Proc. Nat. Acad. Sci. USA Vol. 69, No. 11, pp. 3287-3291, November 1972 Role of Muscarinic Cholinergic Receptors in Regulation of Guanosine 3':5'-Cyclic Monophosphate Content in Mammalian Brain, Heart Muscle, and Intestinal Smooth Muscle (cyclic AMP/nicotine receptors/atropine) 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 acetylcholine 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 methacholine, bethanechol, and pilocarpine, 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 oxotremorine, 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 choline. 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 acetylcholine receptor. 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.