genetic aspects of OCD in terms of the PI-linked 5HT receptor subtypes and relates these to the behavioural and therapeutic effects of MI. Since OCD often is poorly responsive to current drug treatment, understanding the neuropharmacology of MI holds great promise for understanding the neuropathology of this and other MI-responsive disorders.

RT-PCR reveals muscarinic acetylcholine receptor mRNA in the pre-Bötzinger complex.

Muscarinic receptors mediate the postsynaptic excitatory effects of acetylcholine (ACh) on inspiratory neurons in the pre-Bötzinger complex (pre-BötC), the hypothesized site for respiratory rhythm generation. Because pharmacological tools for identifying the subtypes of the muscarinic receptors that underlie these effects are limited, we probed for mRNA for these receptors in the pre-BötC. We used RT-PCR to determine the expression of muscarinic receptor subtypes in tissue punches of the pre-BötC taken from rat medullary slices. Cholinergic receptor subtype M(2) and M(3) mRNAs were observed in the first round of PCR amplification. All five subtypes, M(1)-M(5), were observed in the second round of amplification. Our results suggest that the majority of muscarinic receptor subtypes in the pre-BötC are M(2) and M(3), with minor expression of M(1), M(4), and M(5).