Muscarinic binding sites on bovine pulmonary arterial endothelial cells in culture.

We have investigated the presence and nature of muscarinic binding sites on membranes from cultured bovine pulmonary arterial endothelial cells (BPAE). BPAE were harvested and subcultured nonenzymatically; experiments were performed 3-5 days postconfluence and between 10 and 25 passage numbers. Utilizing radioligand binding techniques with the muscarinic receptor antagonists [3H]3-quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]MS) as probes, we identified a small population of atropine-sensitive muscarinic sites (1,800-2,000 sites/cell or 7-8 fmol/mg protein). Muscarinic binding sites on BPAE membranes resembled classical muscarinic receptors in that (a) the binding of 2 nM [3H]QNB was inhibited by muscarinic agonists and antagonists, (b) [3H]QNB binding was 30 times more sensitive to R(-)- than to S(+)-QNB, (c) binding of the muscarinic receptor agonist carbamylcholine involved high and low affinity components, (d) the stable GTP analog, Gpp(NH)p (100 microM) shifted agonist binding curves to the right by a factor of three, and (e) the high affinity binding of the agonist [3H]oxotremorine-M to muscarinic receptors was depressed by Gpp(NH)p. On the other hand, gallamine, which allosterically regulates muscarinic receptor binding in other tissues, did not affect the rates of dissociation of [3H]QNB, [3H]MS or [3H]oxotremorine-M from BPAE binding sites. We concluded that BPAE in culture exhibit muscarinic binding sites which possess many but not all of the properties associated with classical muscarinic receptors.     

Properties of the muscarinic cholinergic receptors in rat atrium

Summary 1. Properties of the muscarinic cholinergic receptor sites in the rat atrial homogenate and microsomal fraction were studied by the use of tritium labelled 3-quinuclidinyl benzilate ([³H]-QNB), a potent muscarinic antagonist. 2. The specific [³H]-QNB binding to the receptor sites displayed saturability, stereospecificity as well as reversibility. 3. The competition studies showed that muscarinic antagonists were more potent than muscarinic agonists. 4. Certain neuromuscular blocking agents, antipsychotics, antiarrhythmics and antihistamines also were capable of interacting with the [³H]-QNB binding sites. However, - and -adrenergic agents, calcium antagonist (D-600) and ionophore (A-23187) failed to show any effect. 5. Analyses by the double reciprocal plot, Hill plot and Scatchard plot of the dependence of the specific [³H]-QNB binding on the concentration of QNB suggested that binding was occurring to a single population of receptor sites in the atrial homogenate or microsomal fraction. Further, there was no evidence of any detectable site to site interactions (positive or negative cooperative type). From the Scatchard plot, the equilibrium dissociation constant (K_D) of 1.1 nM was calculated and the Hill coefficient was close to 1.0 6. Interaction of the muscarinic antagonists with the [³H]-QNB sites showed the Hill coefficients close to 1.0 whereas for the agonists, the coefficients were much less than 1.0 indicating that agonist-receptor site interactions have some different characteristics from those following antagonist-receptor site interaction. 7. The rate and the maximal level of QNB binding to the receptor sites was markedly influenced by the temperature; various cations, on the other hand, displayed no effect either on the association or dissociation of QNB binding. The specific QNB binding exhibited a broad pH optimum from pH 6.0–8.5. 8. Treatment of the membrane fraction (or homogenate) with either phospholipase A or C and with p-chloromercuribenzoate caused significant inhibition of [³H]-QNB binding. 9. The QNB binding site was resistant to tryptic digestion. Even when about 40% of the membrane proteins were removed by the tryptic proteolysis, the [³H]-QNB binding ability of the membrane remained unaffected; in fact, the removal of tryptic proteolytic products by centrifugation markedly increased the specific QNB binding to the membrane.     

Alterations in muscarinic receptors of ventricular muscle in carbachol-induced short-term desensitization

The inhibitory action of a muscarinic agonist on the contractile response of cardiac muscle is transient due to short-term desensitization of muscarinic cholinergic receptors. Studies were made on the binding of the muscarinic antagonist L-[3H]-quinuclidinyl benzilate ([3H]QNB) to the muscarinic receptor in the membrane fraction of ventricular muscle of guinea pigs desensitized by perfusion with carbachol for 10 min. Desensitization did not change the maximum binding or equilibrium dissociation constant (Kd) of [3H]QNB, but shifted the inhibition curves of [3H]QNB binding by carbachol to the right both in the presence and absence of 5-guanylyl imidodiphosphate (GppNHp). Analysis of these inhibition curves with a multiple site model suggested that superhigh and high affinity agonist binding sites were converted to low affinity sites in the desensitized state. GppNHp has additive effects to the prior exposure to carbachol, suggesting a different site of action from short-term exposure of agonist. We conclude that agonist-induced short-term desensitization of the muscarinic receptor of ventricular muscle is caused by reduction in the affinity of the receptor for agonist without reduction in its amount or affinity for antagonist.     

Muscarinic cholinoceptors in the rabbit's myometrium: a study of the relationship between binding and response

The relationship between [3H](-)-quinuclinidyl benzilate [( 3H](-)QNB) binding and muscarinic cholinoceptors in the myometrium of the oestrogen treated rabbit was studied. [3H](-)QNB binding was specific, saturable and reversible. The ability of muscarinic agonists and antagonists and some of their stereoisomers to inhibit [3H](-)QNB binding and to stimulate or inhibit contraction of the myometrium were almost identical suggesting that [3H](-)QNB binds specifically to the muscarinic cholinoceptor in this tissue. There appeared to be no receptor reserve in this tissue and whilst structural and stereospecific requirements for binding were almost identical to those for the muscarinic cholinoceptor in the guinea pig ileum requirements for activation were not. Competition binding experiments with pirenzepine suggested that antagonist binding sites were heterogeneous whilst those with 4-diphenylacetoxy-N-methyl-piperidine MeBr did not. Results were discussed with regard to simple and complex models of muscarinic cholinoceptor interactions.     

Heterogeneity of binding sites on cardiac muscarinic receptors induced by the neuromuscular blocking agents gallamine and pancuronium

The binding of [3H]quinuclidinyl benzilate ([3H]QNB) to cardiac muscarinic receptors was inhibited not only by classical muscarinic antagonists but also by nicotinic blocking agents and inhibitors of acetylcholinesterase. Gallamine, pancuronium, ambenonium, and decamethonium were the most potent of these agents examined. All of the nicotinic antagonists with significant muscarinic receptor activity had two or three quaternary nitrogens, and the potency of a series of these compounds was a function of the distance between quaternary nitrogens. The effects of gallamine and pancuronium were studied in detail because these neuromuscular blocking agents showed heterogeneity in their binding to cardiac muscarinic receptors, whereas classical muscarinic antagonists such as QNB and atropine did not. Gallamine did not compete for all of the [3H]QNB binding sites on atrial membranes, but left at least 20% of [3H]QNB binding unaffected. Curves of pancuronium competition for [3H]QNB binding were shallow, consistent with two binding sites for pancuronium, with approximately 20% having low affinity. Additionally, in the presence of gallamine or pancuronium, [3H]QNB binding sites were no longer homogeneous, and Scatchard plots became nonlinear. Guanine nucleotides did not alter the effect of gallamine or pancuronium on [3H]QNB binding. Gallamine and pancuronium showed no agonist activity but, like atropine, completely antagonized muscarinic receptor-mediated inhibition of cyclic AMP formation. However, differences in the behavior of gallamine and atropine suggested that gallamine was not a purely competitive antagonist. Gallamine did not protect against receptor alkylation by propylbenzilylcholine mustard, and [3H]QNB dissociation was apparently slowed by gallamine. We interpret our data to suggest that gallamine not only competes for [3H]QNB binding sites, but also binds at a secondary site on the receptor, forming a ternary complex with [3H]QNB. Heterogeneity in ligand binding is proposed to result from the dual actions of gallamine and pancuronium as ligands at both primary and secondary sites on the cardiac muscarinic receptor.     

Guanosine 5'-triphosphate converts some populations of propylbenzilylcholine mustard-sensitive muscarinic cholinoceptor sites to sites resistant to the drug in intestinal smooth muscle.

From functional studies with propylbenzilylcholine mustard (PrBCM), we reported that there coexist PrBCM-sensitive and PrBCM-resistant muscarinic cholinoceptor mechanisms in guinea pig taenia caecum. We investigated the interrelationship between these two cholinoceptor mechanisms using an in vitro receptor binding assay with [3H]quinuclidinyl benzilate (QNB) and [3H]PrBCM. Pretreatment of the muscle strips with 300 nM PrBCM (in vivo alkylation) for 10-50 min resulted in progressive decreases of the number of the maximum [3H]QNB binding sites. However, a prolongation of the period of in vivo alkylation up to 90 min was accompanied with no further loss in the binding sites. Under these conditions, there is no significant change in the affinity of [3H]QNB for the binding sites. The concentration of carbachol required to displace 50% of the bound [3H]QNB was larger in membranes obtained from the tissues that had been alkylated in vivo with PrBCM for 50 min than that from control strips, but was not altered when the pretreatment with the drug was carried out after homogenization (in vitro alkylation). When GTP was added during in vitro alkylation, the affinity of carbachol was lower than that in control membranes, as observed when in vivo alkylation was carried out. In the presence of guanine nucleotide, PrBCM thus appears to recognize two distinct populations or states of muscarinic receptors.     

[3H]-Quinuclidinyl benzilate binding to muscarinic receptors in rat brain: comparison of results from intact brain slices and homogenates.

1. The binding of [3H]-( +/- )-quinuclidinyl benzilate ([3H]-( +/- )-QNB) to muscarinic sites in rat brain slice and homogenate preparations was compared. 2. Evidence is presented in support of the view that only the (-)-enantiomer of QNB binds with high affinity to muscarinic sites. 3. The Kd value for [3H]-(-)-QNB binding in slices was eight times higher than that measured in homogenates. 4. Similarly, the potencies of various muscarinic ligands as inhibitors of [3H]-(-)-QNB binding were consistently lower in slices than in homogenates. 5. It is proposed that the results may reflect differences in the binding properties of muscarinic receptors in intact tissue slice and homogenate preparations.     

The effects of noradrenergic denervation on muscarinic receptors of smooth muscle.

1 Changes in the response to acetylcholine of expansor secundariorum muscles from chicks have been analyzed by pharmacological techniques and by [3-3H)-quinuclidinyl benzilate ([3-3H]-QNB) binding to quantify the muscarinic receptor population. 2 The expansor secundariorum muscle responded to acetylcholine up to the age of 30 days; the response declined thereafter. This developmental decrease in response to acetylcholine was prevented by surgical denervation. 3 In chicks aged less than 25 days, denervation did not affect the sensitivity of the expansor muscle to acetylcholine. In order chicks (above 40 days) denervation gradually restored the sensitivity of the expansor muscle to acetylcholine. Responses of the expansor muscle were always abolished by atropine (1 microM) indicating they were mediated by muscarinic receptors. 4 Binding studies with [3-3H]-QNB showed that changes in response of expansor muscle to acetylcholine were primarily due to changes in the muscarinic receptor population. 5 It is suggested that the noradrenergic innervation of the expansor muscle influences the number of muscarinic receptors expressed in the tissue.     

Regulation of muscarinic receptor subtypes and their responsiveness in rat brain following chronic atropine administration

Chronic administration of l-atropine to rats caused a dose-dependent increase (30%) in the density of muscarinic receptors, as measured with [3H]quinuclidinyl benzilate ([3H]QNB], in cortex (CTX), dorsal hippocampus (DH), and heart but not the corpus striatum. Serum concentrations of l-atropine reached 80 to 160 nM within 6 hr, whereas densities of binding sites for [3H]QNB did not show a significant increase until after the second day of infusion and receptor densities did not reach new steady state levels until after the fourth day of infusion. The density of binding sites for the M1-selective muscarinic receptor antagonist, [3H]pirenzepine ([3H]PZ) was also measured. As noted previously, the density of binding sites for [3H]PZ (defined as M1) was lower than the density of binding sites for [3H]QNB (defined as M1 plus M2). When the densities of binding sites for [3H]QNB and [3H]PZ in CTX plus DH were determined after 14 days of treatment, [3H]QNB binding sites showed a 28% increase, whereas [3H]PZ binding sites did not show any increase. The difference between the densities of binding sites for [3H]QNB and [3H]PZ, an estimate of the density of M2 sites, doubled. The density of binding sites for [3H]QNB appeared to be stable for at least 64 hr after the withdrawal of the drug. The increase in the density of binding sites for [3H]QNB was not reflected in the binding of [3H]oxotremorine-M ([3H]OXO-M), a muscarinic agonist, which was unchanged by l-atropine administration. Because the binding of [3H]OXO-M is sensitive to GTP, this observation suggests that the "induced" receptors may not be coupled to a guanine nucleotide-binding protein. In spite of the fact that there was a doubling of the density of M2 sites, no significant differences in dose-response curves for carbachol-induced inhibition of [3H]cAMP accumulation were observed in slices of CTX plus DH from control and l-atropine-treated rats. Similarity, acetylcholine-stimulated accumulation of [3H]inositol phosphates in slices of CTX plus DH showed no significant differences between the tissues from control and treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of muscarinic receptors of bovine adrenal chromaffin cells: binding, secretion and anti-microtubule drug effects.

1. Binding of [3H]QNB to adrenal membranes is saturable, specific and to a single class of receptors. 2. Tubulozole, and not other microtubule drugs, inhibits [3H]QNB binding. 3. Pretreating cultured chromaffin cells with oxotremorine, a muscarinic receptor agonist, has no effect on either basal, nicotine (10 microM) or K(+)-stimulated catecholamine release and failed to enhance secretion of submaximal concentrations of nicotine (3-5 microM). 4. These results confirm that binding of [3H]QNB is associated with muscarinic receptors on bovine adrenal medullary tissue. 5. These studies also demonstrate that although bovine adrenal chromaffin cells possess muscarinic receptors, these receptors do not appear to be coupled to secretory processes.     

Muscarinic receptors in slice cultures of rat brain

Muscarinic acetylcholine receptors in organotypic slice cultures of hippocampus of the rat, have been examined using the tritiated muscarinic antagonist quinuclidinylbenzilate [( 3H]QNB) as a as a marker. Maximum specific binding of [3H]QNB in mature explants of hippocampus amounted to 316 fmol/mg protein and a dissociation constant (KD) of 185 pM was determined. Scatchard analysis suggested binding to one single binding site. In younger cultures smaller KDs were registered. This decrease in ligand affinity in maturer cultures possibly reflects a decrease in the turnover of acetylcholine. Muscarinic antagonists inhibited the total binding of [3H]QNB significantly, whereas muscarinic agonist, nicotinic antagonists and cholinesterase inhibitors had no influence whatsoever on the total binding of [3H]QNB. The content of muscarinic acetylcholine receptors varied between cultures with explants from different brain areas: hippocampus greater than striatum greater than septum greater than spinal cord greater than cerebellum. These in vitro results are generally in good agreement with results obtained in situ by other investigators and suggest that the binding of [3H]QNB observed in these cultures is indeed correlated to specific muscarinic receptor sites.     

Molecular characterization of muscarinic receptor subtypes in bovine cerebral cortex by radiation inactivation and molecular exclusion h.p.l.c.

Muscarinic receptor subtypes in bovine cerebral cortex were investigated by means of radiation inactivation and molecular exclusion high performance liquid chromatography (h.p.l.c.). The functional molecular size of the muscarinic receptor in situ was determined by the radiation inactivation method. The value for the muscarinic receptor labelled with [3H]-quinuclidinylbenzilate ([3H]-QNB) was 91,000 daltons, while that labelled with [3H]-pirenzepine [( 3H]-PZ) was 157,000 daltons. The muscarinic receptor solubilized with digitonin could be labelled with [3H]-PZ as well as with [3H]-QNB. 3-[(3-Cholamidopropyl)-dimethylammonio] - propane sulphonate (CHAPS) solubilized the muscarinic receptor labelled with [3H]-QNB but not that labelled with [3H]-PZ, in agreement with the low affinity of pirenzepine for inhibiting [3H]-QNB binding in CHAPS-solubilized preparations. The size of the muscarinic receptor in solution was estimated by molecular exclusion h.p.l.c. The digitonin-solubilized muscarinic receptor had a molecular weight of 290,000 and the [3H]-QNB and [3H]-PZ binding activities behaved identically. The CHAPS-solubilized muscarinic receptor labelled with [3H]-QNB was apparently of high molecular weight (greater than 1,000,000 Mr), indicating the formation of aggregates and/or micelles. In the presence of digitonin this form was dissociated into a lower molecular weight species (580,000 Mr). These data indicate that the ligand binding component of the muscarinic receptor species labelled by both [3H]-QNB and [3H]-PZ exists on the same receptor protein, but that the [3H]-PZ binding component in situ is probably coupled to other components in the membrane.     

Correlation between cholinergic histamine release and quinuclidinyl-benzilate ([3H]-QNB) binding in mast cell membranes

Isolated purified rat mast cells release histamine when exposed to acetylcholine according to a different pattern of sensitivity. No correlation was found between the release of histamine evoked by acetylcholine and the high affinity binding of [3H]-quinuclidinyl-benzilate (QNB), a specific cholinergic muscarinic ligand, to rat mast cell membranes, since QNB binding was the same in membrane isolated from cells which were sensitive or insensitive to acetylcholine. In murine neoplastic mast cells, a negative correlation was found between histamine release and [3H]-QNB binding, as no evidence of specific [3H]-QNB binding was present in murine neoplastic mast cell membranes which, accordingly, do not release histamine when exposed to acetylcholine. It is concluded that murine neoplastic mast cells are not provided with muscarinic cholinergic receptors. In rat mast cells, muscarinic cholinergic receptors are always present, but not always coupled with the effector mechanisms triggering the exocytosis.     

Peripheral blood lymphocyte muscarinic cholinergic receptors in airway hyperresponsiveness: a marker of cholinergic dysfunction?

1 Muscarinic cholinergic receptors were assayed in human peripheral blood lymphocytes of healthy control and airway hyperresponsive subjects using a radioligand binding assay technique and the muscarinic cholinergic receptor antagonist [³H]-quinuclidinyl benzilate (QNB) as a radioligand. Subjects investigated were divided in four different groups based on threshold responses to methacholine inhaled as challenge test. 2 [³H]-QNB was bound to human peripheral blood lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. Dissociation constant (K_d) values of [³H]-QNB binding were similar in the different groups examined, whereas maximum density of binding sites (B_max) was increased in airway hyperresponsive subjects in comparison with healthy controls. 3 The above findings indicate that the density of muscarinic cholinergic receptors is increased in peripheral blood lymphocytes of airway hyperresponsive subjects. 4 This suggests that airway hyperresponsiveness is associated with cholinergic hyperreactivity and is probably a systemic cholinergic dysfunction since it is accompanied by changes in the density of muscarinic cholinergic receptors expressed by peripheral blood lymphocytes.     

Sialic acid residues as catalysts for M2-muscarinic agonist-receptor interactions.

The role of sialic acid residues in the interactions of muscarinic agonists with the cardiac M2 muscarinic receptor was investigated by competitive binding experiments using the lipophilic radioligand (-)-[benzilic-4,4-3H]quinuclidinyl benzilate ([3H]QNB) and the hydrophilic ligand [N-methyl-3H]scopolamine methyl chloride ([3H]NMS). Direct labeling of the agonist binding sites was performed with the radiolabeled agonist [methyl-3H]oxotremorine M acetate ([3H]oxo-M). Neuraminidase decreased the affinity of the M2-selective agonist carbamylcholine in competitive binding experiments performed with [3H]QNB and [3H]NMS. The binding of the M1-selective agonist (4hydroxy-2-butynyl)trimethylammonium chloride m-chlorocarbanilate (McN-A-343), of the M1-selective antagonist pirenzepine, and of the M2-selective antagonist 11-([2-[(diethylamino)methyl]-1 piperidinyl]acetyl)-5,11-dihydro-6H-pyrido(2,3b)(1,4)benzodiazepin -6-on (AF-DX-116) were not affected by neuraminidase. Neuraminidase did not modify the binding parameters of 3H-antagonists but reduced the number of agonist binding sites revealed by [3H]oxo-M. The removal of sialic acid decreased the half-life of the receptor-agonist complex. The present results suggest that removal of sialic acid reduces the formation of super-high affinity agonist-receptor complexes. Sialic acid may catalyze macroscopic binding by enhancing accumulation of the agonist at the membrane surface.     

Muscarinic receptors of the pancreas: a correlation between displacement of (3 H)-quinuclidinyl benzilate binding and amylase secretion.

(3H)-quinuclidinyl benzilate [(3H)-QNB], A potent muscarinic antagonist, was used as a tool to reveal the presence of muscarinic receptors in rat pancreas. Binding assays were performed on a 49,000 g pellet. Known muscarinic antagonists and agonists were used for competition assays on (3N)-QNB binding and studies on amylase secretion in vitro. A correlation was established between the binding assays and the studies on amylase release which suggests that the binding sites and those involved in the cholinergic control of pancreatic enzyme secretion are similar.     

Relations between muscimol, quinuclidinyl benzilate and nicotine binding sites in brain after very long treatment with ethanol in rats

Rats were treated with ethanol in the drinking fluid 2 X 1 h daily for 83 weeks. [3H]Muscimol, [3H]quinuclidinyl benzilate ( [3H]QNB) and [3H]nicotine binding was measured in selected brain areas 7, 14 and 21 days after withdrawal of ethanol. A significant increase (P less than 0.05) when compared to controls was found on day 7 in both [3H]-QNB binding in the cortex and high affinity [3H]muscimol binding in the cerebellum. Furthermore, on day 7 of abstinence there was a positive correlation in number of binding sites between [3H]muscimol (high affinity sites; cerebellum) and [3H]QNB (cortex) while a negative correlation was found between [3H]muscimol (low affinity sites; cerebellum) and [3H]nicotine (cortex). The first correlation might indicate a relation between increased excitation (QNB) and increased inhibition (muscimol) in two brain areas.     

Muscarinic receptors of the vascular bed: radioligand binding studies on bovine splenic veins

Despite an obvious lack of parasympathetic innervation to the spleen, pharmacological evidence suggests the presence of cholinergic receptors in isolated bovine splenic veins. We therefore studied muscarinic cholinergic binding sites in a bovine splenic vein preparation by direct radioligand binding techniques using [3H]quinuclidinyl benzilate ([3H]QNB) as radioactive probe. Saturation experiments indicated one homogeneous class of high-affinity binding sites, with a KD of 0.11 nM and a binding site density Bmax of 55 fmol/mg protein. The rate constants at 37 degrees C for formation and dissociation of the [3H]QNB receptor complex were 2.7 X 10(9) M-1 h-1 and 0.38 h-1, respectively, yielding a KD of 0.14 nM. The binding sites showed a high stereospecificity, which was evident from competition experiments with dexetimide (KI = 1.3 nM) and levetimide (KI = 4.6 microM). In competition experiments with muscarinic and nicotinic antagonists and some antidepressants, only one binding site was found, whereas with muscarinic agonists, two binding sites were detected. In the presence of 0.1 mM guanyl-imido-diphosphate, only one binding site could be identified with the muscarinic agonist carbamylcholine. The affinity of [3H]QNB, on the other hand, was slightly decreased, and Bmax values were unchanged. It is concluded that specific, saturable, high-affinity muscarinic binding sites in the bovine splenic vein have been identified and characterized that exhibit properties similar to cholinergic receptors of brain and peripheral tissues and probably mediate acetylcholine-induced relaxation of splenic veins.     

Chronic scopolamine treatment and brain cholinergic function

Scopolamine was either continuously infused or injected once daily into C3H mice. Chronic infusion resulted in mice that were supersensitive to the hypothermia and tremor produced by the muscarinic agonist, oxotremorine. Chronic scopolamine infusion did not alter brain acetylcholinesterase (AChE) or choline acetyltransferase (ChAT) activities but it did produce an increase in brain muscarinic receptors, as measured by quinuclidinyl benzilate (QNB) binding. The maximal increase in QNB binding was seen at the 0.2 mg/kg/hr dose. Further increase in dose resulted in a return to control QNB binding in all brain regions studied except cortex. These animals were still supersensitive to oxotremorine, suggesting a dissociation between receptor number and response to agonist. Animals injected once daily for 10 days with 5 mg/kg exhibited an increase in QNB binding while no increase was seen at 20 mg/kg/day. Chronic oxotremorine infusion resulted in tolerance to the hypothermia-producing effects of oxotremorine. This was accompanied by a decrease in brain QNB binding. Coinfusion of scopolamine with oxotremorine blocked both the tolerance development and receptor changes. These experiments demonstrate that chronic scopolamine treatment can elicit an increase in brain muscarinic receptors which is accompanied by supersensitivity to agonists. However, this effect is not clearly dose related, and a strict relationship between receptor number and agonist response does not exist.