Proc. Nat. Acad. Sci. USAVol. 71, No. 5, pp. 1725-1729, May 1974

Muscarinic Cholinergic Binding in Rat Brain(quinuclidinyl benzilate/receptors)

HENRY I. YAMAMURA AND SOLOMON H. SNYDERt

Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, Johns HopkinsUniversity School of Medicine, Baltimore, Maryland 21205

Communicated by Seymour S. Kety, January 28, 1974

ABSTRACT Binding sites with high affinity and spec-ificity for [3Hiquinuclidinyl benzilate (QNB) are presentin homogenates of rat brain. The characteristics of thebinding sites resemble those of muscarinic cholinergicreceptors. Specific binding is saturable with respect to[3H]QNB and tissue concentration and is time-, tempera-ture-, and pH-dependent. The bimolecular rate of asso-ciation (2.0 X 108 M-1 min-) and dissociation (1.2 X 10-2min-) at 350 indicate a dissociation constant of 60 pM anda density of 65 pmol/g of brain. Muscarinic antagonistsand agonists displace specific [3H]QNB binding, while ni-cotinic and non-cholinergic drugs possess little affinity for[3HJQNB-binding sites.

Identification of neurotransmitter receptor sites by chemicalmeasurements of direct binding has been reported for nico-tinic cholinergic receptors of invertebrate electric organs (1),mammalian neuromuscular junction (2) and central nervoussystem (3), and for the glycine receptor in the mammaliancentral nervous system (4). Attempts to study the muscariniccholinergic receptor biochemically have involved measuringthe binding of atropine to the guinea pig intestine (5) andsubcellular fractions of rat brain (6). Recently an alkylatingagent derived from the muscarinic antagonist benzilylcholinehas been employed in the guinea pig intestine (7) and ratbrain (8). 3-Quinuclidinyl benzilate (QNB) has been reportedto be a potent central muscarinic antagonist (9, 10). More-over, in the periphery, QNB has been shown to antagonize theacetylcholine-induced contractile response of the guinea pigileum 50% at 0.01 AoMI (11). Since QNB possesses potency,specificity, and persistence of action, it appears to be a suitableagent for receptor labeling.We now report a simple and sensitive assay for specific

muscarinic cholinergic binding in the rat central nervoussystem using QNB, and describe kinetic properties of thebinding, its regional and subcellular localization, and the rela-tive affinity of cholinergic and noncholinergic drugs.

MATERIALS AND METHODS

QNB was labeled by catalytic tritium exchange at New En-gland Nuclear Corp., Boston, Mass. Fifty milligrams of QNBdissolved in 0.3 ml of glacial acetic acid were mixed with 25mg of platinum catalyst and 10 Ci of 3H20. After stirring 18 hrat 800, labile 3H was removed in vacuo with methanol as asolvent and, after filtration from the solvent, the product wasdissolved in 10 ml of methanol. In our laboratory, the productwas purified by thin-layer chromatography in Silica-Gel F-254plates, 0.25-mm thickness (EM Laboratories, Inc., Elmsford,N.Y.) in 1-butanol: glacial acetic acid: water [4: 1: 1 ] and the

Abbreviation: QNB, 3-quinuclidinyl benzilate.

t To whom reprint requests should be sent at the Department ofPharmacology.

1725

purity determined in three solvent systems (1-butanol: glacialacetic acid: water [4: 1: 1 ]; chloroform: acetone: diethylamine[50:40:10] and chloroform: diethylamine [90:10]). Purified[3H]QNB (90-95%) moved as a single peak with authenticQNB in all three systems. The specific activity of the [3H]-QNB was 1.6-4.0 Ci/mmol as determined by comparison withthe ultraviolet absorption of standard solutions at 258 nm.Male Sprague-Dawley rats (100-150 g) were decapitated

and their brains were rapidly removed. After excision of thecerebella, which were nearly devoid of receptor activity asdetermined in preliminary experiments, each brain was ho-mogenized in 10 volumes of ice-cold 0.32M sucrose in a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle. Thewhole homogenate was centrifuged for 10 min at 1000 X g.The pellet (crude nuclear fraction) was discarded and theresultant supernatant fluid (SI) was homogenized with a Poly-tron (setting no. 5, 60 see) and used for [3H]QNB-bindingstudies.To assay specific binding of [3H]QNB, 25-50 Ml of this

preparation were incubated at 250 with 2 ml of 0.05 M so-dium-potassium (Na-K) phosphate buffer, pH 7.4, containing[3H]QNB. After a 60-min incubation 3 ml of ice-coldNa-K phosphate buffer were added and the contents werepassed through a glass filter (GF/B) positioned over a vac-uum. The filters were washed three times under vacuum with3 ml of ice-cold buffer. Every determination of binding wasperformed in triplicate, together with triplicate samples con-taining unlabeled QNB (0.01 ,AM) or oxotremorine (100 MAM)to determine nonspecific [3H]QNB binding. The filters wereplaced in vials containing 12 ml of Triton X-100:toluene-phosphor, maintained at 250 for 8-12 hr and the radioactivitythen assayed by liquid scintillation spectrometry (PackardTri-Carb models 3375, 3385), at a counting efficiency of 40%.

Choline acetyltransferase (EC 2.3.1.6) and cholinesterase(EC 3.1.1.7) activities were assayed by a modification (12) ofradiometric assays (13). Protein was determined by themethod of Lowry et al. (14) using bovine-serum albumin as astandard.Compounds were obtained as follows: acetylcholine (East-

man Organic Chem, Rochester, N.Y.); acetyl-,3-methylcho-line, physostigmine, (Calbiochem, Los Angeles); isoprop-amide, d-chlorpheniramine (Smith, Kline & French, Phila.,Pa.), atropine, neostigmine (Sigma Chem, St. Louis, Mo.);oxotremorine, scopolamine pilocarpine (Aldrich Chem. Co.,Milwaukee, Wis.); l-chlorpheniramine, l-brompheniramine,(Schering Corp., Bloomfield, N.J.); d-brompheniramine (A. H.Robins Co., Richmond, Va.); carbamylcholine, (Merck, Sharp& Dohme, West Point, Pa.); 3-quinuclidinyl benzilate, andneurotoxin and corticotoxin from the cobra (Naja naja) were

gifts from Edgewood Arsenal, Md.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

021

1726 Physiology: Yamamura and Snyder

/ /

0

I I

6 7

pH

0.6 0.7 0.8

8

FIG. 1. Specific binding of [3H]QNB as a function of concen-

tration of tissue and pH. Bottom: Rat brain homogenates were

incubated with 0.05 M Na-K phosphate buffers (pH 5.0-8.0)and 0.6 ni\I [3H]QNB. After incubation at, 25, for 60 min, specificbinding was determined as described in the text. Top: Variousamounts of tissue were incubated 60 min at 250 in 2.0 ml of 0.05MI Na-K phosphate buffer (pH 7.4) with 0.6 nMl [3H]QNB.

RESULTS

Subcellular Localization of Specific QNB Binding. In typicalexperiments in which 0.5 mg of whole brain protein is incu-bated with 2000 cpm of ['H]QNB, about 400 cpm total bind-ing is observed. In the presence of 0.01 ,M unlabeled QNB or

100 4M oxotremorine, binding is reduced to 50-70 cpm. Es-sentially no binding (<10 cpm) to the filters occurs when tis-sue is omitted from the incubation. Specific ['H ]QNB bindingis defined as the total binding minus the binding in the pres-

ence of 0.01 MIM unlabeled QNB or 100,MM oxotremorine, bothof which give the same values. Under these conditions theratio of specific to nonspecific binding is 8-10:1. About 10-20%of the ['H]QNB in the medium is bound in a specific fashion.To measure the relative amounts of specific ['H ]QNB-

binding activity in various subcellular fractions, homogenatesof the whole brain without the cerebellum have been sub-jected to differential centrifugation (Table 1). Only about 11%of the total binding is recovered in the crude nuclear fraction.The crude mitochondrial fraction, which includes synapto-somes (pinched-off nerve endings) and mitochondria, containsabout 57% of the total binding, while the crude microsomalfraction, which contains a variety of membranes and some

myelin, displays about 35% of total binding. Although thecrude mitochondrial fraction contains the largest total amount

c0

E0.6

EO.S

2E 0.4cm

i 0.3

0

2

D 0.1

0 2 4 6 8 10 12 14[3H]QNB, nM

FIG. 2. Specific binding of [3H]QNB to rat brain homogenatesas a function of the concentration of QNB. Six-tenths milligramof tissue was incubated at 250 for 60 min in 2.0 ml of 0.05 MNa-K phosphate buffer (pH 7.4) with various concentrations of[3H]QNB. Specific binding (a--*) and nonspecific binding(o--o) of QNB to tissue were determined as described in thetext.

of binding, the specific activity of [3H ]QNB binding is greatestin the crude microsomal fraction, whose specific activity isabout 2.5 times that of the crude mitochondrial fraction.Virtually all the binding of the whole homogenate is recoveredin the three particulate subcellular fractions. No direct studieshave been performed to rule out QNB binding in the solublesupernatant fraction. This pattern of subcellular localization,with the greatest total amount of binding in the crude mito-chondrial fraction and the highest specific activity in themicrosomal fraction, is similar to results obtained in this lab-oratory for specific opiate receptor (15) and glycine receptorbinding in the rat central nervous system (4).

Because the crude nuclear fraction displays very little spe-

TABLE 1. Subcellular distribution of specific [3H]QNBbinding in rat brain homogenates

Percentof total

Total specificSpecifically [3H]QNB [3H]QNBbound bound/ binding

[3H]QNB fraction of whole(pmol,/g of (nmol/ homog-

Fraction protein) fraction) enates

Whole homogenate 363 ± 74 218 ± 44Crude nuclear pellet

(1000 X g for 10min) 107 +t 72 25.7 + 1.7 11

Crude mitochondrialpellet (17,500 X gfor 20 min) 525 4- 6 12.6 i 0.2 57

Crude microsomalpellet (100,000 X gfor 60 min) 1284 ± 98 7.7 ± 0.6 35

Tissues were homogenized and subjected to differential cen-trifugation. The various pellets were resuspended in 24 ml of0.05 M sodium-potassium phosphate buffer, pH 7.4, and an ali-quot assayed as described in Methods. The data represent themean of three experiments + SEMI.

0E

C]z

0coza

U-

muC)C.)

U.M

0.44 -

0.35

0.22 _

0.11

I_ L I .-.-.-I0.1 0.2 0.3 0.4 0.5

Protein (mg)

0 0.52

n 0.5cE0

E 0.480zD 0.46mz° 0.44I-0U- 0.42

U,0.ucLCn

0.40

II.

Proc. Nat. Acad. Sci. USA 71 (1974)

- I. .12

3 F

IF

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

021

Proc. Nat. Acad. Sci. USA 71 (1974)

0.6 -

E 0.5 QNB OXOTREMORINE

E

0z00In 0.3-

In

0

0.2I

C,)

10-10 10-9 10-11 10-7 10-1 lo-1 10-4PRUG], M

FIG. 3. Displacement of specific ['H]QNB binding by variousconcentrations of QNB and oxotremorine. Six-tenths milligram oftissue in 20 ml of Na-K phosphate buffer (pH 7.4) was incubatedwith 0.06 nM ['H]QNB and the indicated amounts of QNB oroxotremorine for 60 min at 250. Specific QNB binding was de-termined as described in the text.

cific [3H]QNB binding, we have employed the supernatantfluid from the crude nuclear pellet for routine assays.

Specific [3H]QNB binding is linear between 0.1 and 0.6 mgof tissue protein and displays a fairly broad pH optimum be-tween pH 6 and 8, but with a distinct maximum at pH 7.4(Fig. 1).

Ionic manipulations do not markedly affect specific [3H]-QNB binding. Sodium and potassium concentrations as highas 500 mM and 100 mM, respectively, do not alter specific[3H]QNB binding. Calcium and magnesium have no effect at1 mM but lower binding 20% at 10 mM.

Saturability of [3H]QNB Binding to Rat Brain Homogenates.Specific [3H]QNB binding is saturable with increasing con-centrations (Fig. 2), with half-maximal binding at about 0.4nM. By contrast, nonspecific binding, in the presence of 0.01.uM unlabeled QNB or 100 ,AM oxotremorine, is not saturableand increases linearly with increasing [3H]QNB. [3H]QNBbinding is displaced by nonradioactive QNB with half-maxi-mal displacement at about 0.4-0.5 nM nonradioactive QNBand maximal displacement at 0.01 MM (Fig. 3). The fact thathalf-maximal saturation occurs at similar concentrations ofnonradioactive and radiolabeled QNB indicates that [3H ]QNBis biologically equivalent to the nonradioactive drug in terms ofreceptor binding and confirms the validity of the determinedspecific activity of [3H ]QNB.The same maximal displacement of [3H]QNB binding is ob-

tained with oxotremorine as with QNB (Fig. 3). Half-maximaldisplacement occurs with 0.5-0.8 /M oxotremorine, whilemaximal displacement requires 100 1AM oxotremorine.

Association and Dissociation of Specific [3H]QNB Binding.At 350, specific [3H]QNB binding to whole rat brain homog-enates is half maximal at about 4 min and plateaus by 40 min(Fig. 4). The bimolecular rate constant for the [3H]QNB-receptor association, ki, is 2.0 + 0.2 X 108 M-1 min'. Bycontrast, nonspecific QNB binding is not time-dependent andis only about 12% of the total specific [3H]QNB binding.

S

{0

o 0270

0.3

018a

00

60az 4000coCO 20za.T1006

04

21

I I I I I I I I

10 20 30 40 50 60 70 80MINUTES

gI . .30 60 90

MINUTESFIG. 4. Time dependence of specific (.--*) and nonspecific

(o-o) binding of [3H]QNB and dissociation of the [3H]QNBcomplex. Top: The incubation medium contained 0.6 mg of tissuein 2.0 ml of Na-K phosphate buffer (pH 7.4)and 0.6 nMI [3H] QNB.Specific and nonspecific binding were determined at various timesat 350 as described in the text. Bottom: The incubation mediumcontained 0.6 mg of rat brain tissue in 2.0 ml of Na-K phosphatebuffer (pH 7.4) with 0.6 nM [3H]QNB. After incubation for 60 minat 250, nonradioactive QNB (0.01 IAM) or oxotremorine (100 MM)were rapidly added and samples were either filtered immediately(time 0) or maintained for various intervals at 350 before rapidcooling and filtration.

The rate of dissociation of the QNB-receptor complex hasbeen studied by labeling brain preparations with [3H ]QNB at250 and then measuring the decline of bound [3H]QNB afterfurther incubation at 350 with either 0.01 MM nonradioactiveQNB or 100 MM oxotremorine. (Fig. 4). The rate of dissocia-tion was measured at 350, because only negligible dissociationoccurs at 25° for up to 40 min. When plotted semilogarith-mically, the half-life of the QNB-receptor complex at 350 isabout 60 min. The rate constant for dissociation at 350, k- l,is 1.2 X 10-2 min'. The dissociation constant k-,/k, is 0.06nM.

Effects of Cholinergic and Noncholinergic Drugs on Specific[3H ]QNB Binding. If specific QNB binding reflects an interac-tion with the muscarinic receptor, muscarinic cholinergic drugsshould have significant affinity for the binding site, whilenicotinic and noncholinergic drugs should not. As shown inTable 2, the muscarinic antagonists, scopolamine, isoprop-

0 OXOTREMORINE* ONB

Musearinic Receptor 1727

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

021

1728 Physiology: Yamamura and Snyder

amide, and atropine are the most potent agents of the drugsexamined. All three agents inhibit specific [3H]QNB binding50% at 0.4-2 nM. By contrast, the nicotinic antagonists, d-tubocurarine, pempidine, hexamethonium, mecamylamine,and a neurotoxin from the cobra (Naja naja), fail to alterbinding at 10,uM concentration.

Muscarinic agonists have considerably less affinity thantheir antagonists. Acetylcholine and acetyl-f3-methylcholineincubated together with 5 ujM physostigimine to inhibitacetylcholinesterase have ED50 values (the dosage effectivein displacing 50% of the specific binding) of about 2-4 ,uMand 3-5 MM, respectively. Oxotremorine, whose centralmuscarinic cholinergic potency is greater than that of ace-tylcholine, displays 5 times more affinity for QNB bindingsites than acetylcholine. The muscarinic agonists arecoline,pilocarpine, and carbamylcholine have ED50 values of 10MM, 7 MAM, and 20-30 MM, respectively. Nicotinic agonists,nicotine and dimethylphenylpiperazinium (DMPP) inhibitspecific [3H]QNB binding only minimally (10-20%) at 10,uM. Both (+) and (-) isomers of the antihistamines, chlor-pheniramine and brompheniramine, which display mild mus-carinic anticholinergic pharmacologic effects, inhibit specific[3H]QNB binding about 50% at 30-50 MM concentrations.A variety of drugs without known anticholinergic actions,which fail to alter specific [3H ]QNB binding at 10 uM levelsincludes: A9-tetrahydrocannabinol, corticotoxin, glycine, y-aminobutyric acid (GABA), glutamic acid, aspartic acid,proline, naloxone, levorphanol, dextrorphan, diazepam, andchlordiazepoxide.

Regional Localization of Specific [3H ]QNB Binding. Ifspecific [3H]QNB binding involves the brain's muscariniccholinergic receptor, its regional distribution should parallelthat of muscarinic synapses. We have measured specific [3H ]-QNB binding, choline acetyltransferase and cholinesteraseactivities in nine discrete regions of the rat brain (Table 3).Specific [3H]QNB binding and both enzyme activities arehighest in the corpus striatum, the area of the brain which isrichest in acetylcholine (16). The cerebral cortex, which con-tains almost as much specific [3H]QNB binding as the corpusstriatum, is one of the least enriched brain regions for cholineacetyltransferase and cholinesterase. The least specific [3H]-QNB binding is detected in the cerebellar cortex, which alsocontains the least choline acetyltransferase and cholinesteraseactivities of the brain regions. QNB binding in other areastends to parallel choline acetyltransferase better than cho-linesterase.

DISCUSSIONThe specific binding of [3H ]QNB to homogenates of rat brainhas many characteristics which might be expected of inter-actions with muscarinic cholinergic receptors in the brain.Displacement of [3H ]QNB binding is greatest with muscarinicantagonists and the relative affinity of muscarinic cholinergicagonists tends to parallel their pharmacological potency. Bycontrast nicotinic and noncholinergic drugs have much lessaffinity for these binding sites. The extremely high affinity inthe nanomolar range of muscarinic antagonists for QNB-binding sites is quite similar to their molar affinity for mus-

carinic receptors as demonstrated in smooth muscle. Atropinecan interact with the specific choline uptake system of cho-linergic neurons in the brain but only at concentrations severalorders of magnitude greater than its affinity for QNB-bindingsites (18). Muscarinic antagonists have negligible affinity forcholine acetyltransferase, cholinesterase, or acetylcholine

TABLE 2. Relative potencies of drugs in reducing [3H]QNBbinding to rat brain homogenates

Drug EDW,* (MM)Quinuclidinyl benzilate (QNB) 0.0004-0.0005Scopolamine 0.0008-0.0009Isopropamide 0.001-0.002Atropine 0.001-0.002Oxotremorine 0.5-0.8Pilocarpine 7d-Chlorpheniramine 20-30l-Chlorpheniramine 40-50d-Brompheniramine 20-30l-Brompheniramine 40-50Arecoline 1-3Acetylcholinet 2-4Carbamylcholine 20-30Acetyl (3-methylcholinet 3-5

No effect at 10 juMAspartic acid, y-aminobutyric acid, glutamic acid, proline,

naloxone, methylphenidate, glycine, pempidine, levorphanol,dextrorphan, purified corticotoxin and neurotoxin from the cobra(Naja nraja), hexamethonium, d-turbocurarine, mecamylamine,A9-tetrahydrocannabinol, neostigmine, diazepam, and chlordiaze-poxide.

Values are the mean of data from three separate experimentsperformed in triplicate whose results varied less than l5%.

* Concentration of drug which displaced specific [3H]QNBbinding by 50%.

t Corrections for spontaneous hydrolysis were not possible;however physostigmine (5 jMiM) was added to the incubationmedium to prevent enzymatic (cholinesterase) hydrolysis.

storage sites (19). Thus the selectively high affinity of mus-carinic anticholinergic drugs for muscarinic receptors and forQNB-binding sites strongly favors the identity of the bindingsites with muscarinic cholinergic receptors in the brain.

Subcellular fractionation experiments involving disruptionof synaptosomes indicate an association of QNB binding withsynaptic membrane fractions (20). Recently we have identifiedspecific QNB binding to membrane fractions of the guinea pigileum with kinetic properties and affinities for muscarinicantagonists and agonists very similar to values in the brain(22). In the ileum molar pharmacologic potencies (5) andbinding affinities can be directly compared, providing addi-tional evidence that QNB-binding sites involve muscariniccholinergic receptors and suggesting that the receptor respondsto drugs similarly in the peripheral and central nervous sys-tem. After septal lesions interrupting the septal-hippocampalcholinergic tract, QNB binding in the hippocainpus is un-

altered even though hippocampal choline acetyltransferaseactivity is depleted by 80% (21). This indicates that QNBbinding sites are not located on cholinergic pre-synaptic ter-minals.

In some respects the regional distribution of specific [3H]-QNB binding parallels that of choline acetyltransferase andcholinesterase. All three are highest in the corpus striatum andlowest in the cerebellar cortex. The lesser correlation of some

other brain areas may relate to a variety of factors such as

nicotinic (19), in addition to muscarinic (16), synapses andlocalization of enzymes in cell bodies and noncholinergicstructures as well as at synapses. Very similar regional differ-ences in [3H ]QNB binding, choline acetyltransferase andcholinesterase activities have been observed in 30 areas of therhesus monkey brain (23).

Proc. Nat. Acad. Sci. USA 71 (1974)

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

021

Proc. Nat. Acad. Sci. USA 71 (1974)

TABLE 3. Regional distribution of specific [3IHIQNB binding,choline acetyltransferase (C.A.T.), and cholinesterase (ChE)

activities in rat brain homogenates

C.A.T. ChE[3H]QNB (nmol of (nmol ofspecifically acetylcho- acetylcho-bound line/mg line/mg

(pmol/g of of protein of proteinprotein) per hr) per min)

Corpus striatum 478 ± 82 36.0 153Hypothalamus 131 ± 38 7.9 41.1Midbrain and pons 150 ± 61 32.8 55.5Thalmus 137 i 35 15.3 49.0Medulla oblongata-

cervical spinal cord 5.1 i 5 24.3 37.6Superior and inferior

colliculi 230 4 83 11.9 35).4Cerebral cortex 390 41 23 8.6 28.2Cerebellar cortex 34 i 3 2.3 23.9Hippocampus 243 ± 11 10.8 23.8

Specific [3H]QNB binding was assayed as described in the text.Binding values represent the mean ± SEM of three determina-tions. Choline acetyltransferase (C.A.T.) and cholinesterase(ChE) activities are the average of two determinations, each per-formed in duplicate.

Muscarinic antagonists have 3 to 5 orders of magnitudemore affinity for QNB binding sites than do cholinergic ago-nists. Similarly, strychnine, a glycine receptor antagonist, hasabout 1000 times more affinity for the glycine receptor thandoes glycine itself (4). The affinity of acetylcholine for QNB-binding sites is about the same as that of glycine for the gly-cine receptor and similar to that of acetylcholine for nicotinicreceptor binding sites (19) and norepinephrine (24) and dop-amine (25) for receptor-associated adenylate cyclase prepara-tions.

It can be calculated from the direct measurement of specific[3H]QNB binding at saturation that 1 g of rat brain withoutcerebellum has sufficient receptors to bind 65 pmol of [3H]-QNB. Assuming that each [3H]QNB molecule interacts withone receptor site, we calculate the number of receptor units inone rat brain (1.6 g) to be about 4 X 10's. This value is close tothe calculated density of nicotinic cholinergic receptors in theelectric organ of the electric eel but about '/loth the numberof receptors in the electric organ of Torpedo (1, 19). The den-sity of apparent muscarinic receptor sites in rat brain is similarto the number of opiate receptors (15), and to the number ofglycine receptors in the rat spinal cord (26). The number ofreceptor sites in rat brain we have calculated is the same asthat estimated by Hiley et al. (8) for muscarinic sites in ratcerebral cortex by measuring the binding of a radiolabeledhomologue of benzilylcholine mustard and similar to that re-ported for high affinity binding of [3H1jatropine to whole ratbrain homogenates (6).The binding of [3H]QNB appears closely similar to the

binding of a benzilylcholine mustard homologue (8), and mayresemble [3H ]atropine binding to rat brain homogenates (6).The authors are indebted to Dr. C. A. Broomfield and 1).

Greenberg for their helpful suggestions and kind assistance.This research was supported by USPHS Grants MH-18501 andDA-00266 and grants of the Scottish Rite and John A. HartfordFoundations. S.H.S. is the recipient of a USPHS Research

Muscarinic Receptor 1729

Scientist Development Award, MH-33128. H.I.Y. is the recipi-ent of a USPHS Special Research Fellowship Award, MH-54777.

1. Changeux, J. P., Kasai, M. & Lee, C. -Y. (1970) Proc. Nat.Acad. Sci. USA 67, 1241-1247; Eldefrawi, M. E., Britten,A. G. & Eldefrawi, A. T. (1971) Science 173, 338-340; Klett,R. P., Fulpius, B. W., Cooper, D., Smith, M., Reich, E. &Possani, L. D. (1973) J. Biol. Chem. 248, 6841-6853; &Miledi, R., Mlolinoff, P. & Potter, L. T. (1971) Nature 229,554-557; O'Brien, R. D. & Gilmour, L. P. (1969) Proc.Nat. Acad. Sci. USA 63, 496-503; Raftery, M. A., Schmidt,J., Clark, D. G. & Walcott, R. G. (1971) Biochem. Biophys.Res. Commun. 45, 1622-1629.

2. Berg, D. K., Kelly, R. B., Sargent, P. B., Williamson, P. &Hall, Z. W. (1972) Proc. Nat. Acad. Sci. USA 69, 147-152;Fambrough, D. M. & Hartzell, H. C. (1972) Science 176,189-191; Hartzell, H. C. & Fambrough, D. M. (1972) J.Gen. Physiol. 60, 248-262; Miledi, R. & Potter, L. T. (1971)Nature 233, 599-603.

3. Bosmann, H. B. (1972) J. Biol. Chem. 247, 130-145;DeRobertis, E. (1971) Science 171, 963-971; DeRobertis,E., Fiszer, S., Pasquini, J. M. & Soto, E. F. (1969) J. Neuro-biol. 1, 41-52; Eldefrawi, A. T. & O'Brien, It. D. (1970) J.Neurochem. 17, 1287-1293.

4. Young, A. B. & Snyder, S. H. (1973) Proc. Nat. Acad. Sci.USA 70, 2832-2836.

5. Paton, W. D. M. & Rang, H. P. (1965) Proc. Roy. Soc. Ser.B 163, 1-44; Rang, H. P. (1967) Ann. N.Y. Acad. Sci. 144,756-767.

6. Farrow, J. T. & O'Brien, R. D. (1973) Mol. Pharmacol. 9,33-40.

7. Fewtrell, C. & Rang, H. P. (1971) Brit. J. Pharmacol. 43,417P-418P; Gill, E. W. & Rang, R. P. (1966) Mol. Pharma-col. 2, 284-297.

8. Hiley, C. R., Young, J. M. & Burgen, A. S. V. (1972)Biochem. J. 127, 86P; Burgen, A. S. V., Hiley, C. R. &Young, J. \i. (1974) Brit. J. Pharmacol., in press; Hiley,C. B. & Burgen, A. S. V. (1974) J. Neurochem., in press.

9. Albanus, L. (1970) Acta Pharmacol. Toxicol. 28, 305-326.10. Meyerhoffer, A. (1972) J. Med. Chem. 15, 994-995.11. Becker, M. J. (1964) Report No. DA 18-108-CMI-6631,

liT Research Institute, Chicago, Ill.12. Goldberg, A. M1. & Welch, B. L. (1972) Science 178, 319-

320.13. McCaman, R. E. & Hunt, J. i1\. (1965) J. Neurochem. 12,

253-259; McCaman, M. AM., Tomey, L. R. & McCaman,R. E. (1968) Life Sci. 7, 233-244.

14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall,R. J. (1951) J. Biol. Chem. 193, 265-275.

15. Pert, C. B. & Snyder, S. H. (1973) Science 179, 1011-1014;Pert, C. B. & Snyder, S. H. (1973) Proc. Nat. Acad. Sci.USA 70, 2243-2247.

16. Krnjevic, K. & Phillis, J. W. (1963) J. Physiol. London 166,328-350.

17. Campbell, L. B. & Jenden, D. J. (1970) J. Neurochem. 17,1697-1699; Schmidt, D. E., Speth, R. C., Welsch, F. &Schmidt, M. J. (1972) Brain Res. 38, 377-389; Sethy, V. H.,Roth, R. H., Kuhar, Mi. J. & VanWoert, M. (1973) Neuro-pharmacol. 12, 819-824.

18. Yamamura, H. I. & Snyder, S. H. (1972) Science 178, 626-628; Yamamura, H. I. & Snyder, S. H. (1973) J. Neuro-chem. 21, 1355-1374.

19. O'Brien, R. D., Eldefrawi, M. E. & Eldefrawi, A. T. (1972)Annu. Rev. Pharmacol. 12, 19-34.

20. Yamamura, H. I. & Snyder, S. H., in preparation.21. Yamamura, H. I., Kuhar, M. J. & Snyder, S. H., in prepa-

ration.22. Yamamura, H. I. & Snyder, S. H., in preparation.23. Yamamura, H. I., Kuhar, AI. J., Greenberg, D. & Snyder,

S. H. (1974) Brain Res. 66, 541-546.24. Robison, G. A., Butcher, R. W. & Sutherland, E. W. (1971)

Cyclic AMP (Academic Press, New York).25. Kebabian, J. W., Petzold, G. L. & Greengard, P. (1972)

Proc. Nat. Acad. Sci. USA 69, 2145-2149.26. Young, A. B. & Snyder, S. H., in preparation.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

021