In vitro pharmacological properties of 4-bromodexetimide for muscarinic receptors

The decrease of m-AChR density observed hi neurodegenerative disorders has generated considerable interest in non-invasive mapping of muscarinic acetylcholine receptors (m-AChR) in the central nervous system. The ami of our study was to evaluate the selectivity of 4-bromodexetimide for the M1, M2, M3 and M4 m-AChR subtypes using in vitro binding analysis to determine the potential use of the bromine-76 labelled 4-bromodexetimide in the investigation of m-AChR subtypes in human brain with Positron Emission Tomography. Subtype selectivity of 4-bromodexetimide was determined in competition studies against tritiated subtype selective ligands using various rat or rabbit structure homogenates reflecting a single binding site and in optimal saturation and low non specific binding conditions. These conditions were reached for every subtype studied by analyzing the data from the saturation experiments of the tritiated ligands. 4-bromodexetimide displayed nanomolar affinities for the four m-AChR subtypes and a preferential selectivity for the M1 and M4 subtypes. The saturation analysis of [⁷⁶Br]4-bromodexetimide, performed with rat cortex membranes showed high affinity for m-AChR receptors (K_d = 1.8 nM). As in vivo studies of [⁷⁶Br]4-bromodexetimide showed preferential localization in the cortex and the striatum which are M1 and M4 rich structures and since it binds preferentially to the M₁ and M₄ subtypes, this radiotracer can still allow a combined subtype specific measurement of these muscarinic receptors.     

Aflatoxin inhibition of glucocorticoid binding capacity of rat liver nuclei

The effect of aflatoxin B₁ on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [³H]dexamethasonccytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B₁. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 36 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B₁ dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant.Administration of [³H]dexamethasone to aflatoxin B₁-treated rats produced a similar pattern of glucocortocoid binding distribution in vivo to that observed in vitro. No differences in [³H]dexamethasone-cytoplasmic receptor binding between control and aflatoxin B₁-treated rats were found, whereas nuclear [³H]dexamethasone binding was reduced 34% by aflatoxin B₁ treatment.     

Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase

A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammoniumchloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [³H]Diisopropyl fluorophosphate ([³H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [³H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme.A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.Abbreviations AChE Acetylcholinesterase - BW284C51 1,5-bis(4-allyl dimethylammonium phenyl) pentan 3-one-dibromide - DFP Diisobropyl fluorophosphate - TIPP Tetra isopropyl pyrophosphoramide - TPCK N-Tosyl-L-phenylalanylchloromethyl ketone - MAP m-Aminophenyl trimethylammonium chloride - RCA₁ Ricinus communis agglutinin 120 - TEAB Tetraethylammonium bromide - DTT Dithiothreitol     

Albumin binding as a potential biomarker of exposure to moderately low levels of organophosphorus pesticides

We have evaluated the potential of plasma albumin to provide a sensitive biomarker of exposure to commonly used organophosphorus pesticides in order to complement the widely used measure of acetylcholinesterase (AChE) inhibition. Rat or human plasma albumin binding by tritiated-diisopropylfluorophosphate (³H-DFP) was quantified by retention of albumin on glass microfibre filters. Preincubation with unlabelled pesticide in vitro or dosing of F344 rats with pesticide in vivo resulted in a reduction in subsequent albumin radiolabelling with ³H-DFP, the decrease in which was used to quantify pesticide binding. At pesticide exposures producing approximately 30% inhibition of AChE, rat plasma albumin binding in vitro by azamethiphos (oxon), chlorfenvinphos (oxon), chlorpyrifos-oxon, diazinon-oxon and malaoxon was reduced from controls by 9±1%, 67±2%, 56±2%, 54±2% and 8±1%, respectively. After 1 h of incubation with 19 µM ³H-DFP alone, the level of binding to rat or human plasma albumins reached 0.011 or 0.039 moles of DFP per mole of albumin, respectively. This level of binding could be further increased by raising the concentration of ³H-DFP, increasing the ³H-DFP incubation time, or by substitution of commercial albumins for native albumin. Pesticide binding to albumin was presumed covalent since it survived 24 h dialysis. After dosing rats with pirimiphos-methyl (dimethoxy) or chlorfenvinphos (oxon) (diethoxy) pesticides, the resultant albumin binding were still significant 7 days after dosing. As in vitro, dosing of rats with malathion did not result in significant albumin binding in vivo. Our results suggest albumin may be a useful additional biomonitor for moderately low-level exposures to several widely used pesticides, and that this binding differs markedly between pesticides.     

Adaptive processes of the central and autonomic cholinergic neurotransmitter system: age-related differences

Potential age-related differences in the response of the ileum strip longitudinal and circular muscle to repeated treatment with diisopropyl fluorophosphate (DFP) were evaluated in Sprague-Dawley rats. The response was measured in terms of both biochemical parameters (acetylcholinesterase-AChE inhibition, muscarinic acetylcholine receptor binding sites-mAChRs, choline acetyltransferase-ChAT) and functional responsiveness (contractility of the isolated ileum stimulated by cholinergic agonists). The biochemical data were compared with those obtained for the cerebral cortex. Male 3- and 24-month old rats were s.c. injected with DFP on alternate days for 2 weeks (doses in mg/kg: first 1.1, two of 0.7 and four of 0.35). They were killed 48 hr and 7, 14, 21, 28 and 35 days after the last treatment. In the ileum strip of control rats there was a significant age-related decline of AChE, maximal density of 3H-QNB binding sites (Bmax) and ChAT. During the first week of DFP treatment the cholinergic syndrome was more pronounced in aged than in young rats, resulting in 35% and 10% mortality, respectively; subsequently the syndrome attenuated. At the end of DFP treatment ileal AChE were inhibited by about 30%; the down-regulation of mAChRs was about 50% in young and 35% in aged rats. No significant differences in the recovery rate of AChE were noted between young and aged rats (normalization within 7 days). On the contrary, mAChRs normalized within 5 weeks in young and 3 weeks in aged rats. This was probably due to more adaptive decline in the former group. There was a post-treatment increase of ChAT, transitory in young and persistent in aged rats. In spite of age-related marked loss of ileal mAChRs there were only little, although significant, changes in the contractile responsiveness of the isolated ileum to cholinergic agonists. Considerable DFP-induced down-regulation of mAChRs was not accompanied by changes in contractility stimulated by the agonists. The overall data indicate that age- and treatment-induced changes of AChE, mAChRs and ChAT in the ileum strip differ considerably from those observed in the cerebral cortex of the same rats.     

Differential Effects of Cocaine-Induced Seizures and Lethality on M1-Like Muscarinic and Dopaminergic D1- and D2-Like Binding Receptors in Mice Brain

Summary This work was designed to study the changes produced by cocaine-induced seizures and lethality on dopaminergic D₁- and D₂-like receptors, muscarinic M₁-like binding sites, as well as acetylcholinesterase activity in mice prefrontal cortex (PFC) and striatum (ST). Binding assays were performed in brain homogenates from the PFC and ST and ligands used were [³H]-N-methylscopolamine, [³H]-NMS (in the presence of carbachol), [³H]-SCH 23390 and [³H]-spiroperidol (in presence of mianserin), for muscarinic (M₁-like), D₁- and D₂-like receptors, respectively. Brain acetylcholinesterase (AChE) activity was also determined in these brain areas. Cocaine-induced SE decreased [³H]-SCH 23390 binding in both ST and PFC areas. A decrease in [³H]-NMS binding and an increase in [³H]-spiroperidol binding in PFC was also observed. Cocaine-induced lethality increased [³H]-spiroperidol binding in both areas and decreased [³H]-NMS binding only in PFC, while no difference was seen in [³H]-SCH 23390 binding. Neither SE, nor lethality altered [³H]-NMS binding in ST. AChE activity increased after SE in ST while after death the increase occurred in both PFC and ST. In conclusion, cocaine-induced SE and lethality produces differential changes in brain cholinergic and dopaminergic receptors, depending on the brain area studied suggesting an extensive and complex involvement of these with cocaine toxicity in central nervous system.     

Glucocorticoid regulation of two serine hydrolases in rat splenic lymphocytes in vitro

A quantitative assay employing binding of [³H]diisopropylfluorophosphate ([³H]DFP) and SDS-polyacrylamide gel electrophoresis was used to measure serine hydrolases in cell-free extracts from rat splenic lymphocytes. After labeling with [³H]DFP at pH 7, six major serine hydrolases are detected on 10% gels, having molecular weights of 78, 55, 34, 30, 28 and 17 (· 10^?3). When labeled at pH 4, only four activities are measured, with M_r or 79, 55, 33 and 17 (· 10^?3). Incubation of splenic lymphocytes for 8 h in vitro with 1 μM dexamethasone followed by [³H]DFP labeling at pH 7 produces a 91% increase in the 17000 [³H]DFP. Hormone treatment for 8 h with subsequent labeling at pH 4 results in a 15% increase in the largest (78000) species, as well as 73% increase in the 17000 enzyme, compared with lysates from cells incubated without steroid. These effects are not observed after only 4 h of glucocorticoid exposure. Dexamethasone treatment for 8 h does not produce a decrease in any of these serine hydrolases, nor is there an apparent induction of new enzymes (i.e., having a molecular weight different from the preexisting species). Studies examining the effect of protease inhibitors on the [³H]DFP capacity of these proteins, show that the 17000 enzyme is sensitive to the protease inhibitor, pepstatin A, as well as the sulfhydryl reagents dithiothreitol and N-ethylmaleimide. These result suggest that this dexamethasone-responsive enzyme is a protease which requires a free thiol group for optimal activity. These findings are discussed with regard to the mechanism of glucocorticoid action in lymphocytes.     

Decreased muscarinic binding sites in small intestine from mice treated with neostigmine

It has been reported by several authors that animals given repeated sublethal doses of an organophosphate, acetylcholinesterase (AChE) inhibitor, develop tolerance to its toxicity. This phenomenon seems to be due, at least partially, to a decrease of central and peripheral cholinergic receptors. In the present study, we report that a decrease of muscarinic receptors, as measured by [³H]-quinuclidinyl benzilate (³H-QNB) binding, occurs in the small intestine of mice treated with the carbamate, AChE inhibitor, neostigmine. Male mice were given neostigmine in the drinking water at daily increasing concentrations (from 20 to 1000 ppm). Methylatropine (20mg/kg, i.p.) was administered twice a day for the same period to two groups of control and neostigmine-treated animals. AChE activity was inhibited 60–70% in small intestine and diaphragm and [³H]-QNB binding was significantly reduced in the small intestine of neostigmine-treated mice; both the number of receptors and the affinity were lower than control. This decrease was not present in the tissue of mice given methylatropine together with neostigmine. Administration of methylatropine alone caused a significant increase of [³H]-QNB binding in the small intestine.     

Plasticity of Brain α-Adrenoceptors During the Development of Diet-Induced Obesity in the Rat

Male Sprague-Dawley rats, which are prone to develop diet-induced obesity (DIO) on a high energy (HE) diet can be separated from rats which are diet-resistant (DR) by several prospective tests. Using such tests, chow-fed DR-prone rats have higher binding of ³H paraminoclonidine (PAC) to brain α₂-adrenoceptors than do DIO-prone rats. These differences disappear after 3 months on a HE diet. To study the predictive value of these tests and possible associated changes in presynaptic membrane composition, brain α₁-(1nM ³H prazosin) and α₂-adrenoceptor (1nM ³H PAC) binding and synaptosomal fatty acid composition were assessed in 3-month-old male rats separated by weight gain into DR and DIO groups after 1 month on a HE diet. DIO had comparable total caloric intake but gained 30% and 43% more weight and were hyperinsulinemic compared to DR and chow-fed rats, respectively. After 1 month on a HE diet, DR rats still had 15%-53% higher ³H PAC binding than DIO and/or chow-fed rats in 14 of 16 brain areas assessed. A phenotype effect was present primarily in the amygdala where DR rats had higher ³H PAC binding than DIO rats. A diet effect was seen in some hypothalamic nuclei where both DR and DIO rats had higher ³H PAC binding than chow-fed rats. Conversely, DIO rats had 14%–21% higher ³H prazosin binding than DR rats in 3 brain areas. Changes in brain synaptosomal membranes' fatty acids reflected both phenotype and diet effects. Thus, while diet composition affects presynaptic membrane composition and α₂-adrenoceptor binding in both DR and DIO rats, the predominance of plasticity of these parameters is limited to the brains of DR rats. This suggests that such plasticity may be an important determinant of the ability to resist the development of diet-induced obesity on a HE diet.     

Desensitization of the nicotinic acetylcholine receptor by diisopropylfluorophosphate

The interaction of diisopropylfluorophosphate (DFP) with the nicotinic acetylcholine (ACh) receptor of Torpedo electric organ was studied, using [³H]-phencyclidine ([³H]-PCP) as a reporter probe. Phencyclidine binds with different kinetics to resting, activated, and desensitized receptor conformations. Although DFP did not inhibit binding of [³H]-ACh or ¹²⁵I-α-bungarotoxin (BGT) to the receptor recognition sites and potentiated in a time-dependent manner [³H]-PCP binding to the receptor's high-affinity allosteric site, it inhibited the ACh or carbamylcholine-stimulated [³H]-PCP binding. This suggested that DFP bound to a third kind of site on the receptor and affected receptor conformation. Preincubation of the membranes with DFP increased the receptor's affinity for carbamylcholine by eightfold and raised the pseudo-first-order rate of [³H]-PCP binding to that of an agonist-desensitized receptor. Accordingly, it is suggested that DFP induces receptor desensitization by binding to a site that is distinct from the recognition or high-affinity noncompetitive sites.     

Effects of Chronic Basic Fibroblast Growth Factor Administration to Rats with Partial Flmbrial Transections on Presynaptic Cholinergic Parameters and Muscarinic Receptors in the Hippocampus: Comparison with Nerve Growth Factor

Abstract: The present study compares the effects of chronic administration of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on various hippocampal cholinergic parameters in rats with partial unilateral fimbrial transections. Lesions resulted in marked reductions of several presynaptic cholinergic parameters: choline acetyltransferase (ChAT) activity (by 50%), [³H]-acetylcholine ([³H]ACh) synthesis (by 59%), basal and ve-ratridine (1 μM)-evoked [³H]ACh release (by 44 and 57%, respectively), and [³H]vesamicol binding site densities (by 35%). In addition, [³H]AF-DX 116/muscarinic M₂ binding site densities were also modestly decreased (by 23%). In contrast, [³H]pirenzepine/muscarinic M₁ and [³H]AF-DX 384/muscarinic M₂/M₄ binding site densities were not altered by the lesions, nor were they affected by any of the treatments. Intracerebroventricular administration of bFGF (10 ng, every other day, for 21 days) partially prevented the lesion-induced deficit in hippocampal ChAT activity, an effect that was not markedly different from that measured in the NGF-treated (1 μg intracerebroventricularly, every other day, for 21 days) rats. In rats treated with a combination of bFGF and NGF, ChAT activity was not different from that in rats treated with the individual factors alone. In contrast, the lesion-induced deficits in the other cholinergic parameters were not attenuated by bFGF treatment, although they were at least partially prevented by NGF administration. To determine whether higher concentrations of bFGF are necessary to affect cholinergic parameters other than hippocampal ChAT activity, rats were treated with 1 μg (every other day, 21 days) of the growth factor. In this group of rats, detrimental effects of bFGF, manifested by an increased death rate (46%), and marked reductions in body weight of the survivors, were observed. In addition, this concentration of bFGF appeared to exacerbate the lesion-induced reduction in [³H]ACh synthesis by hippocampal slices; [³H]ACh synthesis in lesioned hippocampi represented 36 and 52% of that in contralateral unlesioned hippocampi for the bFGF-treated and control groups, respectively. In conclusion, although bFGF administration attenuates the deficit in hippocampal ChAT activity induced by partial fimbrial transections, this does not appear to translate into enhanced functional capacity of the cholinergic terminals. This is clearly in contrast to NGF, which enhances not only hippocampal ChAT activity, but also other parameters indicative of increased function in the cholinergic terminals.     

Aging-Associated Changes in the Pharmacological Properties of the Benzodiazepine (ω) Receptor Isotypes in the Rat Hippocampus

Abstract: The aging-associated changes in hippocampal benzodiazepine (ω) receptor isotypes have been investigated in rats of the Wistar and Fischer 344 strains. Displacement experiments of [³H]flunitrazepam binding by zolpidem demonstrated that in hippocampal membranes from adult (3-month-old) Wistar strain rats, high (type I; ω₁)-, intermediate (type II_M; ω₂)-, and low (type II_L; ω₅)- affinity sites for this imidazopyridine account for 27.1 ± 7.5, 44.2 ± 7.5, and 28.8 ± 5.1%, respectively. In hippocampal membranes from aged (24-month-old) rats of the same strain, the relative abundance of these sites was 42.8 ± 9.3, 26.3 ± 4, and 36.0 ± 5.9%, respectively. In contrast, no significant difference was observed in the whole benzodiazepine (ω) binding site density between adult and aged rats. The increase in type I (ω₁) binding site density in the hippocampus of aged rats was also demonstrated in saturation experiments with [³H]zolpidem. This aging-induced increase in [³H]zolpidem binding was also observed in hippocampal membranes from Fischer 344 rats. Moreover, in both rat strains, GABA induced a greater enhancement of [³H]zolpidem (5 nM) binding to type I (ω₁) sites (GABA shift) in aged than in adult hippocampal membranes. Quantitative autoradiographic analysis of [³H]zolpidem binding to coronal brain sections from adult and aged Fischer 344 rats demonstrated that the aging-associated increases in the density of type I (ω₁) binding sites were restricted to the hippocampus. Moreover, increases in binding density were larger in the dentate gyrus and in the CA2 field than in the CA1 and CA3 fields.     

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells

Binding experiments with the specific muscarinic ligand [³H]N-methylscopolamine (³H-NMS) have shown the presence of constitutive muscarinic acetylcho-line receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1–M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K_D) for ³H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B⁺) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B⁺ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells. J. Cell. Physiol. 178:333–340, 1999. © 1999 Wiley-Liss, Inc.     

Decrease or increase in cardiac muscarinic cholinergic receptor number in rats treated with methacholine or atropine

The effects of chronic treatment of the rat with methacholine and atropine on the cardiac muscarinic cholinergic receptors were investigated. [³H]Quinuclidinyl benzilate ([³H]QNB) was used to directly estimate the number and affinity of the receptors in the heart ventricular membrane. Methacholine treatment decreased, in a dose-related and time-dependent manner, the specific binding of [³H]QNB by 34% as compared to the control. Atropine treatment, on the other hand, resulted in a dose-related increase (28 to 66%) in the number of the receptors. The equilibrium dissociation constant (K_D) of the receptors for the ligand was the same (about 200 pM) for the control and the methacholine treated groups of rats, whereas a dose-related increase (39 to 105%) in the K_D was noted for the atropine treated rats. Similarly, the concentration of acetylcholine causing a 50 percent inhibition (IC₅₀) of the [³H]QNB binding was unaltered for the methacholine treated rats (4 μM), but it was increased 43% for the atropine treated rats.     

Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors

The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2–4 months) and aged (22–24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [³H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [³H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.     

Altered Muscarinic and Nicotinic Receptor Densities in Cortical and Subcortical Brain Regions in Parkinson's Disease

Abstract: Muscarinic and nicotinic cholinergic receptors and choline acetyltransferase activity were studied in postmortem brain tissue from patients with histopathologically confirmed Parkinson's disease and matched control subjects. Using washed membrane homogenates from the frontal cortex, hippocampus, caudate nucleus, and putamen, saturation analysis of specific receptor binding was performed for the total number of muscarinic receptors with [³H]quinuclidinyl benzilate, for muscarinic M₁ receptors with [³H]pirenzepine, for muscarinic M₂ receptors with [³H]oxotremorine-M, and for nicotinic receptors with (–)-[³H]nicotine. In comparison with control tissues, choline acetyltransferase activity was reduced in the frontal cortex and hippocampus and unchanged in the caudate nucleus and putamen of parkinsonian patients. In Parkinson's disease the maximal binding site density for [³H]quinuclidinyl benzilate was increased in the frontal cortex and unaltered in the hippocampus, caudate nucleus, and putamen. Specific [³H]pirenzepine binding was increased in the frontal cortex, unaltered in the hippocampus, and decreased in the caudate nucleus and putamen. In parkinsonian patients B_max values for specific [³H]oxotremorine-M binding were reduced in the cortex and unchanged in the hippocampus and striatum compared with controls. Maximal (–)-[³H]nicotine binding was reduced in both the cortex and hippocampus and unaltered in both the caudate nucleus and putamen. Alterations of the equilibrium dissociation constant were not observed for any ligand in any of the brain areas examined. The present results suggest that both the innominatocortical and the septohippocampal cholinergic systems degenerate in Parkinson's disease. The reduction of cortical [³H]oxotremorine-M and (–)-[³H]nicotine binding is compatible with the concept that significant numbers of the binding sites labelled by these ligands are located on presynaptic cholinergic nerve terminals, whereas the increased [³H]pirenzepine binding in the cortex may reflect postsynaptic denervation supersensitivity.     

Central neurotransmitter receptors in hypertensive rats

Muscarinic cholinergic ([³H]QNB), α₁ ? ([³H]WB-4101), and α₂ ? ([³H]clonidine) adrenergic ligand binding was measured in various regions of the brains of adult normotensive, spontaneously hypertensive, and DOCA-salt hypertensive rats. There was a 66% increase in the number of α₁-adrenergic receptors in hypothalamus of the spontaneously hypertensive rats as compared to normotensive controls, with no change in the K_d value. There were no other differences in the spontaneously hypertensive rats and none in the DOCA-salt model. α₁-Adrenergic binding was elevated in hypothalamus of spontaneously hypertensive rats 4–20 weeks of age even though blood pressure in the 4-week old animals was not at hypertensive levels (i.e., <150 mmHg). Treatment of adult spontaneously hypertensive rats with clonidine HCl significantly reduced blood pressure but failed to alter the binding of [³H]WB-4101 in hypothalamus. Thus, it appears that the enhanced number of α₁-adrenergic receptors in hypothalamus of spontaneously hypertensive rats is neither a consequence of the increased blood pressure, nor a phenomenon common to all models of hypertension.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. I. Modulation of Central 5-Hydroxytryptamine Receptors in the Rat

Abstract: The present study indicates that central 5-hydroxytryptamine (5-HT; serotonin) receptors can be modulated in opposite directions by Ca²⁺ and guanine nucleotides [guanosine triphosphate (GTP), β, γ-imidoguanosine 5′-triphosphate (GppNHp)]. Thus CaCl₂ (≥0.5 mm ) inhibited whereas GTP and GppNHp (10 μm ) stimulated the 5-HT-sensitive adenylate cyclase in the hippocampus of newborn rats. Both the affinity (K_d ^?1) and the number (B_max) of [³H]5-HT binding sites in hippocampal membranes from adult rats were increased in the presence of Ca²⁺ (≥0.25 mm ); GTP (≥0.1 mm ) and GppNHp (≥0.3; μm ) produced reverse effects. The efficacy of guanine nucleotides in inhibiting specific [³H]5-HT binding was counteracted by Ca²⁺: the addition of this cation (5mm -CaCl₂) to the assay mixture resulted in a 40-fold increase in the IC₅₀ for GTP; the IC₅₀ for GppNHp increased five-fold under the same condition. The examination of the respective effects of Ca²⁺ and of GTP on the specific binding of [³H]5-HT to various hippocampal membrane preparations (from developing rats, from subcellular fractions of adult tissues, and from adult rats after the selective degeneration of serotoninergic innervation in the forebrain) indicated that the amplitudes of the Ca²⁺-induced increase and of the GTP-induced decrease were generally correlated. This conclusion did not apply to striatal membranes of kainic acid-treated rats because [³H]5-HT binding sites persisting after the intrastriatal injection of kainic acid (i.e., half of the total number in striatal membranes from control rats) were markedly less affected by GTP but at least as responsive as control membranes to the Ca²⁺-induced increase. These data are compatible with the hypothesis of a possible coupling of some–but not all–[³H]5-HT binding sites to adenylate cyclase in the rat brain.     

Changes of NMDA receptor binding in spontaneously epileptic rat and parent strains

We measured the binding of [³H]3-[(±)2-carboxypiperazin-4-yl]propyl-1-phosphonic acid ([³H]CPP), a competitive ligand forN-methyl-d-aspartate (NMDA) receptors, in double mutant spontaneously epileptic rats (SER:zi/zi, tm/tm) and their parent strains, zitter rats, and tremor rats, and WTC rats (control rats from tremor rats derived from Kyoto:Wistar rats) before and after the onset of seizures in tremor rats and SER. Significantly lower [³H]CPP binding receptor density (B_max) was found in the cortex of SER and zitter rats at 12–15 weeks of age than in that of WTC rats and tremor rats, and at 4 weeks of age the Bmax in zitter rats was lower than that in the other strains. The reduction of Bmax in SER at 12–15 weeks of age may reflect a down regulation of NMDA receptors due to repetitive tonic seizures in SER.     

Sequestration of Muscarinic Cholinergic Receptors in Permeabilized Neuroblastoma Cells

Abstract: The feasibility of using a permeabilized preparation of human SH-SY-5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin-O, or the α-toxin from Staphylococcus aureus to oxotremorine-M (Oxo-M) for 30 min resulted in a 25–30% reduction in the number of cell surface mAChRs, as monitored by the loss of N[³H]methyl- scopolamine ([³H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 μM digitonin, the Oxo-M-mediated reduction in [³H]NMS binding was time (t_1/2～ 5 min) and concentration (EC₅₀～ 10 μM) dependent and was agonist specific (Oxo M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [³H]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [³H]NMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca²⁺, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5′-O-(2-thiodiphosphate). mAChRs sequestered in response to Oxo-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 μM). The results indicate (a) that permeabilized SH-SY-5Y cells support an agonist-induced sequestration of mAChRs, the magnitude of which is ～ 65–70% of that observed for intact cells, (b) that when internalized, mAChRs are located in a cellular compartment to which [³H]NMS has only a limited access despite the removal of the plasma membrane barrier, and (c) that the production of phosphoinositide-derived second messengers is not a prerequisite for mAChR sequestration.