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Proc. Nati. Acad. Sci. USA Vol. 88, pp. 4860-4864, June 1991 Neurobiology Mixed nicotinic and muscarinic features of coupled to secretion in bovine chromaffin cells MITCHELL H. SHIRVAN, HARVEY B. POLLARD, AND ELIAHU HELDMAN Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Bernhard Witkop, February 12, 1991 (received for review December 13, 1990)

ABSTRACT evokes release from cultured nists. We conclude -that the cholinergic receptor which is bovine;chromaffin cells by a mechanism that is believed to be coupled to secretion in bovine chromaffin cells possesses classically nicotinic. However, we found that the full musca- mixed nicotinic and muscarinic characteristics. rihic -M (Oxo-M) induced a robust cat- echolamine (CA) secretion. By contrast, , pilo- carpine, , and McN-A-343 did not elicit any secre- MATERIALS AND METHODS tory response. Desensitization of the response to by Preparation ofCultured Chromaffn Cells. Primary cultures Oxo-M and desensitization of the response to Oxo-M by of chromaffin cells were prepared from fresh bovine adrenal nicotine suggest that both nicotine and Oxo-M were acting at medulla as described (5). the same receptor. Additional experiments supporting this Preparation of Chromaffin Cell Membranes. Cells grown in conclusion show that nicotine-induced secretion and Oxo-M- tissue culture dishes were washed twice with standard release induced secretion were similarly blocked by various muscarinic medium (SRM). SRM consisted of 125 mM NaCl, 4.75 mM and nicotinic antagonists. Moreover, secretion induced by KCI, 1.4 mM MgCl2, 2 mM CaC12, 10 mM glucose, and 25 mM nicotine and Oxo-M were Ca2+ dependent, and both Hepes, and the final pH was adjusted to 7.35. After the induced 4Ca2+ uptake. Equilibrium binding studies showed washes, the cells were resuspended in 50 mM phosphate that [3HJOxo-M bound to chromaffin cell membranes with aKd buffer (pH 7.4), homogenized in a glass-on-glass homoge- value of3.08 x 1A- M and a Hill coefficient of 1.00, suggesting nizer in 2-3 ml of the buffer per dish, and frozen at -70TC. one for this ligand. Nicotine inhibited Oxo-M For binding assays the homogenate was thawed, and the binding in a noncompetitive manner, suggesting that both membranes were collected by centrifugation at 30,000 x g for ligands bind at two different sites on the same receptor. We 10 min. In a typical experiment, membranes from 1.5 x 108 propose that the receptor on bovine chromaffin cells that is cells were resuspended in 1 ml of cold phosphate buffer and coupled to secretion represents an unusual cholinergic receptor immediately used for the binding assay. that has both nicotinic and muscarinic features. Binding Assays and Saturation Curves. Approximately 0.1 mg of membrane protein was incubated in a 1-ml volume of Chromaffin cells ofthe adrenal medulla are innervated by the phosphate buffer containing various concentrations (80 pM to cholinergic splanchnic nerve and secrete catecholamine (CA) 60 nM) of either N-[3H]methylscopolamine .([3H]NMS) or in response to acetylcholine and other cholinomimetic com- [3H]Oxo-M. The mixture was incubated for 1 hr at 37°C. For pounds. Chromaffin cells from most species respond to nonspecific binding, 1 mM sulfate or Oxo-M, was muscarinic agonists (1, 2) and in some cases also to nicotinic added to each concentration of [3H]NMS or [3H]Oxo-M, agonists (1-3). However, chromaffin cells from bovine adre- respectively. Membranes were collected and washed exten- nal medulla are known to secrete only in response to nicotinic sively by vacuum filtration over glass filters (Whatman stimulation (1-3), even though they possess functional mus- GF/B), which were presoaked in 0.1% polyethyleneimine, by carinic receptors. Evidence for functional muscarinic recep- using a Brandel cell harvester. Protein analysis was done by tors on bovine chromaffin cells comes from studies showing the method of Bradford with bovine serum albumin as a that stimulation with muscarine evokes an increase in phos- standard (6). Saturation curves were analyzed by three phatidylinositol turnover (1, 4) as well as potentiates secre- curve-fitting programs, ACCUFIT COMPETITION (Lundon tion evoked by nicotine or high K+ (4). Both processes are Software, Chagrin Falls, OH), GRAPHPAD INPLOT (GraphPad sensitive to atropine. Thus, bovine chromaffin cells contain Software, San Diego), and EQUILIBRIUM BINDING DATA functional nicotinic and muscarinic receptors that may work ANALYSIS [EBDA] (G. A. McPherson). All three programs in concert, although the mechanism of their cooperative gave similar analysis values. action remains poorly defined. Displacement of Labeled Ligands by Various Cholinergic To understand better the basis for this cooperativity, we Compounds. The assays were performed in a similar fashion studied the effect of a series of cholinergic ligands on phys- to the experiments determining the saturation curves except iological processes related to secretion. In addition, we also that one concentration of the labeled ligand, 0.22 nM studied the binding properties of these ligands and correlated [3H]NMS or 2.0 nM [3H]Oxo-M, was added to each tube in them with the physiological observations. Our principal the presence of various concentrations (1 pM to 1 mM) ofthe observation is that the muscaninic agonist oxotremorine-M displacing ligand. (Oxo-M) evokes CA secretion in bovine chromaffin cells, Displacement experiments with intact cells were per- similar in extent to that induced by nicotine. Oxo-M also formed in 24-well plates. Cells were washed once with SRM stimulates Ca2+ influx in these cells, although to a lesser and incubated at 37°C for 1 hr with 0.25 nM [3H]NMS in the extent than nicotine. The secretory response to both agonists presence of various concentrations (1 pM to 1 mM) of the were antagonized by both nicotinic and muscaninic antago- Abbreviations: Oxo-M, oxotremorine-M; CA, catecholamine; QNB, The publication costs of this article were defrayed in part by page charge quinuclidinyl benzylate; NMS, N-methylscopolamine; DMPP, 1,1- payment. This article must therefore be hereby marked "advertisement" dimethyl4phenylpiperazinium; aBTX and KBTX, a- and K-bunga- in accordance with 18 U.S.C. §1734 solely to indicate this fact. rotoxin; dTC, d-tubocurarine. 4860 Downloaded by guest on October 3, 2021 Neurobiology: Shirvan et al. Proc. Natl. Acad. Sci. USA 88 (1991) 4861

displacing ligand. The plated cells were then washed five mixed muscarinic/ induced a sim- times with SRM. The cells, still attached to the bottom ofthe ilar extent of secretion only at higher concentrations ranging wells, were removed by two consecutive washes with 0.5 ml from 300 to 500 ,uM (Table 1). On the other hand, the mixed of2.5% Triton X-100 and transferred to a scintillation vial for muscarinic/nicotinic agonist (which bears a tertiary measurements of bound radioactivity. Displacement of la- nitrogen moiety) at concentrations from 500 to 1000 ,M beled ligand in intact cells was performed only with induced only a partial secretory response (Table 1). The [3H]NMS, since the nonspecific binding of [3H]Oxo-M was secretion was about 35% of that induced by the full nicotinic high. Similar results were obtained with intact cells and agonists. Quaternary analogs of this compound such as membrane preparations. arecolone methiodide (9) at 100 AM and isoarecolone me- Secretion of Catecholamines. Plated chromaffin cell cul- thiodide (10) at 50 ,M induced a robust secretion similar to tures were washed with SRM and then incubated in the same that found with nicotine and DMPP. medium for 15 min at 370C. The medium was then removed, Stimulation of secretion was observed not only with nic- and the cells were incubated with SRM containing the tested otinic agonists but also with some muscarinic agonists. For compound for an additional 15 min. Where appropriate, a example, the specific Oxo-M (11-13) was second incubation containing tested compounds was carried found to be a full agonist (Table 1), inducing secretion in a out for an additional 15 min. The supernatants were col- dose-dependent manner (Fig. 1 Top). A significant level of lected, and total CA was determined by lysing the cells in the secretion was observed at a concentration as low as 25 AuM, well with 5% acetic acid and assaying the lysate. For Ca2+- and a maximum secretory response was observed at 300 AtM. dependency studies, cells were washed in Ca2+-free SRM, Nicotine-induced secretion was marginally faster than Oxo- and the reaction was carried out in various concentrations of M-induced secretion (Fig. 1 Middle). Unlike the full agonist Ca2+ (0-2 mM) and tested compounds. CA was measured by Oxo-M, oxotremorine behaved as a weak , the modified trihydroxyindole method (7) with epinephrine as inducing 13% ofthe maximum response obtained with Oxo-M a standard. at 1 mM (Table 1). Also, 1 mM cis- induced a weak Uptake of 4Ca2". Plated cells in 24-well plates were and partial secretory response (about 25% of the maximum washed with 0.5 ml of SRM and then preincubated in this secretion induced by nicotine and Oxo-M) (Table 1). Other medium for 15 min at 370C. SRM containing 45Ca2+ [1 ILCi (37 muscarinic agonists, such as muscarine, , betha- kBq) per well] with or without either nicotine or Oxo-M was nacol, and McN-A-343, did not elicit any significant secretion added to each well. At the appropriate time (5-60 sec), the up to a concentration of 1 mM. cells were washed quickly five times with cold SRM con- Ca2' Dependency of Oxo-M-Induced Secretion. Since nic- taining 2 mM LaCl3 and were lysed as described above in otinic activation of CA secretion in chromaffin cells was 5% acetic acid. The lysate was then assayed for radio- found to be Ca2l dependent (14-16), we tested whether activity. secretion by Oxo-M also displayed this Ca2' dependency. Our results show that, as with nicotine, Ca2+ is also required RESULTS when cells are stimulated to secrete by Oxo-M. However at Ca2+ concentrations up to =0.80 mM, Oxo-M gave a some- Effect of Various Cholinergic Agonists on Catecholamine what higher secretion than nicotine (Fig. 1 Bottom), while at Secretion. Various muscarinic and nicotinic agonists were higher Ca2+ concentrations, Oxo-M and nicotine induced tested for their ability to induce secretion from cultured similar secretion responses. bovine chromaffin cells. This study was consistent with The dependency of Oxo-M- and nicotine-induced secre- previous studies (1, 4, 8) in findings that the specific nicotinic tions on external Ca2+ suggests that cholinergic stimulation agonists nicotine and 1,1-dimethyl-4-phenylpiperazinium by both cholinergic agonists are accompanied by a Ca2+ (DMPP) and the mixed nicotinic/muscarinic agonist carba- influx. Indeed, stimulation of chromaffin cells with either chol behaved as full agonists with respect to the induction of nicotine or Oxo-M resulted in an increase of Ca2+ influx. CA secretion (Table 1). However, not all agonists of the However, nicotine induced a greater degree of Ca2+ influx nicotinic receptor were found to induce secretion with a than did Oxo-M (Fig. 2 Upper). The initial rates of nicotine- similar potency. For example, whereas nicotine and DMPP at and Oxo-M-induced Ca2+ influx were compared with their concentrations greater than 5 ,AM induced significant secre- corresponding release rates and were found to fit a straight tion with a maximum secretory response at 20-40 uM, the line with a correlation coefficient of 0.986 (Fig. 2 Lower). Thus, secretion induced by nicotine and Oxo-M appeared to Table 1. Percent stimulation of CA release by parallel closely the respective receptor-stimulated Ca2+ up- cholinergic agonists take processes. Concentration range, ,LM Desensitization of the Secretory Response by Oxo-M and Nicotine. Nicotinic agonists bind to their receptor in chro- of 40-50 80-100 300-500 1000 Type agonist maffin cells and induce a time-dependent desensitization to a Nicotinic subsequent dose of agonist (17). Inasmuch as Oxo-M has Nicotine 100 much the same effects as nicotine in terms of induction of DMPP 100 secretion and activation of 45Ca2+ influx (vide supra), we Isoarecolone Mel 123 91 questioned whether both nicotine and Oxo-M might be bind- Arecolone Mel 71 88 ing to the same receptor. Our first approach was to test Mixed nicotinic/muscarinic whether nicotine and Oxo-M exhibited reciprocal desensiti- Carbachol 22 47 100 zation ofthe secretory response. As previously reported (18), Arecoline 5 11 35 repeated treatments of cells with nicotine was found to Muscarinic reduce significantly the amount of CA that was released with Oxo-M 41 94 each repeated dose (homologous desensitization) (Fig. 3). We cis-Dioxolane 12 22 25 also found that homologous desensitization occurred with Oxotremorine 2 12 repeated doses of Oxo-M (Fig. 3). We then performed the Release of CA was measured with plated chromaffin cells as tests of heterologous desensitization, with sequential doses described. Results are expressed as the percentage of the maximal of nicotine followed by Oxo-M or sequential doses of Oxo-M stimulated release, normalized to the percent release at 40 ,uM followed by nicotine. Heterologous desensitization was ap- nicotine. Mel, methiodide. proximately half as efficacious as the homologous desensi- Downloaded by guest on October 3, 2021 4862 Neurobiology: Shirvan et al. Proc. Natl. Acad. Sci. USA 88 (1991)

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Time, min 0 0 1000 2000 3000 4000 45Ca2+ uptake, cpm per well FIG. 2. 45Ca2l uptake in plated cells stimulated to secrete by 60 ,uM nicotine and 300 uM Oxo-M. (Upper) Time course of 45Ca2+ uptake. Each point represents the mean ± SEM of three determi- nations. (Lower) Correlation between 45Ca2+ uptake and CA release. concentrations of the muscarinic antagonists quinuclidinyl benzylate (QNB) and NMS (not shown). However, the most potent antagonist was the mecam-

_ * lNicotine:Nicotine FE 100 0 Oxo-M/Oxo-M 0.0 1.0 2.0 LZ Nicotine/Oxo-M Ca2+, mM 0 El Oxo-MINIcotine a) 80 Ei Nicotine'K+. FIG. 1. Stimulation ofCA release from plated chromaffin cells by _ Oxo-MU K+.. C) Oxo-M and nicotine. (Top) Concentration-response curve ofOxo-M. (Middle) Time course of 300 A&M Oxo-M- and 60 ,M nicotine- 660 stimulated release. (Bottom) Ca2l dependence of 300 A&M Oxo-M- and 60 AM nicotine-stimulated release. Each point represents the mean ± SEM of three determinations. 20 tization (Fig. 3). However, since treatment with K+ after C' exposure of chromaffin cells to either nicotine or Oxo-M yielded maximal secretion (Fig. 3), we concluded that nico- tine and Oxo-M shared a common desensitization mechanism that could yet proveto be localized to a common receptor. Effect of Cholinergic Antagonists on Catecholamine Secre- WN O N0 O ON N K *~K tion Induced by Nicotine and Oxo-M. One way to test this First stit-ulus -second stitnUlUlS common receptor hypothesis would be to study the effect of specific nicotinic and muscarinic antagonists on secretion FIG. 3. The effect ofsequential treatments ofOxo-M and nicotine induced by both agonists nicotine and Oxo-M. Secretion on CA release. Cells were first stimulated to secrete by either 40 t&M nicotine (N) or 300 IAM Oxo-M (o), the medium was removed by induced by nicotine and Oxo-M were similarly inhibited by suction, and the cells immediately were stimulated to secrete a the same concentrations of the nicotinic antagonist hexa- second time by nicotine, Oxo-M, or high (50 mM) K+ (K). Results methonium bromide and the atropine were normalized to the percent CA release obtained with the first (Fig. 4). Similar inhibition was also observed with similar stimulus. Data represent the mean ± SEM of three determinations. Downloaded by guest on October 3, 2021 Neurobiology: Shirvan et al. Proc. NatL. Acad. Sci. USA 88 (1991) 4863

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10 30 100 10 30 100 0.1 0.5 1.0 1 5 10 10 10 20 Antagonist, uM FIG. 4. The effect of various antagonists on 60 /AM nicotine- stimulated (hatched bars) and 300,uM Oxo-M-stimulated (solid bars) secretion. Hex, bromide; Atr, atropine; Mec, ; aBtx and kBtx, aBTX and KBTX. ylamine, which inhibited both Oxo-M- and nicotine-induced secretion at concentrations as low as 0.1 AM (Fig. 4). d-Tubocurarine (dTC) was also more effective than either atropine or hexamethonium bromide in blocking secretion induced by both nicotine and Oxo-M (Fig. 4). By comparison, the neural nicotinic antagonist, K- (KBTX), and the antagonist ofthe nicotinic receptors ofthe skeletal muscle, a-bungarotoxin (aBTX), were much less effective than mecamylamine and dTC in blocking both nicotine-induced and Oxo-M-induced secretion. A significant inhibition by these 0 9--' Id,--I . *--n . .--' .i*0--4.. .*--.*- two compounds started to appear only at 10 ,M (Fig. 4). 1010~1O 7 1O-6 RO-5 10-4 1O-3 10- 11 1- Binding Properties of PH]NMS and PH]Oxo-M in Chro- Displacing ligand, M maffin Cell Membranes and Intact Cells. The data, which FIG. 5. Displacement of[3H]NMS from intact chromaffin cells by show similar potencies ofeach antagonist as blockers ofboth various cholinergic ligands. (Upper) Antagonists. (Lower) Agonists. nicotine- and Oxo-M-induced CA secretion, suggest that the Chromaffin cells were incubated for 1 hr at 37°C in multiwell plates nicotinic agonist, nicotine, and the muscarinic agonist, with [3H]NMS in the presence or absence of various concentrations Oxo-M, activate a common receptor. To further test the of cholinergic ligands as described. common-receptor hypothesis, we carried out binding studies with [3H]NMS (a specific muscarinic antagonist) and poorly displaced by the muscarinic antagonist atropine or by [3H]Oxo-M (the actual agonist in question) as labeled ligands. the nicotinic antagonist dTC (Fig. 6). Agonists, on the other [3H]NMS was found to bind to chromaffin cell membranes hand, effectively displaced [3H]Oxo-M (Fig. 6) with the with an apparent Kd value of 3.8 x 10-9 M and with a Hill following order of potency: Oxo-M > oxotremorine > nic- coefficient of 0.999 (data not shown), suggesting a single otine. When we examined the binding parameters of Oxo-M binding site for this compound. The density of these binding in the presence of 100 ,uM nicotine (a concentration of sites, as estimated from the Bm, value, is 1.95 x 10-11 nicotine that partially displaces [3H]Oxo-M from its binding mol/mg of protein. Similar results were also obtained with sites), the Kd was only slightly changed, but the Bmax was intact cells. The muscarinic antagonists QNB and atropine significantly reduced (Fig. 7). These data suggest that Oxo-M effectively displaced [3H]NMS with an IC50 value of about 4 and nicotine do not compete for the same site. We concluded nM. Complete displacements by these antagonists occurred that nicotine and Oxo-M bind two distinct binding sites on the at a concentration of 1 ,uM (Fig. 5 Upper). By contrast to bovine chromaffin cell membrane. Taken together, the phys- muscarinic antagonists, the nicotinic antagonists dTC, hex- iological experiments and the binding results suggest that amethonium bromide, and KBTX at the concentration range Oxo-M and nicotine activate a common receptor to induce used for the muscarinic ligands did not displace [3H]NMS at CA secretion. However, each of these agonists binds to a all. However, cholinergic agonists of both types, muscarinic distinct binding site on the same receptor. In addition, and nicotinic, displaced [3H]NMS, but at much higher concen- chromaffin cells from bovine adrenal medulla contain another trations than the antagonists (Fig. 5 Lower). Thefollowing order type of cholinergic receptor, which resembles in its pharma- of potency was found for the displacement of [3H]NMS by the cological features the classical muscarinic binding site. The agonists: oxotremorine > muscarine 2 Oxo-M > nicotine. latter receptor is most probably not directly associated with The muscarinic agonist [3H]Oxo-M also binds specifically an activation of the secretory mechanism. to a single binding site on chromaffin cell membranes. Oxo-M binding was observed with a Kd value of 3.08 x 10-8 M and a Hill coefficient of 1.00 (not shown). However, in this case, DISCUSSION there were -20 times more binding sites compared with those An unusual cholinergic receptor, having both nicotinic and for [3H]NMS, as the Bma,, value for Oxo-M was 4.35 x 10-1° muscarinic properties, appears to be responsible for the mol/mg of protein. Also unlike [3H]NMS, [3H]Oxo-M was initiation of exocytotic CA secretion from cultured bovine Downloaded by guest on October 3, 2021 4864 Neurobiology: Shirvan et al. Proc. Natl. Acad Sci. USA 88 (1991)

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7 Oxo-M bound, pmol I o'10I o9 108 I 0 106 1 0oS 1 0 4 1 0 3 1 0 2 Displacing ligand, M FIG. 7. Scatchard plot of [3H]Oxo-M in the presence and absence of nicotine. Chromaffin cell membranes were incubated for 1 hr at FIG. 6. Displacement of [3H]Oxo-M from chromaffin cell mem- 37TC with [3H]Oxo-M in the presence or absence of0.1 mM nicotine. branes by various cholinergic ligands. Membrane preparations were For nonspecific binding, 1 mM Oxo-M was present in the incubation. incubated for 1 hr at 37TC with [3H]Oxo-M in the presence or absence of various concentrations of cholinergic ligands as described. receptor on the bovine chromaffin cell appears to express both nicotinic and muscarinic properties. Accordingly, we chromaffin cells. Until the present study, the cholinergic have assigned this receptor the name "M-type nicotinic" receptor that activates secretion had been viewed as "nico- receptor, where "M" stands for mixed and muscarinic char- tinic," since nicotinic agonists such as nicotine and DMPP acter. We also refer to this receptor as the "muscatinic could evoke secretion (1-3). By contrast, the classical mus- receptor." carinic agonist, muscarine, was inactive, except that it could potentiate nicotine-induced secretion by action at a classical, We are grateful to Dr. John Daly for providing the agonists atropine-sensitive muscarinic receptor (4). Furthermore, nic- arecolone methiodide and isoarecolone methiodide. otine-induced secretion was blocked by hexamethonium bro- mide and dTC, which are classical inhibitors of the nicotinic 1. Schneider, A. S. (1987) in Stimulus-Secretion Coupling in Chromaffin Cells, eds. Rosenheck, K. & Lelkes, P. I. (CRC, on as cholinergic receptors skeletal muscle. However, de- Boca Raton, FL), pp. 51-70. scribed here, chromaffin cells could be induced to secrete CA 2. Pollard, H. B., Orenberg, R., Levine, M., Kelner, K., Morita, and to take up concomitantly '5Ca2l in response to not only K., Levine, R., Forsberg, E. J., Brocklehurst, K., Duong, L., traditional nicotinic agonists but also the muscarinic agonist Lelkes, P., Heldman, E. & Youdim, M. (1985) Vitam. Horm. Oxo-M. Furthermore, both nicotine and Oxo-M could de- 42, 109-1%. to a 3. Knight, D. E. & Kesteven, N. T. (1983) Proc. R. Soc. London sensitize the cholinergic receptor of the chromaffin cell Ser. B 218, 177-199. second challenge by either agonist. In addition, we found that 4. Forsberg, E. J., Rojas, E. & Pollard, H. B. (1986) J. Biol. the response to both agonists was inhibited not only by the Chem. 261, 4915-4920. classical nicotinic agonists but also by atropine, QNB, and 5. Pollard, H. B., Burns, A. L., Rojas, E., Schlaepfer, D. D., NMS. This result strongly indicates that both nicotine and Haigler, H. & Brocklehurst, K. (1989) Methods Cell Biol. 31, Oxo-M could effect secretion at a common receptor and that 207-227. this receptor bears mixed nicotinic and muscarinic features. 6. Pollard, H. B. & Weingrad, D. (1976) Anal. Biochem. 76, 382-384. A cholinergic receptor with mixed muscarinic and nicotinic 7. Anton, A. H. & Sayre, D. F. (1962) J. Pharmacol. Exp. Ther. features has been described also in insect neurosecretory 138, 360-375. cells (19). The conclusion that the cholinergic receptor that 8. Frye, R. A. & Holz, R. W. (1984) J. Neurochem. 43, 146-150. activates secretion in chromaffin cells bears muscarinic fea- 9. Spivak, C. E., Waters, J. A. & Aronstam, R. S. (1989) Mol. tures is also supported by findings that another muscarinic Pharmacol. 36, 177-184. compound, McN-A-343 (20), similarly inhibits both nicotine- 10. Waters, J. A., Spivak, C. E., Hermsmeier, M., Yadav, J. S., and Oxo-M-induced secretion (unpublished observations). Liang, R. F. & Gund, T. M. (1988) J. Med. Chem. 31, 545-554. This inhibition by McN-A-343 was not reversed by 1 ,uM 11. Atack, J. R., Wenk, G. L., Wagster, M. V., Kellar, K. J., atropine, a concentration that has been shown in the present Whitehouse, P. J. & Rapoport, S. I. (1989) Brain Res. 483, 367-372. study to completely displace [3H]NMS and hence saturate 12. Raiteri, M., Marchi, M., Paudice, P. & Pittaluga, A. (1990) J. the muscarinic receptors. These results indicate a direct Pharmacol. Exp. Ther. 254, 496-501. action of the muscarinic compound McN-A-343 on this 13. Freedman, S. B., Harley, E. A. & Iversen, L. L. (1988) Br. J. cholinergic receptor. Pharmacol. 93, 437-445. Finally, binding studies on intact cells and isolated mem- 14. Kilpatrick, D. L., Slepetis, R. J., Corcoran, J. J. & Kirshner, branes clearly delineate this unusual cholinergic receptor N. (1982) J. Neurochem. 38, 427-435. from the classical muscarinic receptor also present on the 15. Holz, R. W., Senter, R. A. & Frye, R. A. (1982) J. Neuro- cell. The binding studies described here show that a receptor chem. 39, 635-646. exists on chromaffin cell membranes that binds both nicotine 16. Heldman, E., Levine, M., Raveh, L. & Pollard, H. B. (1989) J. Biol. Chem. 264, 7914-7920. and Oxo-M. Competition studies showed that nicotine was an B. G. Neurochem. 607-617. apparent noncompetitive inhibitor of Oxo-M binding to this 17. Boksa, P. & Livett, (1984) J. 42, 18. Heldman, E., Levine, M., Morita, K. & Pollard, H. B. (1991) receptor. By contrast, the classical muscarinic receptor could Biochim. Biophys. Acta, in press. be detected by the specific ligand [3H]NMS for which Oxo-M 19. Lapied, B., Le Corrone, H. & Hue, B. (1990) Brain Res. 533, and nicotine had a relatively low affinity. Thus, on the basis 132-136. of both physiologic and binding studies, the cholinergic 20. Hammer, R. & Giachetti, A. (1982) Life Sci. 31, 2991-2998. Downloaded by guest on October 3, 2021