Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

CHARACTERIZATION AND SUBCELLULAR LOCALIZATION OF BRAIN MUSCARINIC RECEPTORS LABELLED IN VIVO BY [3H]DEXETIMIDE

Abstract— [³H]Dexetimide specifically labels brain muscarinic receptors in vivo . After i.v. injection of labelled drug into rats, radioactivity specifically accumulates in brain regions containing muscarinic receptors but not in cerebellum. This accumulation is stereospecific, saturable and displaceable by unhbelled dexetimide. In contrast, [³H]levetimide, the inactive enantiomer, does not show such preferential uptake or stereospecific displacement.
An analytical approach was used to study the subcellular distribution of [³H]dexetimide binding sites. After differential centrifugation the binding sites are mainly recovered in the microsomal fraction from different brain regions but not from the cerebellum. After displacement the radioactivity is found in the supernatant. After equilibration in a density gradient the distribution pattern of [³H]dexetimide is bimodal, like that of 5'-nucleotidase, with a major peak in a region of low density.
When the microsomal fraction was treated with digitcnin, three groups of membrane were characterized by isopycnic centrifugation on the basis of their differential shift to higher densities. Evidence is provided that the postsynaptic membranes bearing muscarinic receptors belong to the class of plasma membranes. Finally, digitonin treatment may represent a useful tool to produce subfractions enriched in postsynaptic membranes which can now be identified biochemically in binding experiments.     

Agonist and Antagonist Binding of Muscarinic Acetylcholine Receptors Purified from Porcine Brain: Interconversion of High- and Low-Affinity Sites by Sulfhydryl Reagents

The affinity for muscarinic ligands of a preparation of muscarinic acetylcholine receptors purified from porcine brain was examined by means of competitive binding of [3H]quinuclidinylbenzylate and unlabeled ligands, followed by computer-assisted nonlinear regression analysis. The displacements by antagonists fitted a single-site model. In contrast, the displacements by agonists did not fit the single-site model and could be explained by assuming two populations of binding sites. The proportion of the sites with high affinity for muscarinic agonists (H-sites) ranged from 25 to 35% of the total number of sites. GTP had no effect on the displacements by agonists, a finding indicating that H-sites did not result from interaction between receptors and GTP-binding proteins. In the presence of dithiothreitol, the affinity for muscarinic ligands decreased. The largest effects were observed on the affinity for pirenzepine and that of H-sites for carbachol. Preincubation of the preparation with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in an increase in the proportion of H-sites to 75% of the total number of binding sites. The results of sucrose density gradient centrifugation of the preparation indicated apparent heterogeneity as to molecular size of the receptors, but this heterogeneity did not correlate with that of the affinity for agonists. In addition, the receptors were detected as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation, regardless of the presence or absence of disulfide-reducing reagents. These results suggest that the redox state of thiol groups in the receptor molecules is relevant to their affinities for ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of quinidine and lidocaine with rat brain and heart muscarinic receptors

We have studied the effect of quinidine and lidocaine on binding to rat brain and cardiac muscarinic receptors. Both drugs had a higher affinity to brain stem and cardiac receptors, as compared with cerebral cortex, coinciding with the distribution of high-affinity agonist binding sites in the above tissues. The effects of the drugs on muscarinic antagonist and agonist binding did not fit simple competition to one receptor site, suggesting either preferential binding to high affinity agonist binding sites, or allosteric interactions. Batrachotoxin, which opens voltage sensitive sodium channels, had an opposite effect on agonist binding. The possibility of allosteric interactions between the muscarinic receptors and a site analogous to the sodium channel is discussed.     

Muscarinic acetylcholine receptor: thermodynamic analysis of the interaction of agonists and antagonists

The thermodynamic parameters of the interaction of agonists and antagonists with heart and brain muscarinic receptors were determined. The binding of quinuclidinyl [3H]benzilate and the inhibition of quinuclidinyl benzilate (QNB) binding by agonists and antagonists were examined at temperatures between 2 degrees C and 27 degrees C. The density of specific binding sites and the relative proportions of high- and low-affinity binding components of drugs were unaffected by the temperature changes. The binding of atropine was entropy driven in brain and heart membranes. In contrast, net values of these thermodynamic parameters for QNB binding and for the high-affinity binding component of pirenzepine to brain membranes were decreased with the enhancement of the temperature. The low-affinity binding component of the agonists carbachol, oxotremorine and pilocarpine was enthalpy driven. Their high-affinity binding component was entropy driven at 2 degrees C and became enthalpy driven when the incubation temperature was increased. The guanine nucleotide Gpp[NH]p partly prevented the temperature-dependent decrease of net entropy and enthalpy values. Considering that the net changes of thermodynamic parameters are relevant of the interactions between the ligand, the receptor protein and the adjoining membranous molecules, a three-state conformational model is proposed for the muscarinic receptor protein. The receptor selectivity is reappreciated owing to these three states of the receptor protein and the different components of the muscarinic receptor complexes.     

Properties of putative nicotinic and muscarinic cholinergic receptors in the central nervous system of Locusta migratoria

Nervous tissue preparations from Locusta migratoria specifically bind potent nicotinic (α-bungarotoxin) and muscarinic (quinuclidinyl benzilate) ligands. Binding properties and pharmacological data indicate that the central nervous system of the locust contains at least two distinct classes of receptors. Subcellular fractionation experiments revealed that the receptor activity is enriched in the synaptosomal fraction. In the head as well as in the thoracic ganglia the nicotinic acetylcholine receptors were found to be much more abundant than the muscarinic binding sites; whereas in mouse brain the muscarinic receptor type predominates.     

Muscarinic Cholinergic Receptor Subtypes in Hippocampus in Human Cognitive Disorders

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

Heterogeneity of solubilized muscarinic cholinergic receptors: binding and hydrodynamic properties

Previous studies have described the conversion, after detergent solubilization, of the multiple populations of membrane-bound muscarinic agonist binding sites to a population of uniform affinity. This paper describes the solubilization of at least two receptor species, distinct in their agonist binding characteristics, which are capable of interconversion by transition metal ions. This finding enabled a more detailed examination of the molecular properties and regional differences of brain muscarinic receptors than was previously possible. Muscarinic receptors (mAChR) obtained from the rat cerebral cortex or medulla pons were solubilized using digitonin or the zwitterion detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). The equilibrium binding of the antagonist [3H]-4-N-methylpiperidyl benzilate ([3H]4NMPB) to detergent-solubilized receptors resembled binding to neural membranes and exhibited subnanomolar affinity, saturability, and simple mass action kinetics. Agonist binding to soluble preparations was measured by competition of [3H]4NMPB binding sites. Saturation isotherms for agonist binding to digitonin- and Chaps-solubilized mAChR obtained from various brain regions appear flattened and have Hill coefficients in the range 0.52-0.78. Computerized modelling techniques indicate that the best fit to the experimental data is provided by a model specifying two soluble muscarinic agonist binding sites with differing dissociation constants, KH and KL, respectively. Solubilization of cerebral cortex membranes with Chaps or digitonin resulted in a population with a composition of high- and low-affinity sites similar to that found in the membrane-bound state. In contrast, solubilization of the medulla pons resulted in an approximately 40% loss of high-affinity sites. Solubilized receptors retained the sensitivity to transition metals ions, but were insensitive to guanine nucleotides. Density gradient centrifugation indicated that Chaps-solubilized mAChR are composed of two molecular forms with S20,W equal to 9.9 S and 14.9 S. The 14.9 S species comprises approximately 30% of the total binding activity in the cortex and approximately 40% in the medulla. We identify the 14.9 S species as being associated with a guanylnucleotide binding protein because treatment of medulla membranes with guanylylimidodiphosphate prior to solubilization results in disappearance of 14.9 S with 9.9 S unchanged. Sedimentation of cortical mAChR in the presence of Cu+2 leads to an increase in 14.9 S to almost 50% of the total binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective interaction of tricyclic antidepressants with a subclass of rat brain cholinergic muscarinic receptors

Experiments were undertaken to determine whether the anticholinergic actions of tricyclic antidepressants are mediated by a selective interaction with a subclass of muscarinic receptors. To this end, the potencies of these antidepressants to inhibit [3H]-QNB binding to rat brain cerebral cortical membranes was compared to their potencies as antagonists of carbachol-stimulated inositol phosphate accumulation in cerebral cortical slices and carbachol-induced inhibition of GTP-stimulated adenylate cyclase in striatal membranes. Whereas amitriptyline was more potent than pirenzepine, a selective muscarinic M1 receptor antagonist, in competing for [3H]-QNB binding sites and as an antagonist of carbachol-induced inhibition of adenylate cyclase, pirenzepine was substantially more active (ten-fold) than amitriptyline in blocking carbachol-stimulated phosphatidyl inositol turnover. Atropine was more potent than all other agents in these assays, failing to display any significant degree of selectivity. The results suggest that the tricyclic antidepressants, in particular amitriptyline, appear to be selective antagonists for muscarinic receptors associated with adenylate cyclase in striatal membranes. Given the current classification of cholinergic receptors, these findings indicate that the tricyclic antidepressants may be useful for defining the properties of M2 receptors in brain.     

Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

Heterogeneity of muscarinic receptors coupled to phosphoinositide breakdown in guinea pig brain and peripheral tissues

Muscarinic receptors coupled to phosphoinositide hydrolysis (PI) are present in guinea pig bladder and colon. Compared to rat cerebral cortex, an extensively studied muscarinic/PI turnover system, all agonists were more potent and efficacious in both bladder and colon. The "M1-selective antagonists", pirenzepine and dicyclomine, were much more potent (Ki = 1-5 nM) and selective (300 to 500-fold) at both rat and guinea pig brain and guinea pig colon receptors, compared to PI-coupled receptors in guinea pig bladder. In contrast, "M2-selective antagonists", AF-DX 116 and HHSiD, were 2-6 fold more potent in bladder than in brain, while HHSiD was very potent in the colon (50 times more potent than in brain). These results suggest a pharmacological heterogeneity of PI-linked muscarinic receptors. If muscarinic receptors with a low affinity for pirenzepine are defined as M2, these results show that the guinea pig bladder contains PI-linked M2 muscarinic receptors, whereas the guinea pig colon contains PI-linked M1 receptors.     

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Striatal Muscarinic Cholinergic Receptors and High-Affinity Choline Uptake Sites: A Quantitative Autoradiographic Study

The binding characteristics and distribution of M1 and M2 muscarinic cholinergic receptors and high-affinity choline uptake sites were studied in the striatum of the rat at 3-4 and 9-12 weeks of age after exposure to unilateral perinatal hypoxic-ischemic brain injury. High-affinity choline uptake sites were labeled with [3H]hemicholinium-3, M1 receptors with [3H]pirenzepine, and M2 receptors with [3H]AF-DX 116. Saturation experiments revealed a significant decrease in the maximal binding capacity (Bmax) for [3H]pirenzepine-labeled M1 receptors in the lesioned caudate/putamen complex in immature rats with moderate brain injury, in comparison with controls. In contrast, the Bmax value for [3H]hemicholinium-3-labeled high-affinity choline uptake sites was significantly increased. No changes in dissociation constants (KD) were observed. These changes were most pronounced in the dorsolateral region of striatum. Striatal regional distribution of [3H]AF-DX 116 was not affected. In mature rats, binding of [3H]pirenzepine returned to control values, whereas [3H]hemicholinium binding showed a persistent increase (23%). The increase in [3H]hemicholinium-3 binding, as a specific marker of cholinergic nerve terminals, is consistent with our prior morphologic studies demonstrating relative preservation of cholinergic neurons and neuropil, and supports the concept that striatal cholinergic systems are resistant to hypoxic-ischemic injury.     

A comparison of the antimuscarinic effects of pirenzepine and N-methylatropine on ganglionic and vascular muscarinic receptors in the rat

The antimuscarinic properties of pirenzepine and N-methylatropine were evaluated in two intact preparations by measuring A) the inhibition of increase in mean arterial pressure evoked by McN-A-343 in pithed rats through activation of ganglionic muscarinic receptors and B) the inhibition of fall in arterial pressure evoked by methacholine in anaesthetized rats through activation of vascular muscarinic receptors. To characterize the antimuscarinic potencies of pirenzepine and N-methylatropine, for both antagonists doses were calculated that produce a 10-fold shift to the right of the dose-response curves for A) the pressor response to McN-A-343 (i.v. administration) in pithed rats (D10-p.r.) and B) for the depressor effect to methacholine (i.v. administration) in anaesthetized rats (D10-an.r.), respectively. Whereas N-methylatropine was virtually equieffective in blocking both muscarinic responses (D10-an.r./D10-p.r. approximately equal to 1), pirenzepine, however, was considerably more potent at ganglionic than at vascular muscarinic receptors (D10-an.r./D10-p.r. approximately equal to 16). These data confirm the existence of excitatory ganglionic muscarinic receptors with high affinity for pirenzepine (M1) and provide evidence for the presence of M2 receptors - receptors which show a low sensitivity to pirenzepine - on vascular smooth muscle cells. To further characterize the anticholinergic properties of pirenzepine, its effect on the pressor response to DMPP, a nicotinic ganglionic stimulant, was investigated in pithed rats. A high dose of pirenzepine (1.13 mumol/kg), given i.v., did not affect nicotinic ganglionic transmission.     

High affinity acylating antagonists for muscarinic receptors.

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

Solubilization and partial purification of the thiazide diuretic receptor from rabbit renal cortex

This study was designed to solubilize, characterize and begin to purify the thiazide-sensitive Na/Cl transporter from mammalian kidney. Metolazone, a thiazide-like diuretic drug, binds to receptors in rat renal cortex closely related to the thiazide-sensitive Na/Cl transport pathway of the renal distal tubule. In the current study, [3H]metolazone bound to receptors in rabbit renal cortical microsomes. The portion of [3H]metolazone binding that was inhibited by hydrochlorothiazide reflected binding to a high-affinity class of receptor. The affinity (Kd 2.0 +/- 0.1 nM) and number (Bmax = 0.9 +/- 0.4 pmol/mg protein) of high-affinity receptors in rabbit renal cortical membranes were similar to values reported previously for rat. When proximal and distal tubule fragments were separated by Percoll gradient centrifugation, receptors were restricted to the fraction that contained distal tubules. When compared with cortical homogenates, receptor density was enriched 12-fold by magnesium precipitation and differential centrifugation. The zwitterionic detergent CHAPS solubilized 25-35% of the receptors (at 6 mM). Chloride inhibited and Na stimulated binding of [3H]metolazone to solubilized high-affinity receptors. The receptors could be purified significantly by hydroxyapatite chromatography and size exclusion high performance liquid chromatography (HPLC). The combination of magnesium precipitation and differential centrifugation, hydroxyapatite chromatography, and size exclusion HPLC resulted in a 213-fold enrichment of receptors, compared to renal cortical homogenate. The current results indicate that thiazide receptors from rabbit kidney share characteristics with receptors from rat, and that rabbit receptors can be solubilized in CHAPS and purified significantly by hydroxyapatite chromatography and size exclusion HPLC.     

The Intact Human Neuroblastoma Cell (SH-SY5Y) Exhibits High-Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols

The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.     

Agonist binding to multiple muscarinic receptors

The binding of agonists to muscarinic cholinergic receptors is well described by a binding model of multiple affinity states (superhigh, high, and low) in most central and peripheral tissues. Although previous studies of the influences by divalent cations, guanine nucleotides, and sulfhydryl reagents support the concept that these regulators act through closely related sites to alter the relative proportions of muscarinic agonist affinity states, it has become apparent that muscarinic receptor subtypes (as defined with the nonclassical antagonist pirenzepine) are differentially affected by the regulators. For example, in tissues that have few high-affinity [3H]pirenzepine-binding sites (heart, ileum, cerebellum), magnesium ions promote the formation of a high agonist affinity state, whereas exposure of these tissues to the sulfhydryl reagent N-ethylmaleimide (NEM) or guanine nucleotides promotes the formation of a low agonist affinity state. Conversely, tissues rich in high-affinity [3H]pirenzepine-binding sites (cerebral cortex, corpus striatum, hippocampus) show little, if any, change in agonist binding site affinity when magnesium ions or guanine nucleotides are present. Furthermore, NEM enhances the muscarinic binding site affinity for agonists in these tissues. Taken together, these results support the concept of muscarinic receptor heterogeneity, as proposed from previous physiological studies, and indicate that the aforementioned regulators (guanine nucleotides, magnesium ions, NEM) differentially alter the agonist-binding properties of these muscarinic receptor subtypes.     

Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists

Heterogeneity of the muscarinic receptor population in the rat central and peripheral lung was found in competition binding experiments against [3H]quinuclidinyl benzilate [( 3H]QNB) using the selective antagonists pirenzepine, AF-DX 116 and hexahydrosiladifenidol (HHSiD). Pirenzepine displaced [3H]QNB with low affinity from preparations of central airways indicating the absence of M1 receptors in the trachea and bronchi. Muscarinic receptors in the central airways are comprised of both M2 and M3 receptors since AF-DX 116, an M2-selective antagonist, bound with high affinity to 70% of the available sites while HHSiD, an M3-selective antagonist bound with high affinity to the remaining binding sites. In the peripheral lung, pirenzepine bound with high affinity to 14% of the receptor population, AF-DX 116 bound with high affinity to 79% of the binding sites while HHSiD bound with high affinity to 18% of the binding sites. The presence of M1 receptors in the peripheral airways but not in the central airways was confirmed using [3H]telenzepine, an M1 receptor ligand. [3H]Telenzepine showed specific saturable binding to 8% of [3H]QNB labeled binding sites in homogenates of rat peripheral lung, while there was no detectable specific binding in homogenates of rat trachea or heart. The results presented here demonstrate that there are three muscarinic receptor subtypes in rat lungs, and that the distribution of the different subtypes varies within the lungs. Throughout the airways, the dominant muscarinic receptor subtype is M2. In the trachea and bronchi the remaining receptors are M3, while in the peripheral lungs, the remaining receptors are both M1 and M3.     

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

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

Different Subcellular Localization of Muscarinic and Serotonin (S2) Receptors in Human, Dog, and Rat Brain

Cortex from rat, dog, and human brain was submitted to subcellular fractionation using an analytical approach consisting of a two-step procedure. First, fractions were obtained by differential centrifugation and were analyzed for their content of serotonin S2 and muscarinic receptors, serotonin uptake, and marker enzymes. Second, the cytoplasmic extracts were subfractionated by equilibration in sucrose density gradient. In human brain, serotonin and muscarinic receptors were found associated mostly with mitochondrial fractions which contain synaptosomes, whereas in rat brain they were concentrated mainly in the microsomal fractions. Density gradient centrifugation confirmed a more marked synaptosomal localization of receptors in human than in rat brain, the dog displaying an intermediate profile. In human brain, indeed, more receptor sites were found to be associated with the second peak characterized in electron microscopy by the largest number of nerve terminals. In addition, synaptosomes from human brain are denser than those from rat brain and some marker enzymes reveal different subcellular distribution in the three species. These data indicate that more receptors are of synaptosomal nature in human brain than in other species and this finding is compatible with a larger amount of synaptic contacts in human brain.