Muscarinic drugs regulate the PKG-II-dependent phosphorylation of M3 muscarinic acetylcholine receptors at plasma membranes from airway smooth muscle.

Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M2/M3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [(3)H]QNB binding at plasma membranes from bovine tracheal smooth muscle (BTSM), increased with cGMP and was augmented significantly cGMP plus ATP but diminished with the PKG-II inhibitor, Sp-8-pCPT-cGMPS. The [(3)H]-QNB binding was accelerated by okadaic acid, (OKA), a protein phosphatase (PPase) inhibitor. These two results indicated the involvement of a membrane-bound PPase. Moreover, a cGMP-dependent-[(32)P]γATP phosphorylation of plasma membranes from BTSM was stimulated at low concentrations of muscarinic agonist carbamylcholine (CC). However, higher amounts of CC produced a significant decrement of [(32)P]-labeling. A selective M3mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [(32)P]-labeling. The [(32)P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M2/M3mAChR antibodies. The M3mAChR immuno-precipitates exhibited the highest cGMP-dependent [(32)P]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i3) of both M2mAChR (356-369) and M3mAChR (480-493) as external PKG-II substrates resulted in the i3M3-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M3mAChR at the i3M3 domain ((480)MSLIKEKK(485)), suggesting that Ser(481) may be the target. Finally, this phosphorylation site seems to be regulated by a membrane-bound PPase linked to muscarinic receptor. These findings are important to understand the role of M3mAChR in the patho-physiology of ASM involved in asthma and COPD.

Biological sex influences learning strategy preference and muscarinic receptor binding in specific brain regions of prepubertal rats.

According to the theory of multiple memory systems, specific brain regions interact to determine how the locations of goals are learned when rodents navigate a spatial environment. A number of factors influence the type of strategy used by rodents to remember the location of a given goal in space, including the biological sex of the learner. We recently found that prior to puberty male rats preferred a striatum-dependent stimulus-response strategy over a hippocampus-dependent place strategy when solving a dual-solution task, while age-matched females showed no strategy preference. Because the cholinergic system has been implicated in learning strategy and is known to be sexually dimorphic prior to puberty, we explored the relationship between learning strategy and muscarinic receptor binding in specific brain regions of prepubertal males and female rats. We confirmed our previous finding that at 28 days of age a significantly higher proportion of prepubertal males preferred a stimulus-response learning strategy than a place strategy to solve a dual-solution visible platform water maze task. Equal proportions of prepubertal females preferred stimulus-response or place strategies. Profiles of muscarinic receptor binding as assessed by autoradiography varied according to strategy preference. Regardless of biological sex, prepubertal rats that preferred stimulus-response strategy exhibited lower ratios of muscarinic receptor binding in the hippocampus relative to the dorsolateral striatum compared to rats that preferred place strategy. Importantly, much of the variance in this ratio was related to differences in the ventral hippocampus to a greater extent than the dorsal hippocampus. The ratios of muscarinic receptors in the hippocampus relative to the basolateral amygdala also were lower in rats that preferred stimulus-response strategy over place strategy. Results confirm that learning strategy preference varies with biological sex in prepubertal rats with males biased toward a stimulus-response strategy, and that stimulus-response strategy is associated with lower ratios of muscarinic binding in the hippocampus relative to either the striatum or amygdala.

Neonatal hypoxic insult-mediated cholinergic disturbances in the brain stem: effect of glucose, oxygen and epinephrine resuscitation.

Molecular processes regulating cholinergic functions play an important role in the control of respiration under neonatal hypoxia. The present study evaluates neonatal hypoxic insult-mediated cholinergic alterations and the protective role of glucose, oxygen and epinephrine resuscitation. The changes in total muscarinic, muscarinic M1, M2, M3 receptors and the enzymes involved in acetylcholine metabolism--cholineacetyl transferase and acetylcholine easterase in the brain stem were analyzed. Hypoxic stress decreased total muscarinic receptors along with a reduction in muscarinic M1, M2 and M3 receptor genes in the brain stem. The reduction in acetylcholine metabolism is indicated by the down regulated cholineacetyl transferase and up regulated acetylcholine easterase expression. These cholinergic disturbances in the brain stem were reversed by glucose resuscitation to hypoxic neonates. The adverse effects of immediate oxygenation and epinephrine administration were also reported. This has immense clinical significance in establishing a proper resuscitation for the management of neonatal hypoxia.

Expanding the multipotent profile of huprine-tacrine heterodimers as disease-modifying anti-Alzheimer agents.

BACKGROUND: Multifactorial diseases such as Alzheimer's disease (AD) should be more efficiently tackled by drugs which hit multiple biological targets involved in their pathogenesis. We have recently developed a new family of huprine-tacrine heterodimers, rationally designed to hit multiple targets involved upstream and downstream in the neurotoxic cascade of AD, namely ß-amyloid aggregation and formation as well as acetylcholinesterase catalytic activity. OBJECTIVE: In this study, the aim was to expand the pharmacological profiling of huprine-tacrine heterodimers investigating their effect on muscarinic M(1) receptors as well as their neuroprotective effects against an oxidative insult. METHODS: Sprague-Dawley rat hippocampus homogenates were used to assess the specific binding of two selected compounds in competition with 1 nM [(3)H]pirenzepine (for M(1) receptors) or 0.8 nM [(3)H]quinuclidinyl benzilate (for M(2) receptors). For neuroprotection studies, SHSY5Y cell cultures were subjected to 250 µM hydrogen peroxide insult with or without preincubation with some huprine-tacrine heterodimers. RESULTS: A low nanomolar affinity and M(1)/M(2) selectivity has been found for the selected compounds. Huprine-tacrine heterodimers are not neurotoxic to SHSY5Y cells at a range of concentrations from 1 to 0.001 µM, and some of them can protect cells from the oxidative damage produced by hydrogen peroxide at concentrations as low as 0.001 µM. CONCLUSION: Even though it remains to be determined if these compounds act as agonists at M(1) receptors, as it is the case of the parent huprine Y, their low nanomolar M(1) affinity and neuroprotective effects expand their multitarget profile and increase their interest as disease-modifying anti-Alzheimer agents.

Mechanisms of M3 muscarinic receptor regulation by wash-resistant xanomeline binding.

BACKGROUND/AIMS: Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect. METHODS: Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments. RESULTS: The above treatment protocol with xanomeline resulted in similar effects of the binding of [(3)H]NMS and [(3)H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent. CONCLUSION: Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.

Sodium valproate at therapeutic concentrations changes Ca2+ response accompanied with its weak inhibition of protein kinase C in human astrocytoma cells.

Sodium valproate (VPA) has been used clinically for treatment of not only epilepsy but also mood disorder. Although VPA is effective for treatment of epilepsy via inhibition of gamma-aminobutyric acid transaminase, it remains unknown why VPA is effective for the treatment of mood disorder. The authors examined the effect of VPA at therapeutic concentrations (300 and 600 microM) on the elevation of intracellular free calcium concentration ([Ca(2+)](i)) induced by carbachol, a muscarinic receptor agonist, in 1321N1 human astrocytoma cells. Treatment of the cells with 300 and 600 microM VPA for 2 min did not change the carbachol-induced [Ca(2+)](i) elevation. Treatment with 300 and 600 microM VPA for 48 h, however, reduced the elevation. Since we have shown that Li(+) reduced carbachol-induced [Ca(2+)](i) elevation in protein kinase C (PKC)-downregulated 1321N1 cells [Kurita, M., Mashiko, H., Rai, M., Kumasaka, T., Kouno, S., Niwa, S., Nakahata, N., 2002. Lithium chloride at a therapeutic concentration reduces Ca(2+)response in protein kinase C down-regulated human astrocytoma cells, Eur. J. Pharmacol. 442, 17-22.], the activity of PKC was examined. Treatment with VPA at the same concentrations for 24 or 48 h weakly reduced protein kinase C activity in membrane and cytosol fractions from the cells. On the other hand, the treatment of the cells with 600 microM VPA for 24 or 48 h slightly increased the B(max) value, but not the K(d) value, in the binding of [(3)H]quinuclidinyl benzylate, a muscarinic receptor ligand, to the membranes, suggesting that the number or affinity of muscarinic receptor did not decrease after VPA treatment. These results indicate that VPA at therapeutic concentrations slightly decreases the PKC activity and inhibits muscarinic receptor-mediated [Ca(2+)](i) elevation probably through change in the intracellular signaling pathway. VPA-induced reduction of PKC activity and [Ca(2+)](i) elevation may play a role in the treatment of mood disorder.

Regulation of m1 muscarinic receptors and nNOS mRNA levels by autoantibodies from schizophrenic patients.

In this paper we demonstrate that, circulating antibodies from schizophrenic patients interacting with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), can act as an inducer of m1 mAChR-mRNA, and neuronal nitric oxide synthase (nNOS) mRNA gene expression of rat frontal cortex. The different signaling pathways involved in the autoantibody's actions, were characterized. As previously reported serum autoantibodies from schizophrenic patients reacted against neural cells surface inhibiting the binding of the specific mAChR radioligand to rat cerebral frontal cortex membrane. Moreover, by ELISA using M1 synthetic peptide (with identical aminoacid sequence to human M1 mAChR) as coating antigen we demonstrated the reactivity against the second extracellular loop of human cerebral M1 mAChR. The corresponding affinity-purified anti M1 peptide IgG (anti M1 peptide IgG) from schizophrenic patients by stimulation of M1 mAChR exerted an increase in m1 mAChR-mRNA and nNOS-mRNA levels, that significantly correlated with the accumulation of phosphoinositides (IPs) and activation of NOS (alpha = 0.05). All these effects were blunted by pirenzepine and mimicked the action of the authentic agonist. Concurrent analysis of the effects of nNOS, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition on both, m1 mAChR-mRNA and nNOS-mRNA levels, showing that antibody up-regulation mRNA level is under the control of endogenous nitric oxide (NO) signaling system. On the basis of our results, the activation of M1 mAChR by schizophrenic autoantibody appears to induce nNOS-mRNA expression and reciprocally, the activation of NOS up-regulates m1 mAChR gene expression. These results gave support to the participation of an autoimmune process in a particular group of chronic schizophrenic patients.

Effects of developmental co-exposure to methylmercury and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) on cholinergic muscarinic receptors in rat brain.

The developing nervous system is thought to be particularly sensitive to polychlorinated biphenyls (PCBs) present as food contaminants together with methylmercury (MeHg). Effects of perinatal co-exposure to PCB153 and MeHg on brain cholinergic muscarinic receptors (MRs) were investigated by saturation binding studies in mature and immature rats. MeHg alone (1mg/kg/day, GD7-PND7) enhanced cerebral MRs more in dams (87% and 60% in cerebellum and cerebral cortex, respectively) than in PND21 pups (0-50%) in accordance with the higher Hg levels detected in the adult brain (7-9 microg/g) than in the male and female offspring's brain (1.5-2.8 microg/g). Prenatal administration of PCB153 (20mg/kg/day, GD10-GD16), leading to higher contaminant levels in the offspring brain than in that of adults (25-66 microg/g versus 3 microg/g), induced cerebral MR changes of similar extent at both ages, namely decreased cerebellar (20-30%) and increased cortical MR density (40-50%). Co-exposure to PCB and MeHg had no more effect than exposure to either compound alone on cerebral cortex MRs, whereas, in the cerebellum, the combined treatment induced a PCB-like lowering of the MR density that masked the MeHg-induced receptor increase. None of the treatments affected the striatal and hippocampal MRs. A lower MeHg dose (0.5 mg/kg/day) was without any effect on cerebral MRs. These results show that MRs are one of the sensitive biochemical endpoints of the central nervous system altered by developmental exposure to MeHg and PCB153. Cerebral cortex and cerebellum were the most susceptible targets in the response to these neurotoxicants. MR changes were detected in both immature and adult animals and the interaction of MeHg and PCB153 at the level of these receptors occurred in a non-additive manner.

Up-regulation of M1 muscarinic receptors expressed in CHOm1 cells by panaxynol via cAMP pathway.

Loss of cholinergic neurons along with muscarinic acetylcholine receptors (mAChRs) in cerebral cortex and hippocampus is closely associated with Alzheimer's disease (AD). Recent drug development for AD treatment focuses heavily on identifying M(1) receptor agonists. However, mAChRs undergo down-regulation in response to agonist-induced sustained activation. Therefore, therapeutic effectiveness wanes during continuous use. Thus, another potentially effective approach, which overcomes this drawback is to develop compounds, which instead up-regulate M(1) receptor expression. In the present study, we took this alternative approach and contrasted in Chinese hamster ovary cells transfected with human m(1) subtype gene (CHOm(1) cells) changes of M(1) receptor expression levels caused by muscarinic agonists and upregulators of its expression. The muscarinic agonists carbachol and pilocarpine reduced M(1) receptor number in CHOm(1) cells by 29 and 46%, respectively, at 100muM, whereas panaxynol, a polyacetylene compound isolated from the lipophilic fraction of Panax notoginseng, concentration-dependently up-regulated the M(1) receptor number after pre-incubation with CHOm(1) cells for 48 h, reaching a plateau at 1 microM, and was accompanied by enhanced M(1) mRNA levels. Moreover, the protein kinase A (PKA) inhibitor RP-adenosine-3',5'-cyclic mono-phosphoro-thioate triethylamine salt (RP-cAMPs) 5 microM completely prevented panaxynol-induced up-regulation of M(1) receptors. Panaxynol (1muM) caused a significant and consistent stimulation of cAMP accumulation (27% increase above basal at 40 min). These results suggest that in CHOm(1) cells panaxynol up-regulates M(1) receptor number through cAMP pathway-mediated stimulation of gene transcription.

Decreased (3H) quinuclidinyl benzilate binding to lymphocytes in Gilles de la Tourette syndrome.

In spite of an unknown pathophysiology, it has been suggested that central dopaminergic hyperactivity exists in Gilles de la Tourette syndrome (TS). Cholinergic influences have also been postulated as a dopaminergic-cholinergic balance seems to be important in other movement disorders. If TS is due to alterations of cholinergic activity, this may also be expressed at postsynaptic levels. Recently, we showed that circulating lymphocytes may serve as useful peripheral markers reflecting induced alterations or inherent changes in muscarinic receptors in the central nervous system (CNS). In the present study, we compared the muscarinic binding characteristics in peripheral lymphocytes as measured by (3H) quinuclidinyl benzilate [(3H)-QNB] in 27 unmedicated TS patients, against 22 healthy (age and gender-matched) controls. B(max) and Kd values were determined using Lineweaver-Burke plots. The mean B(max) values in nontreated TS patients was markedly and significantly lower than in controls (10.59 +/- 8.4 versus 40.16 +/- 9.2 fmole/10(6) cells, p less than 10(-6), while Kd values were similar in both groups. Our findings suggest that changes in cholinergic receptors may play a role in the pathophysiology of Tourette syndrome.

Neuroleptic binding to sigma receptors: possible involvement in neuroleptic-induced acute dystonia.

Several antipsychotic drugs, belonging to various chemical classes, were compared for their affinity for the sigma, dopamine-D2, and muscarinic receptors. Many neuroleptic drugs were found to bind with high affinity to sigma 2 receptors, and the binding affinity was clearly different from that observed for dopamine-D2 receptors. The dopaminergic and muscarinic theories for the physiopathology of acute dystonia are not completely satisfactory. Since the sigma receptors were reported to play a role in the control of movement, the high affinity of some neuroleptics for these sites suggests their possible involvement in some side effects, such as drug-induced dystonia. There was a correlation between the clinical incidence of neuroleptic-induced acute dystonia and binding affinity of drugs for the sigma receptor, except for some drugs, with a lower incidence, displaying significant affinity for the cholinergic muscarinic receptor. Therefore, we conclude that the affinity for the sigma receptor might be involved in neuroleptic-induced acute dystonia, but this might be partially corrected by the intrinsic anticholinergic properties of the drug.

Chronic lithium treatment prevents atropine-induced supersensitivity of the muscarinic phosphoinositide response in rat hippocampus.

Rats were placed on a lithium diet for 3 weeks or an identical diet without lithium for the same period. During the third week, atropine sulfate (10 mg/kg/day) or saline was infused via subcutaneously implanted osmotic pumps. Twenty-four hours following the removal of the pumps, brain slices and membranes were prepared from the cortex and hippocampus for determination of muscarinic stimulation of phosphoinositide turnover and for receptor-binding studies. Treatment with lithium alone did not significantly affect any of the binding or response parameters measured. Administration of atropine led to (1) an upregulation of muscarinic binding sites in both cortex and hippocampus without significant alteration in the proportion of muscarinic receptor subtypes; and (2) an enhancement of the muscarinic phosphoinositide response in hippocampal slices. However, atropine did not induce supersensitivity of the hippocampal response in rats undergoing lithium administration. These results are consistent with recent suggestions that lithium's efficacy in affective disorders may be related to a dampening of muscarinic supersensitivity.

Kinetic analysis of 3-quinuclidinyl 4-[125I]iodobenzilate transport and specific binding to muscarinic acetylcholine receptor in rat brain in vivo: implications for human studies.

Radioiodinated R- and S-Quinuclidinyl derivatives of RS-benzilate (R- and S-125IQNB) have been synthesized for quantitative evaluation of muscarinic acetylcholine receptor binding in vivo. Two sets of experiments were performed in rats. The first involved determining the metabolite-corrected blood concentration and tissue distribution of tracer R-IQNB (active enantiomer) and S-IQNB (inactive enantiomer) in brain 1 min to 26 h after intravenous injection. The second involved the measurement of brain tissue washout over a 2-min period after loading the brain by an intracarotid artery injection of the ligands. Various pharmacokinetic models were tested, which included transport across the blood-brain barrier (BBB), nonspecific binding, low-affinity binding, and high-affinity binding. Our analysis demonstrated that the assumptions of rapid equilibrium across the BBB and rapid nonspecific binding are incorrect and result in erroneous estimates of the forward rate constant for binding at the high-affinity receptor sites (k3). The estimated values for influx across the BBB (K1), the steady-state accumulation rate in cerebrum (K), and the dissociation rate constant at the high-affinity site (k4) of R-IQNB were independent of the specific compartmental model used to analyze these data (K1 approximately 0.23 ml/min/g, K approximately 0.13 ml/min/g, and k4 approximately 0.0019 min-1 for caudate). In contrast, the estimated values of k3 and the efflux rate constant (k2) varied over a 10-fold range between different compartmental models (k3 approximately 2.3-22 min-1 and k2 approximately 1.6-16 min-1 in caudate), but their ratios were constant (k3/k2 approximately 1.4). Our analysis demonstrates that the estimates of k3 (and derived values such as the binding potential) are model dependent, that the rate of R-IQNB accumulation in cerebrum depends on transport across the BBB as well as the rate of binding, and that uptake in cerebrum is essentially irreversible during the first 360 min after intravenous administration. Graphical analysis was consistent with compartmental analysis of the data and indicated that steady-state uptake of R-IQNB in cerebrum is established within 1-5 min after intravenous injection. We propose a new approach to the analysis of R-IQNB time-activity data that yields reliable quantitative estimates of k3, k4, and the nonspecific binding equilibrium constant (Keq) by either compartmental or graphical analysis. The approach is based on determining the free unbound fraction of radiolabeled ligand in blood and an estimate of K1.(ABSTRACT TRUNCATED AT 400 WORDS)

Chronic nicotine treatment attenuates alpha 7 nicotinic receptor deficits following traumatic brain injury.

Traumatic brain injury (TBI) often causes a persistent and debilitating impairment of cognitive function. Although the neurochemical basis for TBI-induced cognitive dysfunction is not well characterized, some studies suggest prominent involvement of the CNS cholinergic system. Previous studies from our laboratories have shown that alpha 7* nicotinic cholinergic receptors (nAChrs) are especially vulnerable to the pathophysiological effects of TBI. Hippocampal and cortical alpha-[(125)I]-bungarotoxin (BTX) expression of alpha 7* nAChrs is significantly decreased in many brain regions following TBI and this reduction persists for at least 3 weeks following injury. In the present study we evaluated whether chronic nicotine infusion could attenuate TBI-induced deficits in alpha 7* nAChr expression. Male Sprague-Dawley rats were sham-operated, or subjected to mild or moderate unilateral cortical contusion injury. Immediately following brain injury, osmotic mini-pumps that delivered chronic saline or nicotine (0.125 or 0.25 mg/kg/h) were implanted. The animals were euthanatized and the brains prepared for nAChr quantitative autoradiography, 7 days following surgery. Brain injury caused significant decreases in BTX binding in several regions of the hippocampus. TBI-induced deficits in alpha 7* nAChr density were reversed in four of the six hippocampal brain regions evaluated following chronic nicotine administration. If TBI-induced deficits in alpha 7* nAChr expression play a role in post-injury cognitive impairment, pharmacological treatments which restore nAChr binding to control levels may be therapeutically useful.

Modulatory effects on myocardial physiology induced by an anti-Trypanosoma cruzi monoclonal antibody involve recognition of major antigenic epitopes from beta1-adrenergic and M2-muscarinic cholinergic receptors without requiring receptor cross-linking.

It has been proposed that anti-myocardial antibodies (Ab) against neurotransmitter (NT) receptors are involved in the immunopathology of chronic Chagas' heart disease. We demonstrated that an anti-Trypanosoma cruzi monoclonal Ab (mAb), CAK20.12, binds to murine cardiac beta-adrenergic and muscarinic acetyl choline (mACh) receptors eliciting abnormal physiological responses on normal heart. No cross-linking requirement for mAb actions was demonstrated using Fab fragment derived from CAK20.12. mAb binding to synthetic peptides from the second extracellular loop of both beta1-adrenergic and mACh receptors, demonstrated by ELISA, identified the region of NT receptors involved. Cross-reactivity between these peptides and T. cruzi antigen was confirmed by binding inhibition assays. These results support the existence of cross-reactivity due to molecular mimicry between a parasite antigen and the major antigenic epitopes present on both beta1-adrenergic and M2-ACh receptors. Its possible relationship with cardiac dysfunction during chronic stage of Chagas' disease is also discussed.

The change in the threshold for short-term desensitization in isolated smooth muscle cells showing an all-or-none response to acetylcholine.

1. Isolated smooth muscle cells from guinea-pig taenia caecum were prepared by collagenase digestion. The cells showed an all-or-none response to acetylcholine (ACh) under our experimental conditions. 2. Desensitized cells showed an all-or-none response but required a higher concentration of ACh for induction of contraction, indicating that the desensitization was due to a change in the threshold concentration. 3. In [3H]-quinuclidinyl benzilate ([3H]-QNB) binding to the desensitized cells, KD and Bmax were not significantly different from those estimated in the control cells. The competitive inhibition curve for specific binding of [3H]-QNB by ACh in the desensitized cells was in agreement with that of control cells. 4. The ACh-stimulated increase of the 45Ca2+ influx was very rapid and correlated well with the contraction of the cells. The concentrations of ACh inducing the maximal 45Ca2+ influx were increased by desensitization. 5. These results indicated that although the binding of ACh to the receptor was not changed by desensitization, the threshold concentration of ACh for their contraction was raised by desensitization, and the 45Ca2+ influx accompanying the contraction was shifted to the side of high concentration of ACh. 6. These results suggest that the development of short-term desensitization is due to an uncoupling of the receptor from the mechanism for initiation of the contraction.

Muscarinic receptors in basal ganglia in dementia with Lewy bodies, Parkinson's disease and Alzheimer's disease.

Derivatives of the muscarinic antagonist 3-quinuclidinyl-4-iodobenzilate (QNB), particularly [123I]-(R,R)-I-QNB, are currently being assessed as in vivo ligands to monitor muscarinic receptors in Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), relating changes to disease symptoms and to treatment response with cholinergic medication. To assist in the evaluation of in vivo binding, muscarinic receptor density in post-mortem human brain was measured by autoradiography with [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB and compared to M1 ([3H]pirenzepine) and M2 and M4 ([3H]AF-DX 384) receptor binding. Binding was calculated in tissue containing striatum, globus pallidus (GPe), claustrum, and cingulate and insula cortex, in cases of AD, DLB, Parkinson's disease (PD) and normal elderly controls. Pirenzepine, AF-DX 384 and (R,S)-I-QNB binding in the striatum correlated positively with increased Alzheimer-type pathology, and AF-DX 384 and (R,R)-I-QNB cortical binding correlated positively with increased Lewy body (LB) pathology; however, striatal pirenzepine binding correlated negatively with cortical LB pathology. M1 receptors were significantly reduced in striatum in DLB compared to AD, PD, and controls and there was a significant correlation between M1 and dopamine D2 receptor densities. [3H]AF-DX 384 binding was higher in the striatum and GPe in AD. Binding of [125I]-(R,R)-I-QNB, which may reflect increased muscarinic M4 receptors, was higher in cortex and claustrum in DLB and AD. [125I]-(R,S)-I-QNB binding was higher in the GPe in AD. Low M1 and D2 receptors in DLB imply altered regulation of the striatal projection neurons which express these receptors. Low density of striatal M1 receptors may relate to the extent of movement disorder in DLB, and to a reduced risk of parkinsonism with acetylcholinesterase inhibition.

Organizational changes in cholinergic activity and enhanced visuospatial memory as a function of choline administered prenatally or postnatally or both.

This experiment was an examination of the effects of supplemental dietary choline chloride given prenatally (to the diet of pregnant rats) and postnatally (intubed directly into the stomachs of rat pups) on memory function and neurochemical measures of brain cholinergic activity of male albino rats when they became adults. The data demonstrate that perinatal choline supplementation causes (a) long-term facilitative effects on working and reference memory components of a 12-arm radial maze task, and (b) alternations of muscarinic receptor density as indexed by [3H]quinuclidinyl benzilate (QNB) binding and choline acetyltransferase (ChAT) levels in the hippocampus and frontal cortex of adult rats. An analysis of the relationship between these organizational changes in brain and memory function indicated that the ChAT-to-QNB ratio in the hippocampus is highly correlated with working memory errors, and this ratio in the frontal cortex is highly correlated with reference memory errors.

Muscarinic cholinergic receptor binding in rat brain following traumatic brain injury.

Recent evidence suggests that excessive activation of muscarinic cholinergic receptors (mAChRs) contributes significantly to the pathophysiological consequences of traumatic brain injury (TBI). To examine possible alterations in mAChRs after TBI, the affinity (Kd) and maximum number of binding sites (Bmax) of mAChRs in hippocampus, neocortex, brain stem and cerebellum were determined by [3H]QNB binding. Three groups of rats were examined: 1 h post-TBI (n = 21), 24 h post-TBI (n = 21) and sham-injured rats (n = 21). Kd values were significantly higher in hippocampus and brain stem at 1 but not 24 h post-TBI compared with sham-injured controls (P < 0.05). Kd values did not significantly differ in neocortex and cerebellum at 1 or 24 h post-TBI compared with sham-injured controls. Bmax values did not significantly differ in any brain areas at 1 or 24 h post-TBI compared with sham-injured controls. These results show that TBI significantly decreases the affinity of mAChRs in hippocampus and brain stem at an early stage post-TBI, which may contribute to desensitization of mAChRs after TBI. The findings of no change in Bmax values are consistent with a transient elevation in ACh concentrations after TBI.

In vivo dissociation kinetics of [3H]quinuclidinyl benzilate: relationship to muscarinic receptor concentration and in vitro kinetics.

The in vivo washout kinetics of [3H]quinuclidinyl benzilate ([3H]QNB) varies significantly in various structures in the rat brain. The slowest washout rates are from the hippocampus, corpus striatum, and cortex, intermediate rates are exhibited from the thalamus and colliculi, while the fastest washout rate is from the cerebellum. We have also demonstrated a difference in the in vitro dissociation rates (k-1) of [3H]QNB from various structures. The k-1 for the hippocampus, corpus striatum and cortex, is two-fold slower than that observed in the thalamus, colliculi, and cerebellum. The differences in the in vitro dissociation kinetics are not, however, sufficient to explain the differences in the in vivo washout kinetics. We have developed a theoretical formulation which describes conditions under which the washout kinetics are a function of the concentration of receptor in a structure. Furthermore, we present a graphical method in which a plot of the reciprocal of the observed washout rate constant, 1/k(obs), vs receptor concentration is linear. Analysis of the washout kinetics of [3H]QNB from various structures of the CNS of rat were well described by this theory when the differences in in vitro k-1 are included.