Muscarinic Binding Sites Research Articles

Bilateral Symmetry of Cholinergic Deficits in Alzheimer's Disease

The specific activities of the cholinergic enzymes, choline acetyltransferase and acetylcholinesterase, as well as the density of muscarinic binding sites, were determined in five corresponding left and right regions of 16 brains obtained at autopsy from patients with histologically confirmed Alzheimer's disease. While a significant proportion of the individual specimens exhibited left-right asymmetries in cholinergic deficits, bilateral symmetry was the rule for frontal and temporal cortex, basal nucleus, and the prosubiculum of the hippocampus. In contrast to these four regions, left-right asymmetries in choline acetyltransferase activity and muscarinic receptor density appeared to be typical in the entorhinal cortex of the hippocampus.

Different binding properties of muscarinic M2-receptor subtypes for agonists and antagonists in porcine gastric smooth muscle and mucosa

The hypothesis that muscarinic receptors on smooth muscle differ from those in epithelial glands was tested by comparing the properties of muscarinic binding sites in porcine fundic smooth muscle with those in mucosal membranes. The binding of agonists and of antagonists was assessed by displacement of [3H]N-methylscopolamine. Pirenzepine (M1-antagonist) labeled low-affinity binding sites in smooth muscle (KD = 229 nM) and in mucosa (KD = 124 nM) consistent with the presence of M2 sites. Carbachol interacted with a high-affinity (KD = 164 nM) and a low-affinity (KD = 18.2 μM) state of binding sites in smooth muscle. Guanyl 5′-yl-imidodiphosphate converted all sites to the low-affinity state. N-ethylmaleimide pretreatment increased the affinity of carbachol and the proportion of high-affinity sites. In clear contrast, only low-affinity sites of carbachol were detectable in mucosa (KD = 17 μM) that were not modulated by N-ethylmaleimide or guanyl 5′-yl-imidodiphosphate. The cardioselective antagonist AF-DX 116 displayed low affinity to mucosal binding sites (KD = 3.4 μM), whereas its affinity to smooth muscle was 503 nM. The antagonist hexahydro-sila-difenidol had a very high affinity (KD = 2.9 nM) to mucosal receptors, whereas its affinity to smooth muscle sites was 88 nM. These data show that muscarinic M2 binding sites in mucosa and smooth muscle can be distinguished by both agonist and antagonist binding experiments, and suggest the existence of different subtypes of M2-binding sites in these tissues.

Binding of muscarine receptor antagonists to pig coronary smooth muscle.

In order to characterize the muscarinic binding site on coronary smooth muscle, we investigated the binding properties of (3H)quinuclidinyl benzilate (QNB) in membrane preparations of pig coronary arteries and atria. Scatchard analysis and Hill plot showed that (3H)QNB binds to a single population of sites in both tissues. The binding profiles of the muscarine receptor antagonists atropine, 11-[2-[dimethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrid o(2,3- b) (1,4)benzodiazepine-6-one (AF-DX 116), pirenzepine, and 4-diphenylacetoxy-N-methylpiperidine methiobromide (4-DAMP) in both tissues were compared with binding data from other tissues, representative for different muscarinic binding site subtypes. It is concluded that the pig coronary smooth muscle muscarinic binding site is different from M1 and M2 binding sites investigated so far.

Subtypes of muscarinic receptors in insect nervous system

1. 1. High affinity binding sites with muscarinic characteristics have been identified in the nervous system of Locusta migratoria and analyzed with regard to molecular size and modulation by ions or guanylnucleotides. 2. 2. Putative subtypes of muscarinic cholinergic receptors, as defined by differential affinity for pirenzipine, an antagonist for M 1-subtypes, have been detected in locusts nervous tissue. 3. 3. Muscarinic binding sites in synaptosomal membranes displayed a low affinity for pirenzipine, indicating that in the nerve terminals, M 2-subtypes of muscarinic receptors predominate. 4. 4. This is supported by a significant attenuation of cAMP formation in locusts synaptosomes by oxotremorine.

The effect of chronic diazepam and medazepam treatment on the number and affinity of muscarinic receptors in different rat brain structures

1. 1. The effect of chronic (19-day) treatment with the benzodiazepine tranquilizers diazepam (1 mg/kg/day, i.p.) and medazepam (5 mg/kg/day, i.p.) on the binding characteristics of muscarinic receptors in four rat brain structures: cerebral cortex, striatum, hippocampus and hypothalamus were studied using [ 3H]l-quinuclidinyl benzylate ([ 3H]QNB) as radioligand. 2. 2. Diazepam and medazepam treatment caused an overall decrease in muscarinic receptor binding affinity ( K d ). 3. 3. The number ( B max) of muscarinic receptors declined in the hippocampus and striatum and rose in the hypothalamus after both benzodiazepines. The B max of muscarinic receptors in the cerebral cortex was increased after diazepam treatment. 4. 4. The changes in the binding characteristics of muscarinic receptors might be due to benzodiazepine-induced occurrence of two populations of muscarinic binding sites: P 1 (high affinity, low capacity) and P 2 (low affinity, high capacity). 5. 5. The altered brain muscarinic receptor functions after chronic diazepam- or medazepam treatment suggest the role of cholinergic neurotransmission in the mechanism of action of benzodiazepines.

Tetranitromethane Modification of the Muscarinic Receptors in Bovine Adrenal Medulla

Muscarinic receptor binding was examined in bovine adrenal medullary microsomes following exposure to tetranitromethane (TNM) that modifies tyrosine and cysteine residues in proteins. The TNM (10-100 μM) treatment of adrenal medullary microsomes caused a concentration-dependent and irreversible reduction in the maximum number of binding sites (Bmax) for /-(3H)quinuclidinyl benzilate (QNB), with a slight increase in the equilibrium dissociation constant (KD). Typically, about a 36% decrease and a 1.3-fold increase in the corresponding values were obtained at 50 μM of TNM. The alteration in the Bmax was partially prevented by atropine but not carbamylcholine, and it was not reversed by subsequent treatment with dithiothreitol, a disulfide reducing agent. The change in the KD was unaffected by these agents. The TNM (50 μM) treatment also caused a slight decrease in the affinity of atropine and pirenzepine (for both the high and low affinity sites), and it caused a slight decrease in the affinity of carbamylcholine at the high affinity site, with a large loss of the low affinity site. Thus, the results indicate that TNM causes a loss of muscarinic binding sites and a decrease in the binding affinity of muscarinic receptors in bovine adrenal medulla, probably through modifications of functional groups such as tyrosine residues.

Cholinergic enzyme activities and muscarinic binding in the cerebral cortex of rats of different age and sex

1. 1. The choline acetyltransferase and acetylcholinesterase activities in the cerebral cortex and hippocampus and muscarinic binding in the cerebral cortex did not differ significantly between male and female Wistar rats. 2. 2. Choline acetyltransferase activities in the cerebral cortex and hippocampus of rats were not altered during ageing. 3. 3. Acetylcholinesterase activities in these same brain areas were markedly decreased during ageing, possibly reflecting a loss of postsynaptic enzyme activity. 4. 4. When measured using 3H-pirenzepine, binding to the postsynaptic muscarinic receptors was slightly higher in 26-month-old rats than in 12-month-old rats; total muscarinic binding measured using 3H-quinuclidinyl benzilate did not alter during ageing. 5. 5. The present study does not support the hypothesis that in the rat brain the number of postsynaptic muscarinic binding sites decreases during ageing.

Somatostatin, gastrin, and cholinergic muscarinic binding sites in rat gastric, duodenal, and jejunal mucosa.

Clinical and experimental data indicate that the concentration of gastrin-I and somatostatin binding sites in human and rat gastric and duodenal mucosa may be changed in several pathologic conditions, including human peptic ulcer and cancer diseases. There are no data, however, indicating the distribution of receptor binding sites in the normal upper gastrointestinal tract. We studied the regional distribution of somatostatin-14, gastrin-I, and cholinergic muscarinic binding sites in membrane preparations from rat gastric corporeal and antral mucosa and in mucosa obtained from the duodenum and jejunum. The corporeal mucosa contained the most high-affinity gastrin binding sites (Bmax = 39.1 +/- 6.5 fmol/mg protein; Kd = 1.1 +/- 0.4 nM). The antral mucosa contained the most somatostatin and cholinergic muscarinic binding sites (Bmax = 65.7 +/- 6.6 fmol/mg protein and 460.3 +/- 101.8 fmol/mg protein, respectively). The duodenal and jejunal mucosal membranes contained somatostatin, gastrin, and cholinergic muscarinic binding sites in decreasing concentrations. Concentrations of binding sites are characteristic for particular gut regions and may help in analyzing their abnormalities.

Muscarinic stimulation of inositol phosphate formation in rat retina: Developmental changes

The stimulation of inositol phosphates by various cholinergic agonists and the effect of antagonists was studied in rat retinal slices of different ages. The potency order of muscarinic agonists and antagonists for receptors linked to inositol phosphate production was the same for retinas of all ages. The maximum stimulation of inositol phosphate production varied between retinas of different ages, being greatest in the younger animals and lowest in the adult. However, no difference was found in the EC 50 values. This contrasts with the neurotransmitters, noradrenaline and serotonin, both known to stimulate phosphoinositide metabolism; neither was found to have the same developmental profile of carbachol-induced stimulation of inositol phosphates. In contrast to the muscarinic receptors linked to phosphoinositide hydrolysis, the density of muscarinic binding sites measured by [ 3H]QNB binding increased in retinas during development. The affinity constants ( K d) remained constant, however. The results are interpreted as suggesting that muscarinic receptors linked to inositol phosphate production are more efficiently coupled (to a guanine regulator protein) in retinas of young animals than in the adult.

Muscarinic and nicotinic synaptic activation of the developing chicken iris

The development of the mechanical characteristics of contraction and the pharmacology of synaptic activation in chick iris and ciliary body were examined from embryonic day 9 through posthatching. The ciliary ganglion-target muscle system has proven to be a useful model for both in vivo and in vitro studies of neuron-target interactions; one such interaction is involved in neuronal cell death, which in the ciliary ganglion occurs from Stage (St) 34 to 40. To understand the mechanism by which cholinergic blocking agents prevent naturally occurring neuronal death in the chick ciliary ganglion (see the following paper, Meriney et al., 1987), it was necessary to determine the effect of these agents on synaptic transmission at target structures during the cell death period. Initially (St 34-36), iris muscle contraction are synaptically mediated via muscarinic ACh receptors (AChRs) on myoepithelial cells, which have the contractile and structural characteristics of smooth muscle. Close apposition of synaptic terminals, similar to that described for mature synapses, was observed on these myoepithelial cells. Subsequently (St 37), the striated muscle fibers that appear are activated by nicotinic receptors, although muscarinic AChRs are also present. Mechanically, this can be seen as gradually changing from a slow-onset contraction, elicited only by 30 Hz stimulation, to a fast-twitch response (St 37-44). Dilator fibers that develop later in the iris (at about St 39) also possess nicotinic and muscarinic receptors. The ciliary body musculature, although not extensively studied, also appears to have dual cholinergic activation during development. The mature iris has predominately striated muscle fibers that have both junctional nicotinic and muscarinic (mostly extrajunctional) AChRs. The dual presence of both receptor types in the same muscle fiber was confirmed with intracellular recordings, in which only the initial portion of the ACh-elicited depolarization was sensitive to alpha bungarotoxin (alpha BTX). In addition, specific muscarinic binding sites were described in the developing, as well as in the mature, iris. The developing chick iris was also shown to contract directly in response to light, this response disappearing after hatching. This unique dual-receptor pharmacology (nicotinic-muscarinic) and light response of a striated muscle may be due to the neural crest origin of these cells.

Quantitative autoradiography of muscarinic and benzodiazepine receptors in the forebrain of the turtle, Pseudemys scripta.

The distribution of muscarinic and benzodiazepine receptors was investigated in the turtle forebrain by the technique of in vitro receptor autoradiography. Muscarinic binding sites were labeled with 1 nM 3H-quinuclidinyl benzilate (3H-QNB), and benzodiazepine sites were demonstrated with the aid of 1 nM 3H-flunitrazepam (3H-FLU). Autoradiograms generated on 3H-Ultrofilm apposed to tissue slices revealed regionally specific distributions of muscarinic and benzodiazepine binding sites that are comparable with those for mammalian brain. Dense benzodiazepine binding was found in the anterior olfactory nucleus, the lateral and dorsal cortices, and the dorsal ventricular ridge (DVR), a structure with no clear mammalian homologue. Muscarinic binding sites were most dense in the striatum, accumbens, DVR, lateral geniculate, and the anterior olfactory nucleus. Cortical binding sites were studied in greater detail by quantitative analysis of autoradiograms generated by using emulsion-coated coverslips. Laminar gradients of binding were observed that were specific for each radioligand; 3H-QNB sites were most dense in the inner molecular layer in all cortical regions, whereas 3H-FLU binding was generally most concentrated in the outer molecular layer and was least dense through all layers in the dorsomedial cortex. Because pyramidal cells are arranged in register in turtle cortex, the laminar patterns of receptor binding may reflect different receptor density gradients along pyramidal cell dendrites.

Differential ontogeny of alpha 1-adrenergic and cholinergic receptor sites in the atria and ventricles of the inbred Dahl hypertension-sensitive (S/JR) and -resistant (R/JR) rat.

The ontogeny of atrial and ventricular alpha 1-adrenergic and muscarinic cholinergic receptor sites was investigated in inbred Dahl hypertension-sensitive (S/JR) and -resistant (R/JR) rats between 5 and 150 days of age. The density of sites in both cardiac regions was generally greater in the neonate than mature rat. A marked proliferation of sites was observed in neonatal and young adult rats that occurred in the following order: ventricular cholinoceptors----ventricular adrenoceptors----atrial cholinoceptors----atrial adrenoceptors. The density of ventricular adrenoceptors was greater in the S/JR rat than the R/JR rat at 5 days of age. At 150 days of age, the density of sites was less in the S/JR rat than the age-matched R/JR rat or the normotensive 50-day-old S/JR rat. The development of atrial adrenoceptors was similar between the strains, regardless of the blood pressure. The density of ventricular cholinergic receptors was greater in the S/JR strain at 5 and 15 days of age. However, the density of atrial cholinergic sites was consistently greater in the S/JR strain throughout development. The results of this study suggest that: (1) significant prenatal receptor development occurs in the heart; (2) receptor development may precede the functional maturation of postganglionic autonomic efferents; and (3) distinguishing differences in the regional density of alpha 1-adrenergic and muscarinic cholinergic binding sites are present between S/JR and R/JR rats at much earlier points in development than previously shown.

Binding of the S(+)- and R(−)-enantiomers of bethanechol to muscarinic receptors in jejunal smooth muscle, nasal mucosa, atrial and ventricular myocardium of the rat

Both the enantiomers of bethanechol displaced R(−)-[ 3H]quinuclidinyl benzilate from muscarinic binding sites in homogenates from rat jejunum, nasal mucosa, atrial and ventricular myocardium. Stereoselectivity of receptor binding for S(+)- and R(−)-bethanecol was found in all tissues investigated (K i S(+) < K i R(−)). Different proportions of high and low affinity binding sites for bethanechol enantiomers and S(+)-methacholine in jejunal smooth muscle, atrial and ventricular muscle and the presence of only low affinity binding sites in nasal mucosa point to differences in receptor-effector coupling in these tissues. The relative insensitivity of cardiac tissue for bethanechol compared to methacholine might be related to the much smaller proportion of high affinity binding sites found for S(+)-bethanechol in heart ventricular muscle.

Quantitative autoradiographic analysis of muscarinic cholinergic and adenosine A 1 binding sites after transient forebrain ischemia in the gerbil

The influence of transient forebrain ischemia on adenosine A 1 and muscarinic cholinergic receptors in the gerbil brain 1–27 days after recirculation was studied. The topographical distribution and the alteration in the adenosine A 1 and muscarinic receptor sites were analyzed by means of quantitative receptor autoradiography using [ 3H]cyclohexyladenosine ([ 3H]CHA) and [ 3H]quinuclidinyl benzilate ([ 3H]QNB), respectively. In most regions examined, the temporal profiles of the alteration of the receptor density were in accordance with the histopathological findings. [ 3H]CHA binding activity decreased suddenly after neuronal damage, while [ 3H]QNB grain density showed a gradual decrease in the dorsolateral caudate-putamen and in the CA 1 subfield of the hippocampus. In the caudate-putamen, [ 3H]CHA and [ 3H]QNB binding activity in the dorsal aspect was markedly reduced 1–27 days after ischemia. [ 3H]CHA binding activity in the ventromedial region of the caudate-putamen also decreased 1–3 days after ischemia, though neuronal damage was restricted to the dorsolateral aspect. Neuronal death in CA 1 was preceded by the decrease in [ 3H]QNB binding activity in the stratum radiatum 1 and 2 days after ischemia. Marked decrease in [ 3H]QNB and [ 3H]CHA binding activity was noted in the CA 1 subfield 3–27 days after recirculation. Three to 27 days after ischemia, the A 1 binding activities in the CA 3 subfield of the hippocampus and int he dentate gyrus were reduced despite the normal appearance of these areas throughout the reperfusion period. Muscarinic binding sites in the CA 3 subfield were also reduced 27 days after ischemia. Despite minimal neuronal damage in the lateral septal nucleus and in the substantia nigra, the A 1 binding activity in these regions was reduced by 70% and 50%, respectively. These results provide further evidence that the muscarinic receptors in the dorsolateral region of the caudate-putamen are localized postsynaptically on small and medium-sized neurons and that those in the CA 1 subfield of the hippocampus are localized on the CA 1 pyramidal cells.

Muscarinic receptors in human SH-SY5Y neuroblastoma cell line: regulation by phorbol ester and retinoic acid-induced differentiation

The specific binding of the muscarinic ligand [ 3H](-)quinuclidinyl benzilate ([ 3H]QNB) to cell membranes of human SH-SY5Y neuroblastoma cells was studied. Saturation isotherms yielded a K d = 0.28 ± 0.06 nM and a B max of 337 ± 47 pmol/g protein. Pirenzepine inhibited [ 3H]QNB binding; inhibition data showed best fit to a 2-site binding model revealing both a high affinity pirenzepine site (34%, K H = 10 nM) and a low affinity site (66%, K L = 1 μM). These results indicate that muscarinic receptors on SH-SY5Y cells may be subclassified as M 1/M 2 subtypes. Morphological and biochemical differentiation of these cells after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA) resulted in a decrease and an increase in the number of muscarinic binding sites, respectively. Furthermore, TPA- and RA-treated cells showed a significant increase in acetylcholinesterase activity compared with non-treated cells. However, only RA-treated cells showed significant increase in choline acetyltransferase activity compared to nontreated cells. These findings demonstrate that TPA and RA can regulate both the number of muscarinic receptors and the acetylcholinesterase activity in human SH-SY5Y neuroblastoma cells.

Cholinergic stimulation of drinking from the lateral hypothalamus: indications for M2 muscarinic receptor mediation.

Available evidence suggests that muscarinic receptor binding sites may exist in at least two heterogenous subclasses (M1 and M2), distinguished by their affinity for the antagonist pirenzepine. In order to evaluate the role of these receptors in comsummatory behaviour a series of conventional and putatively receptor selective drugs were tested for their effects on water consumption following injection (0.5 microliter/30 sec) into the perifornical hypothalamic area of non-deprived rats. Of the conventional agonists tested, carbachol and oxotremorine were approximately equipotent and arecoline was about 16 X weaker. Of the putative M1 agonists tested, pilocarpine was about 50 X weaker than carbachol and the remainder (MCNA343, AHR602, AH6405) were inactive. Inhibition of carbachol (1 microgram) induced drinking was subsequently measured. The most potent inhibition was found using scopolamine, a non selective antagonist. 4-DAMP was approximately 7 X weaker than scopolamine, but was more potent than the putative M1 antagonists pirenzepine, telenzepine or dicyclomine. In a separate series of experiments the affinity of these drugs for [3H]pirenzepine forebrain receptors (M1) and [3H]QNB brainstem receptors (M2) was determined to confirm their receptor binding selectivity. No systematic relationship was found between agonist potency and M1 or M2 affinities. M2 receptor involvement was indicated by the antagonist data which show a close relationship between rank potency order and M2 receptor affinity. An important role for M1 receptors is excluded by the absence of a clear relationship between potency order and M1 affinity. The data therefore suggest an important role for M2 receptors in mediating drinking stimulated by muscarinic receptor activation.

Muscarinic receptors of rat parotid gland enlarged by gland ablation and bulk diet. Effects of denervation

The density of muscarinic binding sites was increased 10% in the rat parotid gland enlarged (2 times control) as a result of ablation of the submandibular-sublingual glands and maintenance of rats on bulk diet (50% inert cellulose plus 50% solid chow) for 4 weeks. When either the parasympathetic or sympathetic innervation to the gland was unilaterally removed at the time of submandibular-sublingual ablation and introduction of the bulk diet, the density of muscarinic receptors showed an even greater increase from levels of innervated glands of chow-fed controls (29%); with removal of both nerves, the increase was 39%. A 36% increase in cyclic guanosine monophosphate levels accompanied the increase in receptors of the enlarged gland, but when the parotid was denervated, there was no change in cyclic GMP. Absence of either or both nerves led to a maximal decrease of 24–29% in density of muscarinic receptors of parotid gland of chow-fed controls, but to no change in cyclic GMP levels. While autonomic influences mediate the changes in density of muscarinic receptors of parotid gland of chow-fed rats, some additional factor is apparently involved in their increase in the enlarged gland.

Cholinergic topography in Alzheimer brains: a comparison with changes in the monoaminergic profile

The topographical distribution of the choline acetyltransferase (ChAT) activity and the number of muscarinic binding sites was studied in the anterio-posterior direction of the caudate nucleus, hippocampus, globus pallidus and putamen of four subjects with senile dementia of Alzheimer type (AD/SDAT) and in the same regions in six controls. Intranuclear gradients were observed both for ChAT and muscarinic receptors. The data obtained were compared with data on noradrenaline (NA) concentration. With longer duration of the disease a decrease was found in the noradrenergic and cholinergic systems. One patient died acutely after a rather short duration of the disease. In this patient no change in ChAT activity was found while the NA concentration was decreased in all brain regions. An upregulation of the number of muscarinic receptors was observed in the caudate nucleus and putamen of all cases, except for the case with the shortest duration of the disease.

Serum protein leakage in aged human brain and inhibition of ligand binding at alpha2‐ adrenergic and cholinergic binding sites

Serum proteins are known to extravasate into the brain parenchyma in senile and presenile dementia (Glenner: Hum. Pathol. 16:433-435, 1986; Wisniewski and Kozlowski: Ann. NY Acad. Sci. 396:119-129, 1982). We have recently demonstrated that human serum Cohn fraction IV (alpha-globulin enriched) inhibits ligand binding at putative dopamine and serotonin2 receptors labeled by [3H]spiroperidol in human brain (Andorn, Pappolla, Fox, Klemens, and Martello: Proc. Natl. Acad. Sci. USA 83:4572-4575, 1986). We now demonstrate that serum proteins can be identified in the neuropil and in neuronal cell bodies in normal aged brain, that alpha-globulin-enriched fractions inhibit ligand binding at alpha 2-adrenergic and muscarinic binding sites in human brain as well, and that serum proteins can be identified within neuronal cytoplasm and axons.

Contrasting effects of testicular and ovarian steroids upon muscarinic binding sites in the brain.

In adult rats, testosterone increases the saturation binding capacity of hypothalamic and amygdaloid membrane fragments for the muscarinic antagonist, N-methyl scopolamine. In contrast, both estradiol and progesterone decrease binding capacity. There are sexual differences in muscarinic binding capacity and these are dependent upon perinatal exposure to androgens.