Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: implications for cholinergic-based therapies

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex. This defect is manifested by decreased M1 receptor-stimulated GTPgammaS binding and GTPase activity and by a loss in receptor-stimulated phospholipase C activity. Normal levels of G proteins suggest that the aberrant receptor-G protein interaction may result from an altered form of the m1 receptor in AD. The combined use of radioligand binding and receptor-domain specific antibodies has permitted the re-examination of the status of muscarinic receptor subtypes in the human brain. In AD, normal levels of m1 receptor [3H]-pirenzepine binding contrasted with diminished m1 immunoreactivity, further suggesting that there is an altered form of the m1 receptor in the disease. Reduced m2 immunoreactivity was consistent with decreased numbers of m2 binding sites. Increased levels of m4 receptors were observed in both binding and immunoreactivity measurements. These findings suggest one possible explanation for the relative ineffectiveness of cholinergic replacement therapies used to date and suggest potential new directions for development of effective therapeutic strategies for AD.     

Differential Alteration of Various Cholinergic Markers in Cortical and Subcortical Regions of Human Brain in Alzheimer''s Disease

The main objective of the present study was to determine whether cholinergic markers (choline acetyltransferase activity and nicotinic and muscarinic receptors) are altered in Alzheimer's disease. Choline acetyltransferase activity in Alzheimer's brains was markedly reduced in various cortical areas, in the hippocampus, and in the nucleus basalis of Meynert. The maximal density of nicotinic sites, measured using the novel nicotinic radioligand N-[3H]methylcarbamylcholine, was decreased in cortical areas and hippocampus but not in subcortical regions. M1 muscarinic cholinergic receptor sites were assessed using [3H]pirenzepine as a selective ligand; [3H]pirenzepine binding parameters were not altered in most cortical and subcortical structures, although the density of sites was modestly increased in the hippocampus and striatum. Finally, M2-like muscarinic sites were studied using [3H]-acetylcholine, under muscarinic conditions. In contrast to M1 muscarinic sites, the maximal density of M2-like muscarinic sites was markedly reduced in all cortical areas and hippocampus but was not altered in subcortical structures. These findings reveal an apparently selective alteration in the densities of putative nicotinic and muscarinic M2, but not M1, receptor sites in cortical areas and in the hippocampus in Alzheimer's disease.     

Estrogen therapy and brain muscarinic receptor density in healthy females: a SPET study

Estrogen Therapy (ET) may protect against age-related cognitive decline and neuropsychiatric disorders (e.g. Alzheimer's disease). The biological basis for this putative neuroprotective effect is not fully understood, but may include modulation of cholinergic systems. Cholinergic dysfunction has been implicated in age-related memory impairment and Alzheimer's disease. However, to date no one has investigated the effect of long-term ET on brain cholinergic muscarinic receptor aging, and related this to cognitive function. We used Single Photon Emission Tomography (SPET) and (R,R)[(123)I]-I-QNB, a novel ligand with high affinity for m(1)/m(4) muscarinic receptors, to examine the effect of long-term ET and age on brain m(1)/m(4) receptors in healthy females. We included 10 younger premenopausal subjects and 22 postmenopausal women; 11 long-term ET users (all treated following surgical menopause) and 11 ET never-users (surgical menopause, n=2). Also, verbal memory and executive function was assessed in all postmenopausal subjects. Compared to young women, postmenopausal women (ET users and never-users combined) had significantly lower muscarinic receptor density in all brain regions examined. ET users also had higher muscarinic receptor density than ET never-users in all the brain regions, and this reached statistical significance in left striatum and hippocampus, lateral frontal cortex and thalamus. Moreover, in ET users, (R,R)[(123)I]-I-QNB binding in left hippocampus and temporal cortex was significantly positively correlated with plasma estradiol levels. We also found evidence for improved executive function in ET users as compared to ET never-users. However, there was no significant relationship between receptor binding and cognitive function within any of the groups. In healthy postmenopausal women use of long-term ET is associated with reduced age-related differences in muscarinic receptor binding, and this may be related to serum estradiol levels.     

Rapid Communication: cis-Methyldioxolane Specifically Recognizes the m2 Muscarinic Receptor

Abstract: cis -Methyldioxolane (CD) is a muscarinic receptor agonist. [³H] CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [³H] CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [³H] CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [³H] CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [³H] CD with a K _D of 3.5 ± 0.5 nM. Binding was eliminated in the presence of guanosine 5'- O -(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [³H] CD are those that are associated with GDP-bound G protein. Binding of [³H] CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [³H] CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [³H] CD should aid research in these areas.     

Generation and Characterisation of Stable Cell Lines Expressing Recombinant Human N-Methyl-d-Aspartate Receptor Subtypes

Abstract: Transfection of mouse L(tk-) cells with human N -methyl- d -aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP 39653) had high affinity for NR1a/NR2A receptors ( K _D = 3.93 n M ) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.     

Muscarinic Cholinergic Receptor Subtypes in Hippocampus in Human Cognitive Disorders

Total muscarinic receptor levels, the levels of the subtypes exhibiting high and low affinity for pirenzepine, and the high- and low-affinity agonist states of the receptor were investigated in hippocampal tissue obtained at autopsy from mentally normal individuals and the following pathological groups: Alzheimer's disease, Parkinson's disease, Down's syndrome, alcoholic dementia, Huntington's chorea, and motor-neurone disease. A moderate decrease in the density of both high-affinity pirenzepine and high-affinity agonist subtypes was found in Alzheimer's disease, whereas a trend towards an increase in the overall muscarinic receptor density was apparent in the parkinsonian patients without dementia, mainly due to an increase in the low-affinity agonist state; the differences between the Alzheimer's disease and nondemented parkinsonian cases were highly significant. As previously reported, the levels of both choline acetyltransferase and acetylcholinesterase were markedly reduced in both Alzheimer's disease and Parkinson's disease--with a greater loss of both enzymes in the demented subgroup of parkinsonian patients. Activities of the cholinergic enzymes were also extensively reduced in Down's syndrome, accompanied by a loss of high-affinity pirenzepine binding. There were no significant receptor or enzyme alterations in the other groups studied. These observations suggest that in the human brain, extensive degeneration of cholinergic axons to the hippocampus, as indicated by a loss of cholinergic enzymes, is not necessarily accompanied by extensive muscarinic receptor abnormalities (as might be expected if a major subpopulation were presynaptic). Moreover, the opposite changes in muscarinic binding in Parkinson's and Alzheimer's diseases may be related to the greater severity of dementia in the latter disease.     

Cholinergic receptor alterations in the cerebral cortex of spinal cord injured rat

Many areas of the cerebral cortex process sensory information or coordinate motor output necessary for control of movement. Disturbances in cortical cholinergic system can affect locomotor coordination. Spinal cord injury causes severe motor impairment and disturbances in cholinergic signalling can aggravate the situation. Considering the impact of cortical cholinergic firing in locomotion, we focussed the study in understanding the cholinergic alterations in cerebral cortex during spinal cord injury. The gene expression of key enzymes in cholinergic pathway - acetylcholine esterase and choline acetyl transferase showed significant upregulation in the cerebral cortex of spinal cord injured group compared to control with the fold increase in expression of acetylcholine esterase prominently higher than cholineacetyl transferase. The decreased muscarinic receptor density and reduced immunostaining of muscarinic receptor subtypes along with down regulated gene expression of muscarinic M1 and M3 receptor subtypes accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic receptors and reduced immunostaining of alpha 7 nicotinic receptors in confocal imaging. Our data pin points the disturbances in cortical cholinergic function due to spinal cord injury; which can augment the locomotor deficits. This can be taken into account while devising a proper therapeutic approach to manage spinal cord injury.     

Distribution of muscarinic receptor subtypes in rat brain as determined in binding studies with AF-DX 116 and pirenzepine

In vitro competition binding experiments with the selective muscarinic antagonists AF-DX 116 and pirenzepine (PZ) vs 3H-N-methylscopolamine as radioligand revealed a characteristic distribution of muscarinic receptor subtypes in different regions of rat brain. Based on non linear least squares analysis, the binding data were compatible with the presence of three different subtypes: the M1 receptor (high affinity for PZ), the cardiac M2 receptor (high affinity for AF-DX 116) and the glandular M2 receptor (low affinity for PZ and AF-DX 116). The highest proportion of M1 receptors was found in the hippocampus, whilst the cerebellum and the hypothalamus were the regions with the largest fraction of the cardiac M2 and glandular M2 receptors, respectively. In certain brain areas, depending on the relative proportions of the subtypes, flat binding curves were seen for AF-DX 116 and PZ. Based on these data, an approximate distribution pattern of the subtypes in the various brain regions is presented.     

Muscarinic acetylcholine receptors in the brain of the zebrafish (Danio rerio) measured by radioligand binding techniques

Muscarinic acetylcholine receptors (mAChRs) play a role in learning, memory and behavior in vertebrate animals. We measured the muscarinic cholinergic receptor levels in extracts from zebrafish (Danio rerio) brain by radioligand binding techniques. Saturation binding experiments with the radioligand [3H]-quinuclidinyl benzilate (QNB) were used to determine receptor number and relative affinity for several agonists and antagonists. Affinity at zebrafish brain receptors was relatively high with a K(d) of 40 +/- 5 pM. The number of receptors, represented by Bmax, was 63 +/- 16 fmol/mg protein. Oxotremorine and carbachol, agonists at muscarinic acetylcholine receptors, bound with displacement curves indicating multiple binding sites. In addition, oxotremorine bound with a higher affinity than did carbachol. The antagonist potency profile at zebrafish receptors in brain was determined to be atropine>pirenzipine>p-fluoro-hexahydro-sila-difenidol>otenzepad. The results obtained with zebrafish brain compare favorably to those found in insect, fish and mammalian species. Taken together, the binding results and favorable comparisons to mammalian systems indicate that zebrafish may provide a useful model organism for evaluating the role of cholinergic systems in learning, memory and behavior.     

Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Chronic Activation of Muscarinic and Metabotropic Glutamate Receptors Down-Regulates Type I Inositol 1,4,5-Trisphosphate Receptor Expression in Cerebellar Granule Cells

Abstract: The ability of receptors coupled to phosphoinositide turnover to evoke accumulation of inositol 1,4,5-trisphosphate (InsP₃) over extended incubation periods, and consequently to affect the level of InsP₃ receptor expression, was studied in cultured cerebellar granule cells. The cholinergic agonist carbachol (CCh; 1 m M ) evoked a biphasic accumulation of InsP₃, a rapid three- to fourfold peak increase over control levels at ∼10 s, decreasing within 1 min to a long-lasting plateau elevation. Using an antibody against the type I InsP₃ receptor, it was demonstrated that >50% down-regulation of type I InsP₃ receptor expression in cerebellar granule cells occurred within 1 h of incubation with 1 m M CCh. Over 24 h, 1 m M CCh caused an ∼85% decrease in type I InsP₃ receptor levels, and significant decreases in immunoreactivity were evident at much lower concentrations of CCh. Direct assessment of total InsP₃ receptor expression using a radioligand binding method also detected down-regulation, but to an apparently lesser extent. 1-Aminocyclopentane-1 S ,3 R -dicarboxylic acid (200 µ M ), an agonist of metabotropic glutamate receptors, evoked a marked decrease in type I InsP₃ receptors after 24 h of incubation. These findings demonstrate that a functional consequence of maintained InsP₃ production in cerebellar granule cells is the down-regulation of InsP₃ receptor expression and that this down-regulation may be a common mechanism of action of phosphoinositide-linked receptors during prolonged stimulation.     

Deoxamuscaroneoxime derivatives as useful muscarinic agonists to explore the muscarinic subsite: demox, a modulator of orthosteric and allosteric sites at cardiac muscarinic M2 receptors

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.     

High affinity acylating antagonists for muscarinic receptors.

The muscarinic antagonists pirenzepine and telenzepine were derivatized as alkylamino derivatives at a site on the molecules corresponding to a region of bulk tolerance in receptor binding. The distal primary amino groups were coupled to the cross-linking reagent meta-phenylene diisothiocyanate, resulting in two isothiocyanate derivatives that were found to inhibit muscarinic receptors irreversibly and in a dose-dependent fashion. Preincubation of rat forebrain membranes with an isothiocyanate derivative followed by radioligand binding using [3H]N-methylscopolamine diminished the Bmax value, but did not affect the Kd value. The receptor binding site was not restored upon repeated washing, indicating that irreversible inhibition had occurred. IC50 values for the irreversible inhibition at rat forebrain muscarinic receptors were 0.15 nM and 0.19 nM, for derivatives of pirenzepine and telenzepine, respectively. The isothiocyanate derivative of pirenzepine was non-selective as an irreversible muscarinic inhibitor, and the corresponding derivative prepared from telenzepine was 5-fold selective for forebrain (mainly m1) vs. heart (m2) muscarinic receptors.     

Developmentally Regulated Secreted Factors Control Expression of Muscarinic Receptor Subtypes in Embryonic Chick Retina

Abstract: Two molecular mass subtypes of muscarinic receptor are expressed by the chick retina (72 and 86 kDa). During development, the ratio of subtypes changes, with the 72-kDa form becoming predominant. We have found that subtype switch can occur in retina cell culture, and have investigated factors that influence this in vitro increase in the 72-kDa receptor. Increases similar to those in vivo occurred when cells were cultured at 10⁵ cells/cm², but not at 10-fold lower density. High-density cultures, maintained on coverslips, showed no receptor development when transferred to large volumes of fresh medium, indicating that cell-cell contact alone was not responsible for induction. However, replacement of fresh medium with conditioned medium (from high-density cultures) resulted in normal induction. There were no morphological differences between cultures with high and low levels of the 72-kDa receptor. Conditioned medium also induced 72-kDa receptors in low-density cultures, consistent with a minimal role for cell-cell contact. Efficacy of conditioned medium was markedly dependent on age. Media from cells cultured 1–4 days had no effect, but media from cells cultured 5–8 and 1–8 days elicited 1.6-fold and fourfold increases in the 72-kDa subtype, respectively. The data indicate that maturing retina cells secrete developmentally regulated factors that are necessary for abundant expression of the 72-kDa muscarinic receptor subtype.     

Muscarinic cholinergic and histamine H1 receptor binding of phenothiazine drug metabolites

In vitro binding affinities of chlorpromazine, fluphenazine, levomepromazine, perphenazine and some of their metabolites for dopamine D2 receptors, alpha 1- and alpha 2 adrenoceptors in rat brain were previously reported from our laboratories. The present study reports the in vitro binding affinities of the same compounds for muscarinic cholinergic receptors and for histamine H1 receptors in rat brain, using 3H-quinuclidinyl benzilate and 3H-mepyramine as radioligands. Chlorpromazine, levomepromazine, and their metabolites had 5-30 times higher binding affinities for muscarinic cholinergic receptors than fluphenazine, perphenazine and their metabolites. Levomepromazine was the most potent and fluphenazine the least potent of the four drugs in histamine H1 receptor binding. 7-Hydroxy levomepromazine, 3-hydroxy levomepromazine and 7-hydroxy fluphenazine had only 10% of the potency of the parent drug in histamine H1 receptor binding, while the 7-hydroxy-metabolites of chlorpromazine and perphenazine had about 75% of the potency of the parent drug in this binding system. Their histamine H1 receptor binding affinities indicate that metabolites may contribute to the sedative effects of chlorpromazine and levomepromazine.     

The binding of [3H]AF-DX 384 is reduced in the caudate-putamen of subjects with schizophrenia.

Clinical studies of cholinergic pharmacotherapy, together with the putative role of the muscarinic receptor system in the neurophysiology of human behavior, support a possible muscarinic cholinergic involvement in schizophrenia. The present study has measured the density of [3H]AF-DX 384 labelled receptors (muscarinic M2 and M4) in the caudate-putamen, obtained at autopsy, from 19 subjects who had schizophrenia, and 20 subjects who did not have schizophrenia. [3H]AF-DX 384 binding was reduced in caudate-putamen from schizophrenic subjects (104 +/- 10.3 vs 145 +/- 901 fmol mg(-1) TE; mean +/- s.e.; p = 0.007). Preliminary analysis of patient drug data as well as rat studies suggest that the reduced [3H]AF-DX 384 binding in caudate-putamen of schizophrenic subjects is not wholly due to antipsychotic drug treatment, or anticholinergic medication for the treatment of extrapyramidal effects. These data suggest that the muscarinic cholinergic system may be involved in the pathology of schizophrenia.     

Neurotransmitter receptor expression by peripheral mononuclear cells: possible marker of neuronal damage by exposure to radiations.

Peripheral mononuclear cells (PMC) express several neurotransmitter systems. Increasing evidence suggests that PMC neurotransmitter receptors are involved in modulating immune responses. It is also thought that expression of PMC neurotransmitter receptors may reflect the status of homologous brain receptors. A problem encountered with assay of PMC neurotransmitter receptors was in developing techniques suitable for their assessment in spite of low density. In this paper we summarized findings on the expression of alpha1-adrenoceptor and dopamine receptor subtypes in human peripheral blood lymphocytes characterized by radioligand binding assay techniques and immunocytochemistry. Human lymphocytes express alpha1A-, alpha1B- and alpha1D-adrenoceptor subtypes and dopamine D3, D4 and D5 receptors. Compared to radioligand binding assay, immunocytochemistry applied to cytospin-centrifuged peripheral lymphocytes allowed to assay receptor subtypes investigated in small amounts of blood. The development of sensitive and reproducible techniques for assaying PMC neurotransmitter receptor subtypes even in small amounts of blood such as those used for diagnostic purposes may allow to analyze their sensitivity to different conditions including radiation exposure.