Brain Muscarinic Receptors Research Articles

The effects of repeated oral exposures to methyl parathion on rat brain cholinesterase and muscarinic receptors during postnatal development.

Dimethyl phosphorylated cholinesterase (ChE) is known to be more rapidly reactivated, spontaneously, and have a higher aging rate than diethyl phosphorylated ChE. This may result in differences in toxic signs and tolerance development after treatment of juvenile rats with methyl parathion (MPS), a dimethyl phosphorothionate, than after treatment with chlorpyrifos (CPS), a diethyl phosphorothionate. The effects of repeated MPS exposures on brain ChE activity and surface and total muscarinic acetylcholine receptor (mAChR) density were studied in postnatal rats gavaged daily from postnatal day 1 (PND1) through PND 21. Results of this study were compared to our recent report with CPS (Tang et al., 1999, Toxicol. Sci. 51, 265-272). Rats received MPS daily starting at 0.3 mg/kg and increasing gradually to 0.6 mg/kg (for the medium-dosage groups) and then to 0.9 mg/kg (for the high-dosage group). ChE activity was assayed in brain homogenates. Synaptosomal mAChR densities, surface, and total were assayed using 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB), respectively, as ligands. Developmental increases in brain ChE activities and mAChR densities were observed from PND 6 through PND 22. On PND 22, inhibition of ChE activity was observed in the low (26%)-, medium (42%)-, and high (55%)-dosage groups. Significant inhibition was still present on PND 30 (16-24%) and PND 40 (12-14%), which were 9 and 19 days after the last treatment, respectively. Densities of 3H-NMS and 3H-QNB binding sites in treated groups were significantly reduced by PND 22, 1 day following cessation of treatment, and were significantly increased during the recovery period. After MPS exposure, the initial recovery of phosphorylated ChE was more rapid and the density of 3H-NMS binding sites was less readily reduced than following CPS exposure. The lesser effects on surface mAChR may explain why more severe signs appeared after each treatment with the high dosage of MPS than were observed previously with CPS, indicating little or no tolerance had developed to MPS.

Critical analysis of potential body temperature confounders on neurochemical endpoints caused by direct dosing and maternal separation in neonatal mice: a study of bioallethrin and deltamethrin interactions with temperature on brain muscarinic receptors.

The present investigation was conducted to understand better possible confounding factors caused by direct dosing of neonatal mice during the pre-weaning developmental period. By direct dosing, pups might encounter thermal challenges when temporarily removed from their 'natural habitat'. Typically, this leads to a cold environment and food deprivation (impaired lactation) and modulation of the toxic potency of the substance administered. Growth retardation as a consequence of such behavioural changes in pups makes it increasingly difficult to differentiate specific from non-specific mechanisms. Neonatal NMRI mice were dosed daily by gavage (0.7 mg kg(-1) body wt.) from postnatal day (PND) 10-16 with S-bioallethrin, deltamethrin or the vehicle. Then the pups, including their non-treated foster dams, were subjected temporarily for 6 h day to a hypo-, normo- or hyperthermic environment, which was followed by normal housing. The measured temperatures in the environmental chambers were ca. 21, 25 and 30 degrees C, respectively. Thus, temperatures in the hypo- and normothermic groups are comparable to the temperatures commonly present in testing laboratories, whereas the hyperthermic condition is that temperature typically present in the 'natural habitat' of pups. A deviation from the normal behaviour of both pups and dams was observed in the hypo- and normothermic groups. In these groups the rectal temperatures of pups were markedly decreased, especially in the early phase of the study (PND 10-12). Neonates that received either test substance displayed changes in body weights and brain weights at terminal sacrifice (PND 17) when subjected temporarily to a non-physiological environment. An enormous influence of environmental temperature on the density of muscarinic receptors in the crude synaptosomal fraction of the cerebral cortex was ascertained. In summary, these results demonstrate that the direct dosing of thermolabile neonatal mice by gavage is subject to significant artefacts that render the interpretation of findings from such studies difficult. It appears that if direct dosing of neonatal pups is mandated, and inhalation is a relevant route of exposure, the combined inhalation exposure of dams with their litters is an alternative procedure that does not cause disruption of the 'natural habitat' of pups. However, owing to their higher ventilation, under such conditions the pups may receive dosages at least double those of the dams.

Inactivation of the human brain muscarinic acetylcholine receptor by oxidative damage catalyzed by a low molecular weight endogenous inhibitor from Alzheimer’s brain is prevented by pyrophosphate analogs, bioflavonoids and other antioxidants

Oxidative stress has been implicated as a contributing factor to neurodegeneration in Alzheimer’s disease. An endogenous, low molecular weight (LMW) inhibitor from Alzheimer’s brain inactivates the human brain muscarinic acetylcholine receptor (mAChR). The inhibitor prevents agonist and antagonist binding to the mAChR as assesssed by radioligand binding studies. The LMW endogenous inhibitor, which has components with molecular weights between 100 and 1000 Da, requires dissolved oxygen and glutathione. Prevention of inactivation of the mAChR with peroxidase suggests that the LMW endogenous inhibitor generates peroxide. Heme, previously shown to be present in the LMW endogenous inhibitor, also inactivates the mAChR in the presence of peroxide. Free radical damage to the muscarinic receptor by the endogenous inhibitor can be prevented through the use of naturally occurring antioxidants including bilirubin, biliverdin, carnosol, myricetin and quericetin. In addition, pyrophosphate, imidodiphosphate, bisphosphonates and related compounds also protect the muscarinic receptor from free radical damage. Inactivation of the mAChR by the LMW endogenous inhibitor is likely to be a factor in the continual decline of Alzheimer’s patients, even those taking acetylcholinesterase inhibitors. Natural antioxidants and pyrophosphate analogs may improve the effectiveness of acetylcholinesterase inhibitors and prove useful in the treatment and prevention of Alzheimer’s disease since the muscarinic acetylcholine receptor is required for memory, and decreased cholinergic function is a critical deficit in Alzheimer’s disease.

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.

Amnesic effects of the anticholinergic drugs, trihexyphenidyl and biperiden: differences in binding properties to the brain muscarinic receptor

An amnesic effect of anticholinergic drugs was previously described from several behavioral studies. We examined this effect induced by trihexyphenidyl and biperiden, clinically used in the parkinsonism and schizophrenic patients, by using passive avoidance tasks. Both of these drugs (0.1–10 mg/kg, s.c.) showed dose-dependent amnesic effects in the acquisition and retrieval phases. However, the effect induced by trihexyphenidyl was transient, whereas that of biperiden was long-lasting. To clarify the reason for the different duration of the amnesic activity, binding to the muscarinic receptor was examined. In the Scatchard analysis, trihexyphenidyl competed with [ 3 H ]quinuclidinyl benzilate ([ 3 H ]QNB) on the muscarinic receptor (showed increased K d and unchanged B max value), while biperiden decreased [ 3 H ]QNB binding ( B max value) significantly. Furthermore, in an exchange assay for receptor inactivation, trihexyphenidyl binding to muscarinic receptors was exchanged by [ 3 H ]QNB completely, but biperiden decreased the exchangeable binding of [ 3 H ]QNB in a dose dependent manner (0.1–100 nM). These results suggested that the binding of trihexyphenidyl and biperiden to muscarinic receptor might be completely reversible and partially irreversible, respectively, whereas the K i values of these two drugs were similar. In conclusion, this difference in binding property may explain the difference in the time-course of the amnesic effect induced by trihexyphenidyl and biperiden.

Anandamides Inhibit Binding to the Muscarinic Acetylcholine Receptor

Loss of memory and cholinergic transmission are associated with both Alzheimer's disease (AD) and marijuana use. The human brain muscarinic acetylcholine receptor (mAChR), which is involved in memory function and is inhibited by arachidonic acid, is also inhibited by anandamides. Two agonists of the cannabinoid receptor derived from arachidonic acid, anandamide (AEA) and R-methanandamide, inhibit ligand binding to the mAChR. Binding of the mAChR antagonist [3H]quinuclidinyl benzilate ([3H]QNB) is inhibited up to 89% by AEA (half-maximal inhibition at 50 microM). Binding of the more polar antagonist [N-methyl-3H]scopolamine ([3H]NMS) is inhibited by AEA up to 76% (half-maximal inhibition at 44 microM). R-methanandamide inhibits more than 90% of both [3H]QNB binding (I50 = 34 microM) and [3H]NMS binding (I50 = 15 microM) to the mAChR. Both AEA and R-methanandamide stimulate mAChR binding of the agonist [3H]oxotremorine-M at low concentrations (25-75 microM), but significantly inhibit agonist binding at higher concentrations (I50 = 150 microM). The cannabinoid antagonist SR141716A did not alter AEA or R-methanandamide inhibition of [3H]NMS binding to the mAChR, even at concentrations as high as 1 microM. Further, the cannabinoid agonist WIN 55212-2 does not alter antagonist binding to the mAChR. This demonstrates that mAChR inhibition by the anandamides is not mediated by the cannabinoid receptor. Since AEA and R-methanandamide are structurally similar to arachidonic acid, they may interact with the mAChR in a similar manner to inhibit receptor function.

Alzheimer's disease: relationship between muscarinic cholinergic receptors, beta-amyloid and tau proteins.

Senile dementia is one of the most important health problems in developed countries. The main disease causing dementia is Alzheimer's disease that is characterized by the progressive deterioration of the cholinergic system, beta-amyloid production and deposition, and neurofibrillary tangle formation. Most of the reviewed data, along with data from experiments performed in our laboratory, suggest that there are no changes in the number of muscarinic receptors between Alzheimer and control brains, although the receptors expressed in Alzheimer's disease brains can be anomalous in their function. The muscarinic receptor-G-protein interaction also seems to be impaired in Alzheimer's disease compared with control brains, as well as the G-protein system, with an important decrease in the function of the Gq/11, the most important G-protein stimulating phosphoinositide hydrolysis in human brain; in addition, the second messenger system is also impaired, with a decrease in the synthesis of phosphoinositides and in the number of IP3 receptors. Muscarinic cholinergic receptors are also linked to beta-amyloid production, stimulation of the M1 subtype with agonists results in the processing of the beta-amyloid precursor protein to non-amyloidogenic products and administration of a fraction of the beta-amyloid (beta-amyloid 25-35) to rats, results in a decrease in the number of muscarinic receptors in brain. M1 agonists also decrease the phosphorylation of tau proteins, playing again a modulatory role in the pathogenesis of Alzheimer's disease. The existence of a link between beta-amyloid and tau proteins also has been reported; treatment of hippocampal neurones with beta-amyloid, or the 25-35 residue fragment, resulted in an increase in tau protein phosphorylation. The particular contribution of muscarinic receptors, beta-amyloid and tau proteins in the pathogenesis of Alzheimer's disease remains still unclear. Probably Alzheimer's disease could be due to a progressive degeneration in the relationship between the three components covered in this review.

Pharmacological Profile of CEB-1957 and Atropine toward Brain Muscarinic Receptors and Comparative Study of Their Efficacy against Sarin Poisoning

This study consists of two parts, first to compare the pharmacological profile of atropine and CEB-1957 substance toward muscarinic receptor subtypes. In various rat brain structures, binding properties were determined by competition experiments of [3H]pirenzepine, [3H]AF-DX 384, and [3H]4-DAMP in quantitative autoradiography of M1, M2, and M3 muscarinic receptor subtypes, respectively. Competition curves have shown that atropine presents similar nanomolar inhibition constants toward each subtype, while CEB-1957 has distinct affinities (Kifrom 0.26 to 73 nM) with the following range order: M3 ≥ M2 > M1. The second part is to compare atropine and CEB-1957 (in combination with pralidoxime) for their ability to protect against the lethality induced by 2 × LD50 of the acetylcholinesterase inhibitor sarin. CEB-1957 reduced the mortality at doses 10 times lower than atropine. Finally, from these results, it is proposed that a selective blockade of M2 and M3 receptor subtypes could play a pivotal role in the protective effect against sarin poisoning.

Inhibition of antagonist and agonist binding to the human brain muscarinic receptor by arachidonic acid.

Arachidonic acid (AA) inhibits the binding of [3H]quinclidinyl benzilate ([3H]QNB) to the human brain muscarinic cholinergic receptor (mAChR). AA inhibits at lower concentrations in the absence of glutathione (I50 = 15 microM) than in the presence of glutathione (I50 = 42 microM). Inhibition of mAChR binding shows specificity for AA and is reduced with loss of one or more double bonds or with either a decrease or increase in the length of the fatty acid chain. Metabolism of AA by the lipoxygenase, epoxygenase, or fatty acid cyclooxygenase pathways is not required for the inhibitory activity of AA on mAChR binding. Inhibition of [3H]QNB binding by AA is reversible. While decreasing Bmax, AA increased the apparent KD for [3H]QNB and for the more polar antagonist [3H]NMS. In addition, AA inhibits binding of the agonist [3H]oxotremorine-M (I50 = 60 microM) and is the first mediator of mAChR action to be shown to reversibly inhibit mAChR binding. The feedback inhibition of the mAChR by AA may serve a homeostatic function similar to the reuptake and hydrolysis of acetylcholine following cholinergic nerve transmission.

Quantification of muscarinic cholinergic receptors with [11C]NMPB and positron emission tomography: method development and differentiation of tracer delivery from receptor binding.

Quantification of human brain muscarinic cholinergic receptors was investigated with the use of [11C]N-methyl-4-piperidyl benzylate (NMPB) and positron emission tomography (PET). Whole-brain uptake of NMPB at 90 to 110 minutes after intravenous injection was approximately 10% of the administered dose. The initial cerebral distribution of NMPB corresponded to the pattern of cerebral perfusion; however, at progressively longer postinjection intervals, regional distinctions consistent with muscarinic receptor binding were evident: activity at 90 to 110 minutes postinjection was highest in the striatum and cerebral cortex, intermediate in the thalamus and pons, and lowest in the cerebellum. After the development of a chromatographic system for isolation of authentic [11C]NMPB in plasma, tracer kinetic modeling was used to estimate receptor binding from the cerebral and arterial plasma tracer time-courses. Ligand transport rate and receptor-binding estimates were obtained with the use of compartmental models and analytical methods of varying complexity, including a two-parameter pixel-by-pixel-weighted integral approach and regional least-squares curve-fitting analyses employing both two- and three-compartment model configurations. In test-retest experiments, precision of the methods and their abilities to distinguish altered ligand delivery from binding in occipital cortex during an audiovisual presentation were evaluated. Visual stimulation increased the occipital blood-to-brain NMPB transport rate by 25% to 46% in estimates arising from the various approaches. Weighted integral analyses resulted in lowest apparent transport changes and in a concomitant trend toward apparent binding increases during visual activation. The regional least-squares procedures were superior to the pixel-by-pixel method in isolating the effects of altered tracer delivery from receptor-binding estimates, indicating larger transport effects and unaltered binding. Precision was best (less than 10% test-retest differences) for the weighted integral analyses and was somewhat lower in the least-squares analyses (10-25% differences). The authors conclude that pixel-by-pixel-weighted integral analyses of NMPB distribution introduce transport biases into receptor-binding estimates. Similar confounding effects also are predicted in noncompartmental analyses of delayed radiotracer distribution. The use of regional nonlinear least-squares fitting to two- and three-compartment models, although more labor intensive, provides accurate distinction of receptor-binding estimates from tracer delivery with acceptable precision in both intra- and intersubject comparisons.

Involvement of endogenous nitric oxide signalling system in brain muscarinic acetylcholine receptor activation.

Biochemical signalling events coupled to muscarinic cholinergic receptors (mAChR), specifically those related to nitric oxide (NO) production, were studied on rat cerebral frontal cortex. The mAChR agonist carbachol was found to exert a specific biphasic action on NO synthase (NOS) activity: low doses ranging between 10(-9) M to 10(-7) M lead to NOS activation while higher doses (>10(-6) M) inhibited enzymatic activity. Carbachol stimulatory action was blunted by agents that interfere with calcium-calmodulin while a protein kinase (PKC) inhibitor, staurosporine was able to abrogate the inhibitory effect. Moreover, PKC activity showed maximum translocation to cerebral frontal cortex membranes with carbachol concentrations that inhibited NO production. Products from phosphoinosite (PI) hydrolysis are involved in these actions as carbachol was found to increase PI turnover in a dose dependent manner. These results would serve as an example of cross-talk between both enzymatic pathways.

Nicotine administration decreases the number of binding sites and mRNA of M 1 and M 2 muscarinic receptors in specific brain regions of rat neonates

Nicotine has been shown to delay the developmental increase of muscarinic receptors in brain of rat neonates. In this study, we have examined the muscarinic receptor binding sites and corresponding messenger RNA in rat neonates, whose mothers received nicotine in the drinking water, using competition binding study and northern blot analysis. On postnatal 14th day, nicotine treatment led to a 61% reduction in the proportion of high-affinity sites for pirenzepine (M1-subtype) to total [ 3H]quinuclidinyl benzilate binding sites in cerebral cortex in rat neonate. Nicotine treatment also decreased that for 11-2[[2-[(diethylamino)methyl]-1-piperidinyl]-5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one (AF-DX 116) (M2-subtype) by 77% in cerebellum on postnatal 14th day. The levels of m1 and m2 muscarinic receptor messenger RNAs in the same brain regions were also decreased in the same day after nicotine treatment. On postnatal 35th day, no difference was observed in binding sites or in messenger RNA levels of the brain regions tested (cerebral cortex, midbrain, hippocampus, cerebellum and brainstem). Our results show that nicotine reduces muscarinic receptor subtypes in developing rat brain, in part, through suppression of the messenger RNA expression, but that the effects are different among brain regions and subtypes.

Age-related effects of rolipram on [3H]quinuclidinyl benzilate and [3H]phorbol 12,13-dibutyrate binding in the rat brain.

Cholinergic neurotransmission and protein kinase C (PKC) in the brain play important roles in the processes of cognitive function. In this study, we examined the effect of chronic treatment with rolipram, a 3',5'-cyclic adenosine monophosphate (cyclic AMP)-selective phosphodiesterase inhibitor, on age-related changes in [3H]quinuclidinyl benzilate (QNB) and [3H]phorbol 12,13-dibutyrate (PDBu) binding, which labeled brain muscarinic cholinergic receptors and PKC, respectively. Rolipram was administered per os to young (15 weeks old) and old (80 weeks old) Wistar rats at dosage of 0.01 mg/kg and 0.1 mg/kg once a day over 4 weeks. Then, quantitative in vitro autoradiography was performed. Control old rats showed elevations in [3H]PDBu binding in the hippocampus and the cerebellum compared to young rats, but [3H]QNB binding was largely unchanged. Chronic treatment of the old rats with the higher dose of rolipram led to reductions in [3H]QNB and [3H]PDBu binding in many brain regions. However, the same treatment of the young rats induced no or minimal effect. Thus, the response of the brain to rolipram was different between young and old rats. These results suggest that the cyclic AMP-selective phosphodiesterase system in the brain is modified during aging, modulating subsequently cholinergic neurotransmission and PKC activity exclusively in old rat brains.

Autoradiographic comparison of muscarinic M1 and M2 binding sites in the CNS of spontaneously hypertensive and normotensive rats

Spontaneously hypertensive rats (SHR) respond with exaggerated pressor responses of central origin in response to pharmacologic stimulation of brain muscarinic receptors when compared with those to normotensive Wistar Kyoto (WKY) rats. At least part of the enhanced response to central muscarinic stimulation may be due to alterations in the expression of one or more of the five subtypes of muscarinic receptors. SHR are also known to exhibit regional alterations in the levels of mRNA encoding the M1, M2 and M4 receptors. In this study, we estimated the number of specific muscarinic receptor binding sites in 12-week-old SHR and WKY by measuring the binding of M1- and M2-selective ligands. Using standard autoradiographic techniques, coronal sections obtained from 12-week-old SHR and WKY were incubated with [ 3H]pirenzepine or [ 3H]AFDX 384 to label M1 and M2 receptors, respectively. Although both strains exhibited similar distribution patterns for both binding sites, sections derived from SHR expressed a significant increase in the number of [ 3H]pirenzepine binding sites compared to normotensive WKY in caudate putamen, CA3 region of the hippocampus, cingulate cortex, substantia nigra, posterior hypothalamic area and tuberomammillary nucleus. An increased number of [ 3H]AFDX 384 binding sites in SHR were observed in the olfactory tubercle, nucleus accumbens, basolateral amygdaloid nucleus, rostroventrolateral medulla and nucleus paragigantocellularis. Decreases in the number of [ 3H]AFDX 384 binding sites in SHR were also observed in the parietal cortex, medial geniculate, and lateral hypothalamic area. Statistically significant site-selective differences in binding densities between strains ranged from 4.0% to 35.5% of WKY means. These alterations in the expression of M1 and M2 binding sites in cardiovascular regions may contribute to the strain's hyper-responsiveness to cholinergic drugs and possibly to the appearance of other autonomic or behavioral phenotypes exhibited by SHR, including the hypertensive state itself.

Positron emission tomographic investigations of central muscarinic cholinergic receptors with three isomers of [76Br]BrQNP.

We studied the potential of three radiobrominated isomers of BrQNP, (Z(-,-)-[76Br]BrQNP, E(-,-)-[76Br]BrQNP and E(-,+)-[76Br]BrQNP), as suitable radioligands for imaging of central muscarinic cholinergic receptors in the human brain. These radioligands were stereospecifically prepared by electrophilic radiobromodestannylation of the respective tributylstannyl precursors using no-carrier-added [76Br]BrNH4 and peracetic acid. Preliminary pharmacological characterizations were determined by biodistribution, autoradiography, competition, displacement and metabolite studies in rats. The (-,-)-configuration presented important specific uptakes in brain muscarinic cholinergic receptor (mAChR)-rich structures and in heart, low metabolization rates and an apparent M2 selectivity. The (-,+)-configuration revealed more rapid clearance, lower uptake, a higher metabolization rate and an apparent M1 selectivity. Reversibility of the binding was confirmed for the three radiotracers. Positron emission tomography in the living baboon brain revealed high and rapid uptake in the brain and accumulation in the mAChR-rich structures studied. At 30 min p.i., the E(-,-)-radiotracer reached a plateau in cortex, pons and thalamus with concentrations of 29%, 24% and 19% ID/l, respectively. Z(-,-)-[76Br]BrQNP also accumulated in these structures, reaching a maximal uptake (27% ID/l) in the cortex 2 h p.i. At 5 min p.i. a plateau (17% ID/l) was only observed in the cortex for the E(-, +)-[76Br]BrQNP; by contrast, the other structures showed slow washout. After 3 weeks, the (-,-)-radiotracers were studied in the same baboon pretreated with dexetimide (1 mg/kg), a well-known muscarinic antagonist. In all the mAChR structures, the highly reduced uptake observed after this preloading step indicates that these radiotracers specifically bind to muscarinic receptors. Z(-, -)-[76Br]BrQNP, which is displaced in higher amounts from M2 mAChR-enriched structures, reveals an M2 affinity. The two isomers having the (-,-)-configuration are potential probes for investigating central muscarinic receptors. The absolute configuration on the acetate chiral centre influences their muscarinic subtype selectivity and the cis-trans isomerism of the vinyl moiety affects their specific fixation.

Differential effect of muscarinic toxin 3 (MT3) on brain muscarinic receptors coupled to adenylyl cyclase

Differential effect of muscarinic toxin 3 (MT3) on brain muscarinic receptors coupled to adenylyl cyclase

Regional brain distribution and binding of the muscarinic receptor agonist CI-979 studied by positron emission tomography in the monkey.

The regional brain distribution and selective binding of the cholinergic muscarinic receptor agonist CI-979 labelled with 11C was studied in rhesus monkeys by means of positron emission tomography. The selective binding was measured as displacement of [11C]CI-979-derived radioactivity following constant-rate infusion of CI-979 at doses of 0.5-10 micrograms/kg/h. An extensive and rapid distribution of [11C]CI-979 was observed to the basal ganglia and temporal occipital cortices, i.e., regions of the brain with a high density of muscarinic receptors. The radioactivity was dose-dependently decreased in cortical regions following infusions of unlabelled CI-979 (0.5-10 micrograms/kg/h), indicating selective receptor binding of [11C]CI-979 in these brain regions. The binding of [11C]CI-979 was unaltered or even higher in the striatum following unlabelled CI-979 infusion. The high densities of autoreceptors in the striatum may explain the inhibitory feedback mechanism on endogenous acetylcholine induced by the muscarinic receptor agonist. Since muscarinic receptor agonists release acetylcholine, the higher releasable pool of acetylcholine in the stratum will induce feedback inhibition of acetylcholine release and less competition between acetylcholine and CI-979 at the muscarinic receptors. This may explain the differences between cortex and striatum in [11C]CI-979 binding. The cerebral blood flow was not changed by the infusion of unlabelled CI-979, supporting the assumption that effects of CI-979 in brain may be due to the interaction with brain muscarinic receptors. Despite a relative rapid clearance of [11C]CI-979 radioactivity from binding in the brain characteristic of a receptor agonist, the radioligand might be useful in positron emission tomography studies to reveal changes in cholinergic receptors and functional activity in Alzheimer patients treated with muscarinic agonists.

Chronic administration of Oren-gedoku-to (TJ15) inhibits ischemia-induced changes in brain indoleamine metabolism and muscarinic receptor binding in the Mongolian gerbil.

We examined the effect of Oren-gedoku-to (TJ15), which is a traditional herbal Kampo prescription used as an anti-cerebral apoplexy agent on these changes. Chronic pre- and post-ischemia TJ15 oral administration almost completely abolished the ischemia-induced muscarinic receptor reduction and 5-hydroxyindoleacetic acid level increase. These results suggest that TJ15 prevents cholinergic synaptic dysfunction and serotonergic presynaptic hyperactivity induced by transient ischemia.

O-89 - Effects of chronic nicotine treatment on brain muscarinic receptor subtypes and their mRNA in rat neonates

O-89 - Effects of chronic nicotine treatment on brain muscarinic receptor subtypes and their mRNA in rat neonates

Minireview: Genetic Manipulations of Cholinergic Communication Reveal Trans-Acting Control Mechanisms Over Acetylcholine Receptors

Several approaches have been developed for genetic modulations of receptor expression. These initiated with gene cloning and heterologous expression in microinjected Xenopus oocytes, and proceeded through transgenic expression and genomic disruption of receptor genes in mice. In addition, antisense treatments have reduced receptor levels in a transient, reversible manner. Integration of foreign DNA with host genomic sequences yields both cis- and trans-acting responses. These may depend on the DNA integration site, host cells condition and, most importantly, the affected signal transduction circuit. For example, acetylcholinesterase (AChE) overexpression in microinjected Xenopus tadpoles has been shown to upregulate α-bungarotoxin binding levels, indicating trans-acting control conferring overproduction of muscle nicotinic acetylcholine receptors. In transgenic mice expressing human AChE, the hypothermic response to oxotremorine was suppressed, reflecting modified levels of brain muscarinic receptors. To dissociate the feedback processes occurring in transfected cells from responses related to DNA integration, we examined the endogenous expression of the α7 neuronal nicotinic acetylcholine receptor in PC 12 cells transfected with DNA vectors carrying alternative splicing variants of human AChE mRNA. Our findings demonstrate suppression of α7 receptor levels associated with the accumulation of foreign DNA in the transfected cells. Acetylcholine receptor levels thus depend on multiple elements, each of which should be considered when genetic interventions are employed.