Proc. Nat. Acad. Sci. USA Vol. 69, No. 11, pp. 3287-3291, November 1972

Role of Muscarinic Receptors in Regulation of Guanosine 3':5'-Cyclic Monophosphate Content in Mammalian Brain, Heart Muscle, and Intestinal Smooth Muscle (cyclic AMP/nicotine receptors/)

TEE-PING LEE, J. F. KUO, AND PAUL GREENGARD* Department of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, Connecticut 06510 Communicated by Lewis Thomas, August 22, 1972

ABSTRACT Effects of and of agents that Recent studies indicate that acetylcholine can cause the mimic or block its physiological actions have been studied upon concentrations of guanosine 3':5'-cyclic monophos- accumulation of cyclic GMP in heart (10, 11), brain (11, 12), phate (cyclic GMP) and adenosine 3':5'-cyclic monophos- and ductus deferens (13). Perfusion of isolated rat heart with phate (cyclic AMP) in slices of mammalian cerebral cortex, acetylcholine caused an increase of up to 140% in cyclic heart ventricle, and ileum. Acetylcholine, and cholino- GMP content (10). Moreover, application of low concentra- mimetic agents with a predominantly muscarinic action, tions of acetylcholine to slices of rat heart caused increases of such as , bethanechol, and , induced an increase in the concentration of cyclic GMP, up to 10-fold in the concentration of cyclic GMP (11). The accompanied by no change or a slight decrease in the con- amount of cyclic GMP in brains of rats was increased up to centration of cyclic AMP, in all three tissues studied. 80% (12) by the injection of , an agent known to Tetramethylammonium, a cholinomimetic agent with a affect cholinergic mechanisms in the nervous system. The predominantly nicotinic action, on the other hand, did to slices of rabbit cerebellum or not significantly alter concentrations of either cyclic application of acetylcholine GMP or cyclic AMP. The increase in the tissue content of cerebral cortex increased the cyclic GMP concentration by cyclic GMP induced by acetylcholine and its muscarinic up to 3.5-fold (11). analogs was antagonized by atropine, a muscarinic block- There are two classes of acetylcholine receptors, designated ing agent, but not by hexamethonium, a nicotinic block- muscarinic and nicotinic. These two classes of receptors have ing agent. These and other results suggest the generalization that distinct anatomical distributions and physiological functions. the interaction of acetylcholine with muscarinic recep- They can be distinguished from one another by drugs that tors, but not with nicotinic receptors, causes an increase in either mimic or antagonize the various actions of acetyl- the concentration of cyclic GMP. The data are compatible . It seemed of considerable importance to determine with the hypothesis that cyclic GMP may mediate the on concentrations of muscarinic actions of acetyicholine. whether the effects of acetylcholine the The antagonistic actions of cholinergic and adrenergic cyclic nucleotides are characteristic of one or the other or both agents on the physiology of contraction of intestinal types of . In the present study, the effect smooth muscle and of cardiac muscle are reflected in, and of acetylcholine, and of related agonists and antagonists, on may result from, antagonistic actions of these compounds concentrations of cyclic GMP and cyclic AMP has been on the levels of both cyclic GMP and cyclic AMP in these tissues. studied in several types of mammalian tissue. The data indicate that the increase in the amount of cyclic GMP brought about A substantial amount of evidence now exists indicating that by acetylcholine is attributable to its interaction with mus- several hormones and neurotransmitter agents may exert carinic, rather than with nicotinic, receptors in these tissues. their biological actions by altering the amount of adenosine 3':5'- monophosphate (cyclic AMP) in target cells. In recent MATERIALS AND METHODS years, the possibility has been raised that guanosine 3':5'-cyclic Cyclic GMP-dependent protein kinase from pupal fat body monophosphate (cyclic GMP) may also play a significant role from cecropia silkmoth (8) and cyclic AMP-dependent pro- in the regulation of metabolism and function in animal tissues. tein kinase from bovine heart (14) were purified through the Cyclic GMP has been found in tissues and fluids of several step of ammonium sulfate precipitation and DEAE-cellulose species (e.g., 1-4), and enzymes have been found that catalyze column chromatography, respectively. The fat body from the synthesis (4, 5) and breakdown (6) of cyclic GMP. In silkmoth pupae was generously provided by Dr. G. R. Wyatt addition, a family of protein kinases has been found that is of the Department of Biology. Acetylcholine, bethanechol, specifically activated by cyclic GMP (7-9), rather than by methacholine, pilocarpine, tetramethylammonium, atropine, cyclic AMP, and it has been proposed (7) that the biological hexamethonium, tubocurarine, and isoproterenol were pur- effects of cyclic GMP are mediated through this family of chased from Sigma. Other materials used in the present study protein kinases (7-9). Nevertheless, little is known about the were the same as described in ref. 11. factors that regulate the concentration of cyclic GMP in tissues or about the nature of the biological effects that may Preparation and Incubation of Tissue Slices. Preparation be regulated by cyclic GMP. and incubation of slices of rabbit cerebral cortex (11) and of rat-heart ventricle (11, 15) were as described. Tissues were * To whom correspondence should be addressed. incubated for periods of time found to cause maximal increases 3287 Downloaded by guest on September 26, 2021 3288 Cell Biology: Lee et al. Proc. Nat. Acad. Sci. USA 69 (1972)

in cyclic GMP content. These periods were 3 min for slices of TABLE 2. Effect of cholinomimetic agents and of cholinergic cerebral cortex (11), and 30 sec for slices of heart ventricle blocking agents. present singly or in combination, on the amount (unpublished experiments). of cyclic GMP in rat-heart ventricular slices For the study of intestinal smooth muscle, three male guinea pigs, weighing about 350 g each, were killed by decapi- Cyclic GMP tation; their ilea were quickly dissected and washed with Addition (pmol/mg protein) Tyrode's solution. In a few experiments, the washed ilea were Experiment 1 mounted on a pipette and the longitudinal smooth muscle None (control) 0.60 4 0.09 layer was removed and further prepared as described below. Acetylcholine, 1 MM 1.86 ± 0.54* In most experiments, however, the entire muscularis, includ- Pilocarpine, 1 MM 1.50 ± 0.54t ing longitudinal as well as circular muscle layers, was used, Atropine, 1 AM 0.78 ± 0.03t and was prepared as follows: washed ilea were turned inside Acetylcholine, 1 MAM, plus 1 0.72 :1: out, washed twice more with 100 ml of Tyrode's solution, and Atropine, MM 0.15§ Pilocarpine, 1 MM, plus scraped with a spatula to remove the mucosal layer. With Atropine, 1 MM 0.90 ± 0.24§ either preparation, the tissue was cut into 0.5 mm X 0.5 mm Experiment 2 pieces with a McIlwain tissue chopper. Ileal pieces were sus- None (control) 0. 62 :1 0. 05 pended in 50 ml of Tyrode's solution and collected by centri- Tetramethylammonium, 1 MM 0.57 ± 0.031 fugation at 1000 X g for 30 sec. Washing was repeated two 100 MM 0.67 ± 0. 151 Experiment 3 :1 17 TABLE 1. Effect of cholinomimetic agents and of cholinergic None (control) 0. 62 0. Bethanechol, 1 MM 1.24 :1 0.03* blocking agents, present singly or in combination, on amount of cyclic GMP in slices of rabbit cerebral cortex Tubocurarine, 10MAM 0.59 i 0. 16$ Bethanechol, 1 MAM, plus Tubocurarine, 10MM 1.05 :1 0.19, Cyclic GMP Addition (pmol/mg protein) Ventricular slices were incubated for 30 see in the presence of the Experiment 1 indicated agents. Each value represents the mean ± SEM of None (control) 0.89 0.18 assays on three tissue samples, each of which was analyzed in Acetylcholine, 1 AM 1.77 4 0.54* triplicate. Bethanechol, 1 AM 1.67 4 0. 65t * Significantly different from the control (P < 0.01). Tetramethylammonium, 1 AM 0.72 4 0. 111 t Significantly different from the control (P < 0.05). 10M M 1.02 4 0.17 t Not significantly different from the control. 100MM 1.06±0.171 § Significantly different from acetylcholine or pilocarpine Experiment 2 alone (P < 0.01). None (control) 0.53 0.03 ¶ Not significantly different from bethanechol alone. Atropine, 1 MM 0.43 0.05 Methacholine, 1 AM 0.93 0.03t more times, and the tissue slices were finally suspended in Methacholine, 1 uM, plus Tyrode's solution that had been warmed to 37°. Aliquots Atropine, 1 AM 0.44 -- 0.08§ (2.5 ml) of the tissue suspension, containing about 30 mg of Pilocarpine, 1 MM 1.10 i 0. 25* protein, were transferred directly into glass homogenizers Pilocarpine, 1 MM, plus (12 ml capacity) and incubated in Tyrode's solution at 370, Atropine, 1 AM 0. 67 -- 0.21 for various times, in the presence of various agents, as in- Experiment 3 dicated in each table. The test agents were added in a volume None (control) 0.78 0.16 of 0.15 ml of 1 Bethanechol, 1 MM 2.26 -- 0.20* of 10 MA. Reaction was stopped by addition 00% Atropine, 1 MM 0.99 i 0.14t trichloroacetic acid to the incubation mixture. Tissue samples Bethanechol, 1 MM, plus were immediately homogenized and the precipitates were Atropine, 1 MM 0.87 ±- 0.1611 removed by centrifugation. The supernatant fluids (tissue Hexamethonium, 1 MM 0.70 -- 0.14t extracts) were adjusted to pH 7 with 2 M Tris base, and used Bethanechol, 1 MM, plus for analysis of cyclic nucleotides. Hexamethonium, 1 MM 2.48 0. 39** Protein content of the precipitates was determined by the Hexamethonium, 100 MM 0.85 -- 0.08t method of Lowry et al. (16). Significance of differences was Bethanechol, 1 MM, plus determined by Student's t-test. Hexamethonium, 100 MM 1.77 -- 0.31** Measurement of Concentrations of Cyclic GMP and Cyclic of GMP Slices of rabbit cerebral cortex were incubated for 3 min in the AMP in Tissues. Separation and purification cyclic presence of the indicated agents. Each value represents the mean and cyclic AMP from the trichloroacetic acid extracts of SEM of assays on three tissue samples, each of which was tissues (11, 17), assay for cyclic GMP (11) with cyclic GMP- analyzed in triplicate. dependent protein kinase from silkmoth fat body, and assay * Significantly different from the control (P < 0.01). for cyclic AMP (17) with cyclic AMP-dependent protein t Significantly different from the control (P < 0.05). kinase from bovine heart were as described. t Not significantly different from the control. RESULTS § Significantly different from methacholine alone (P < 0.01). Significantly different from pilocarpine alone (P < 0.05). Rabbit cerebral cortex Significantly different from bethanechol alone (P < 0.01). An increase in the concentration of cyclic GMP in slices of ** Not significantly different from bethanechol alone. rabbit cerebral cortex, upon incubation in the presence of Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Cholinergic Regulation of Cyclic GMP 3289 TABLE 3. Effects of acetylcholine, isoproterenol, and atropine, of this smooth muscle: several seconds were required, after present singly or in combination, on the amount of cyclic AMP application of acetylcholine, until the contraction of this in rat-heart ventricular slices smooth muscle could be observed, and about 30 sec was re- quired for maximal contraction. Cyclic AMP The effect of various concentrations of acetylcholine and of Minus Plus the , bethanechol, on cyclic GMP content atropine atropine, 1 gM in the ileum are shown in Table 4. The greater apparent Addition (pmol/mg protein) effect of bethanechol than of acetylcholine, at the lowest concentration tested, may be attributable to the fact that 6. 1 4 0.2 7.0 400.4 None (control) resistant to Acetylcholine, 1 1uM 5.8 :L 0. 1 5.7 41 0.6 bethanechol is hydrolysis by acetylcholinesterase. effect of tetramethylammo- Isoproterenol, 1,gM 25.4 4 0.7* 25.2 -4- 0.9* In contrast to the bethanechol, Isoproterenol, 1 IAM, plus nium did not increase cyclic GMP content of ileal muscula- Acetylcholine, 1 ,gM 17. 8 4 1. 3t 25.8 ± 1.7t ture (Table 4). As in the case of the experiments with brain and heart, the increase in the concentration of cyclic GMP Conditions were the same as described in Table 2, except for in guinea-pig ileum, brought about by acetylcholine or the the indicated additions. muscarinic agonists, was prevented by atropine but not by * Isoproterenol significantly increased the amount of cyclic hexamethonium (Table 4). , an analog of acetyl- AMP in the absence or presence of atropine (P < 0.01). choline possessing both nicotinic and muscarinic properties, t Significantly different from isoproterenol in the absence of has also been found to increase the cyclic GMP content of atropine (P < 0.01). isolated muscle from guinea-pig ileum (G. Schultz, L. Hur- t Not significantly different from isoproterenol in the presence witz, J. G. Hardman, and E. W. Sutherland, personal com- of atropine. munication). acetylcholine, is shown in Table 1. This increase in the cyclic Acetylcholine and other muscarinic agonists cause the con- GMP content of cerebral cortical slices was also observed in traction of intestinal smooth muscle; in contrast, isoproterenol the presence of each of the muscarinic agonists tested, namely, and related 0-adrenergic agonists induce relaxation of these bethanechol, methacholine, and pilocarpine. In contrast, the muscles. It is of considerable interest, in view of this physi- tetramethylammonium did not cause a ological antagonism, that an antagonism between the actions significant increase in the concentration of cyclic GMP. The of bethanechol and isoproterenol was also observed both on increase in cyclic GMP content observed in the presence of cyclic GMP level and on cyclic AMP level of the ileal muscula- the muscarinic agonists was abolished by low concentrations ture (Tables 5 and 6). The increase in cyclic GMP brought of atropine, the , but not by even high about by bethanechol could be prevented by the inclusion of concentrations of hexamethonium, the isoproterenol in the incubation medium (Table 5). Isopro- (Table 1). No significant change in cyclic AMP content was terenol also lowered the endogenous concentration of cyclic observed with any of the agonists or antagonists tested. GMP slightly, but this effect was not statistically significant. Conversely, isoproterenol caused an increase in the concentra- Rat-heart ventricle tion of cyclic AMP in the' guinea-pig ileum that could be As in the case of the studies on cerebral cortex, we found that partially prevented by bethanechol (Table 6). This action of acetylcholine (Table 2) and the muscarinic agonists, namely bethanechol could be blocked by a low concentration of pilocarpine, bethanechol (Table 2), and methacholine, each atropine, indicating that the effect of bethanechol on cyclic increased the concentration of cyclic GMP in slices of rat- heart ventricle, whereas the nicotinic agonist tetramethyl- ammonium was ineffective. A low concentration of atropine prevented the increase in concentration of cyclic GMP *13 brought about by acetylcholine or the muscarinic agonists, whereas hexamethonium and tubocurarine, the two nicotinic antagonists tested, were not effective in this regard. Little or E no change was observed in the concentration of cyclic AMP 0~ in heart slices in the experiments of Table 2. .9~ Acetylcholine has been found to diminish the increase in C) the concentration of cyclic AMP brought about by isopro- 0~-J terenol in heart slices (15). In the present study, we showed 0 that the ability of acetylcholine to lower cyclic AMP content, GMP content, was blocked by a like its ability to raise cyclic 120 low concentration of atropine (Table 3). TIME OF INCUBATION (sec) Guinea-pig ileum FIG. 1. Effect of acetylcholine on cyclic GMP content in of incuba- in of acetylcholine, for muscularis slices from guinea-pig ileum as a function The effect of incubation the presence tion time. Muscularis slices from guinea-pig ileum were in- various periods of time, on the cyclic GMP content of the cubated with acetylcholine (1 IAM) for various times, as indicated. ileal muscularis is shown in Fig. 1. Acetylcholine caused an Each value represents the mean 4-SEM of assays on three tissue increase in cyclic GMP content within 10 sec, the earliest samples, each of which was analyzed in triplicate. Cyclic GMP time studied. This response was sufficiently rapid that it could content, at all incubation times studied, was significantly dif- account for the ability of acetylcholine to induce contraction ferent from the zero-time control (P < 0.05). Downloaded by guest on September 26, 2021 3290 Cell Biology: Lee et al. Proc. Nat. Acad. Sci. USA 69 (1972)

TABLE 4. Effect of cholinomimetic agents and of cholinergic TABLE 6. Effects of bethanechol, isoproterenol, and atropine, blocking agents, present singly or in combination, on cyclic present singly or in combination, on cyclic AMP content in GMP content in slices of muscle from guinea-pig ileum guinea-pig ileal longitudinal muscle Cyclic GMP Cyclic AMP Addition (pmol/mg protein) Minus Plus Experiment 1 atropine atropine, 1 uM None (control) 0.48 d 0. 10 Addition (pmol/mg protein) Acetylcholine, 0.1 MM 0.64 -- 0.15* Acetylcholine, 1.0 MM 0.81 A 0.09t None (control) 4.15 4- 0.19 4.97 -- 0.18 Acetylcholine, 10.0 MM 0.76 -- 0.14t Bethanechol, 10 MM 4.28 -- 0.26 4.42 It 0.26 Experiment 2 Isoproterenol, 10 MM 13.19 4- 0.64* 12.95 -- 0.55* None (control) 0.73 :- 0.07 Isoproterenol, 10juM, plus Bethanechol, 0.1 AM 2.55 :- 1.35t Bethanechol, 10MUM 8.91 -:- 0.75t 12.48 -- 0.48t Bethanechol, 1.0MM 2.46 4- 1.40t Bethanechol, 10.0 AM 1.20 1- 0.24t Slices of longitudinal muscle were incubated for 2 min in the Experiment 3 presence of the indicated agents. Other conditions were the same None (control) 1.20 -- 0.05 as described in Table 4. Tetrarnethylammonium, 1 uM 1.34 1 0. 12* * Isoproterenol significantly increased cyclic AMP content 100MM 1.40 -- 0.14 in the absence and presence of atropine (P < 0.01). Experiment 4 t Significantly different from isoproterenol in the absence of None (control) 1.03 A= 0.03 atropine (P < 0.01). Bethanechol, 1 AM 5.37 A= 0.51t t Not significantly different from isoproterenol in the presence Atropine, 1MAM 1 . 10 4 0.06* of atropine. Bethanechol, 1 uM, plus Atropine, 1MAM 1.08 i 0.18§ curred in response to acetylcholine or bethanechol; this effect Hexamethonium, 10 MM 1.05 4- 0.11* of the cholinergic agonists was also blocked by atropine. Bethanechol, 1 uM, plus Hexamethonium, 10 MM 6.05 -- 0. 50s DISCUSSION There is a striking similarity between intestinal smooth Muscularis slices (Expts. 1 and 2) or longitudinal muscle slices muscle and cardiac muscle with respect to the effect of cho- (Expts. 3 and 4) were incubated for 30 see in the presence of the linergic and adrenergic agents on cyclic nucleotide content. indicated agents. Each value represents the mean A SEM of In cardiac muscle, cyclic GMP content was increased by assays on three tissue samples, each of which was analyzed in acetylcholine, and this increase was prevented by isopro- triplicate. terenol (11); similar results were obtained with * Not significantly different from the control. intestinal smooth muscle (Table 5). Conversely, in both cardiac muscle t Significantly different from the control (P < 0.05). 1 Significantly different from the control (P < 0.01). (ref. 11; Table 3) and intestinal smooth muscle (Table 6), § Significantly different from bethanechol alone ( P < 0.01). cyclic AMP content was increased by isoproterenol, and this ¶ Not significantly different from bethanechol alone. increase was prevented by acetylcholine or bethanechol. A reasonable hypothesis, consistent with these data, is that the AMP content, as well as that on cyclic GMP content, was physiological effects of cholinergic and adrenergic agents on mediated through muscarinic receptors. In some ileal prep- cardiac and intestinal smooth muscle are mediated through arations, a decrease in endogenous cyclic AMP content oc- regulation of the relative concentrations of cyclic GMP and cyclic AMP. Thus, cholinergic effects would be mediated TABLE 5. Effect of bethanechol and isoproterenol, present through a high cyclic GMP/cyclic AMP ratio and adrenergic singly or in combination, on cyclic GMP content in muscularis effects would be mediated through a high cyclic AMP/cyclic slices from guinea-pig ileum GMP ratio in these tissues. The present studies indicate that, at least in brain, heart Cyclic GMP muscle, and intestinal smooth muscle, the increase in con- Addition (pmol/mg protein) centration of cyclic GMP observed in response to acetyl- None (control) 1. 42 d 0. 37 choline is mediated by muscarinic, rather than by nicotinic, Isoproterenol, 1 MM 0.96 4 0.30* receptors. Mammalian brain contains both muscarinic and 10MM 0.9540.18* nicotinic receptors. In heart and intestinal smooth muscle, Bethanechol, 1 MAM 7.07 d 0. 79t the physiological responses to acetylcholine appear to be Bethanechol, 1 MM, plus mediated solely by muscarinic receptors. Recent studies of Isoproterenol, 1 uM 3.15 0.33t mammalian superior cervical ganglia have led to conclusions Bethanechol, 1 MM, plus Isoproterenol, 10 MM 1.53 d 0. 29t similar to those drawn here. These ganglia contain clearly distinct nicotinic and muscarinic receptors. Muscarinic, but GMP Muscularis slices were incubated for 30 see in the presence of the not nicotinic, agonists caused an increase in the cyclic indicated agents. Other experimental conditions were the same content of the ganglia (J. W. Kebabian, A. L. Steiner, and as described in Table 4. P. Greengard, unpublished experiments). * Not significantly different from the control. Thus, the available evidence is compatible with the general t Significantly different from the control (P < 0.005). hypothesis that the action of acetylcholine on muscarinic t Significantly different from bethanechol alone (P < 0.005). receptors is associated with an increased concentration of cy- Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Cholinergic Regulation of Cyclic GMP 3291

clic GMP. Conceivably, the physiological response to activa- from the United States Public Health Service. Present Address: tion of muscarinic receptors by acetylcholine is mediated Department of Pharmacology, Emory University, Atlanta, Ga. through this increased concentration of cyclic GMP. On the 30322. other hand, the available data suggest that the action of acetylcholine on nicotinic receptors does not involve mediation 1. Ashman, D. F., Lipton, R., Melicow, M. M. & Price, T. D. by a cyclic nucleotide. The biological significance of this dif- (1963) Biochem. Biophys. Res. Commun. 11, 330-334. ference between nicotinic and muscarinic receptors becomes 2. Hardman, J. G., Davis, J. W. & Sutherland, E. W. (1966) J. understandable when one takes into account the difference in Biol. Chem. 241,4812-4815. 3. Goldberg, N. D., Dietz, S. R. & OToole, A. G. (1969) J. Biol. the nature of the physiological responses that are triggered by Chem. 244,4458-4466. activation of nicotinic, compared to muscarinic, receptors. 4. Ishikawa, E., Ishikawa, S., Davis, J. W. & Sutherland, E. W. Activation by acetylcholine of nicotinic receptors is associ- (1969) J. Biol. Chem. 244,6371-6376. ated with explosive physiological responses that take only 5. Hardman, J. G. & Sutherland, E. W. (1969) J. Biol. Chem. 244,6363-6370. a few milliseconds to develop, as seen for example in the exci- 6. Beavo, J. A., Hardman, J. G. & Sutherland, E. W. (1970) J. tation of autonomic ganglia, skeletal muscle, and the electric Biol. Chem. 245,5649-5655. organ of the electric eel. The rapidity of these responses makes 7. Kuo, J. F. & Greengard, P. (1970) J. Biol. Chem. 245, 2493- it seem unlikely that they would be mediated by a complex 2498. 8. Kuo, J. F., Wyatt, G. R. & Greengard, P. (1971) J. Biol. series of biochemical reactions. Activation by acetylcholine Chem. 246,7159-7167. of muscarinic receptors, on the other hand, leads to relatively 9. Greengard, P. & Kuo, J. F. (1970) in Role of Cyclic AMP in slow physiological responses that take at least a few hun- Cell Function, eds. Greengard, P. & Costa, E. (Raven Press, dred milliseconds, and in some cases many seconds, to develop New York), pp. 287-306. fully, as seen, for example, in the contraction of intestinal 10. George, W. J., Polson, J. B., OToole, A. J. & Goldberg, N. D. (1970) Proc. Nat. Acad. Sci. USA 66,398-403. smooth muscle, in the decrease in heart rate and contractility, 11. Kuo, J. F., Lee, T. P., Reyes, P. L., Walton, K. G., Donnel- and in the increased activity of exocrine glands. Thus, these ly, T. E., Jr. & Greengard, P. (1972) J. Biol. Chem. 247, 16- responses to activation by acetylcholine of muscarinic re- 22. ceptors are sufficiently slow that it appears reasonable for them 12. Ferrendelli, J. A., Steiner, A. L., McDougal, D. R. & Kipnis, D. M. (1970) Biochem. Biophys. Res. Commun. 41, 1061- to be mediated by a complex series of biochemical reactions 1067. involving increased cyclic GMP and decreased cyclic AMP 13. Schultz, G., Hardman, J. G., Davis, J. W., Schultz, K. & content. Sutherland, E. W. (1972) Fed. Proc. 31, 440. 14. Kuo, J. F. & Greengard, P. (1969) Proc. Nat. Acad. Sci. This work was supported by Grants HL-13305, NS-08440, USA 64, 1349-1355. and MH-17387 from the United States Public Health Service. 15. Lee, T. P., Kuo, J. F. & Greengard, P. (1971) Biochem. Bio- T. P. L. is a recipient of a Postdoctoral Fellowship (1 F02 HL- phys. Res. Commun. 45,991-997. 50491) from the United States Public Health Service. Present 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. Address: Departments of Medicine and Pharmacology, Uni- J. (1951) J. Biol. Chem. 193, 265-275. versity of Wisconsin, Madison, Wis. 53706. J. F. K. is a recipient 17. Kuo, J. F. & Greengard, P. (1970) J. Biol. Chem. 245, 4067- of Research Career Development Award (1 K4 GM-50165) 4073. Downloaded by guest on September 26, 2021