0270.6474/85/0506-1577$02.00/O The Journal of Neuroscience CopyrIght 0 Smety for Neurosmnce Vol. 5, No. 6, pp. 1577-1582 Prrnted rn U S.A. June 1985 High-affinity Binding of [3H]Acetylcholine to Muscarinic Cholinergic Receptors’ KENNETH J. KELLAR,2 ANDREA M. MARTINO, DONALD P. HALL, Jr., ROCHELLE D. SCHWARTZ,3 AND RICHARD L. TAYLOR Department of Pharmacology, Georgetown University, Schools of Medicine and Dentistry, Washington, DC 20007 Abstract affinities (Birdsall et al., 1978). Evidence for this was obtained using the agonist ligand [3H]oxotremorine-M (Birdsall et al., 1978). High-affinity binding of [3H]acetylcholine to muscarinic Studies of the actions of muscarinic agonists and detailed analy- cholinergic sites in rat CNS and peripheral tissues was meas- ses of binding competition curves between muscarinic agonists and ured in the presence of cytisin, which occupies nicotinic [3H]antagonists have led to the concept of muscarinic receptor cholinergic receptors. The muscarinic sites were character- subtypes (Rattan and Goyal, 1974; Goyal and Rattan, 1978; Birdsall ized with regard to binding kinetics, pharmacology, anatom- et al., 1978). This concept was reinforced by the discovery of the ical distribution, and regulation by guanyl nucleotides. These selective actions and binding properties of the antagonist pirenze- binding sites have characteristics of high-affinity muscarinic pine (Hammer et al., 1980; Hammer and Giachetti, 1982; Watson et cholinergic receptors with a Kd of approximately 30 nM. Most al., 1983; Luthin and Wolfe, 1984). An evolving classification scheme of the muscarinic agonist and antagonist drugs tested have for these muscarinic receptors divides them into M-l and M-2 high affinity for the [3H]acetylcholine binding site, but piren- subtypes (Goyal and Rattan, 1978; for reviews, see Hirschowitz et zepine, an antagonist which is selective for M-l receptors, al., 1984). Although details of the distinctions between M-l and M-2 has relatively low affinity. The ratio of high-affinity [3H]ace- receptor subtypes are still emerging, this classification offers a tylcholine binding sites to total muscarinic binding sites la- convenient framework in which to compare the binding properties beled by [3H]quinuclidinyl benzilate varies from 9 to 90% in of ligands. different tissues, with the highest ratios in the pons, medulla, The M-l subtype has high affinity for pirenzepine and appears to and heart atrium. In the presence of guanyl nucleotides, [3H] be the prevalent muscarinic receptor in brain areas such as cerebral acetylcholine binding is decreased, but the extent of de- cortex, corpus striatum, and hippocampus, as well as in sympathetic crease varies from 40 to 90% in different tissues, with the ganglia. The M-2 subtype has relatively much lower affinity for largest decreases being found in the pons, medulla, cere- pirenzepine and is found in high proportions in brain areas such as bellum, and heart atrium. The results indicate that [3H]ace- pons, medulla, and cerebellum, as well as in heart atrium and the tylcholine binds to high-affinity M-l and M-2 muscarinic re- ileum of the small intestine. In addition, guanyl nucleotides affect the ceptors, and they suggest that most M-2 sites have high binding of agonists at muscarinic receptors, and this effect appears affinity for acetylcholine but that only a small fraction of M-l to be greatest in tissues with a high proportion of M-2 sites. This is sites have such high affinity. consistent with evidence that some muscarinic receptors are cou- pled to adenylate cyclase and inhibit the formation of CAMP (Murad et al., 1962; Brown, 1979). In fact, the differences between M-l and M-2 receptor subtypes could derive largely from the effector mech- Muscarinic cholinergic receptors in brain ,and peripheral tissues anisms to which they are coupled. have been studied extensively using radiolabeled antagonist ligands Attempts to measure muscarinic cholinergic receptor binding sites such as [3H]quinuclidinyl benzilate ([3H]QNB), [3H]N-methylsco- with [3H]acetylcholine ([3H]ACh) have generally met with very limited polamine, and [3H]propylbenzilylcholine (Yamamura and Snyder, success, due to high nonspecific binding, low specific binding, and 1974; Birdsall et al., 1978; Hulme et al., 1978). Muscarinic antago- low specific radioactivity of the available ligand.4 We have circum- nists bind to receptors with Hill coefficients (n,) close to 1; however, vented these technical difficulties by utilizing [3H]ACh of high specific muscarinic agonists compete for [3H]antagonist binding sites with radioactivity and an assay procedure which minimizes nonspecific shallow competition slopes (n,,< 1) which deviate from simple mass binding. Since in brain [3H]ACh also binds to nicotinic cholinergic action predictions for a single class of sites (Birdsall et al., 1978). receptor sites (Schwartz et al., 1982) we have carried out the assay One reasonable explanation for this is that agonists bind to multiple for muscarinic sites in the presence of a saturating concentration of sites or states of muscarinic receptors with varying affinities, while cytisin, a nicotinic agonist. Under conditions of the assay, [3H]ACh most antagonists bind to the different sites or states with equal binds to sites with the characteristics of muscarinic $cholinergic receptor agonist recognition sites in brain and peripheral tissues. The purpose of this study was to characterize the kinetics of binding, Received August 24, 1984; Revised November 8, 1984; pharmacology, anatomical distribution, and regulation of these sites. Accepted November 9, 1984. Materials and Methods ’ We thank Nicholas R. Armstrong for his excellent assistance. This work Synthesis of [3/-/]acety/cho/ine. [3H]ACh (80 Ci/mmol) was synthesized was supported by United States Army Medical Besearch and Development by esterification of [3H]choline (80 Ci/mmol; New England Nuclear) as Command Grant DAMD 17-83-C-31 13 and by the Scottish Rite Schizophrenia Research Program. *To whom correspondence should be addressed. 4 During the preparation of this paper, Gurwitz, Kloog, and Sokolovsky ’ Present address: Laboratory of Molecular Pharmacology, Clinical Neu- (1984) reported the successful use of high specific radioactivity [3H]ACh to roscience Branch, NIMH, NIH, Bethesda, MD 20205. label muscarinic sites in rat brain. 1577 1578 Kellar et al. Vol. 5, No. 6, June 1985 r a. 60,000 - 50,000 - 0.25 0.5 40,000 - 2 Time hid t 30,000 - T l!z..J 0 I I I I I I I I 0 20 40 60 80 Time (min) b I 50 log 150 200 250 300 [Vf]ACh (nM) =-1°hkii5 \ Time (man) --.\ 0.5 I I I b \ IO 20 30 60 Time (mln) 0.4 Figure 2. Rate of association and dissociation of [3H]ACh binding to cerebral cortex. a, The association rate was determined by incubating the tissue as described in the text with 6 nM [3H]ACh for various times before filtration. Specific binding is plotted and is representative of four experiments. 0.3 Inset, pseudo-first order kinetic plot of [3H]ACh specific binding. The asso- ciation rate constant, k,, was determined by the equation h, = (kobs- k-,)/ [3H]ACh concentration. b, The rate of dissociation was determined by initially incubating the tissue with 6 nM [3H]ACh for 60 min and subsequently adding 0.2 1.5 PM atropine. The reaction mixture was filtered at the times shown. Specific binding is plotted and is representative of four experiments. Inset, first order kinetic plot of the dissociation of [3H]ACh specific binding. The 0.1 dissociation rate constant, k,, is given by the slope of the line. Binding assays. Tissues were homogenized with a Brinkmann Polytron in , 1 I I I 50 mM Tris-HCI buffer containing 120 mM NaCI, 5 mM KCI, 1 mM MgClp, and 4 6 12 16 20 2 mM CaC12 (pH 7.4 at 25°C). The tissue homogenates were washed twice Bound(fmol/mg tissue) by centrifugation at 48,000 x g for 10 mm with intermediate homogenization in fresh buffer. The final tissue pellet was resuspended in buffer containing Figure 7. [3H]Acetylcholine binding in rat brain. a, Saturation analysrs of 100 PM diisopropyl fluorophosphate (DFP) to inhibit cholinesterases and 1.5 [3H]ACh binding in rat cerebral cortex. Homogenates of cortex were incu- PM cytisin to occupy nicotinic receptors. Routinely, the binding assays were bated in buffer containing 1 PM cytisin and different concentrations of [3H] initiated by the addition of tissue homogenate (5 to 7 mg of original weight ACh (2 to 300 nM) for 60 min at 25”. Nonspecific binding (0) was determined = 250 to 450 pg of protein) to tubes containing buffer and [3H]ACh. In in the presence of 1.5 PM atropine. Specific binding (0) is the difference pharmacological studies, drugs were dissolved in buffer and added to the between total binding and nonspecific binding. b, Scatchard plot of specific tubes before the tissue. Total volume of the reaction mixture was 500 ~1, and binding of [3H]ACh shown in a. The Kd and B,, were determined by linear all assays were run in triplicate. Nonspecific binding was measured in parallel regression analysis. c, Hill plot of specific binding shown in a. The Hill in the presence of 1.5 PM atropine. The reaction mixtures were incubated at coefficient (na) was determined by linear regression analysis. This experiment 25°C for 60 min and were then filtered under reduced pressure through is representative of 10 separate experiments. Whatman GF/C filters which had been wet with buffer containing 0.05% polyethyleneimine to eliminate displaceable binding to the filters (Schwartz described previously (Schwartz et al., 1982). The procedure yields [3H]ACh et al., 1982). The filters were washed 3 times with 4-ml aliquots of cold of high purity (>98%) that is conveniently and economically repurified or buffer, placed in vials to which Liquiscint scintillation fluid was added, and resynthesized weekly (Schwartz et al., 1982).