Ultrastructural Changes Induced by 12-O-Tetradecanoylphorbol 13-Acetate in Pure Cholinergic Synaptosomes of Torpedo Electric Organ

We have studied the morphological changes induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment on pure cholinergic synaptosomes from Torpedo electric organ. These changes were studied by both ultrathin sections and freeze-fracture techniques. We found that after a treatment with TPA, a redistribution of synaptic vesicles inside the nerve endings and exocytotic images could be observed. Also, TPA, under conditions that induced the acetylcholine release, did not change the density of intramembrane particles at the synaptosomal protoplasmic hemimembrane leaflet. Similar results were found when calcium was not present in the extrasynaptosomal medium, and our results suggest that acetylcholine release induced by phorbol ester is probably mediated by exocytosis of synaptic vesicles.     

Phorbol Ester Enhancement of Neurotransmitter Release from Rat Brain Synaptosomes

Neurotransmitter release from rat brain synaptosomes was measured following pretreatment with various phorbol esters. Ca2+-dependent, evoked neurotransmitter release was increased by phorbol esters that were active in stimulating protein kinase C. Protein kinase C activation was demonstrated by increased incorporation of 32P into 87-kilodalton phosphoprotein, a specific substrate for that kinase. Inactive phorbol esters had no effect on neurotransmitter release or on the phosphorylation of 87-kilodalton phosphoprotein. The increased release was observed in either crude cortical synaptosomal fractions (P2) or purified cortical synaptosomal fractions. The enhancement was found for all neurotransmitters (norepinephrine, acetylcholine, gamma-aminobutyric acid, serotonin, dopamine, and aspartate), all brain regions (cerebral cortex, hippocampus, and corpus striatum), and all secretagogues (elevated extracellular K+ level, veratridine, or A23187) examined. It was also observed at all calcium concentrations present during stimulation of release. The phorbol ester enhancement of Ca2+-dependent release occurred whether or not calcium was present during pretreatment. These results indicate that stimulation of protein kinase C leads to an enhanced sensitivity of the stimulus-secretion coupling processes to calcium within the nerve terminal. The results support the possibility that presynaptic activation of protein kinase C modulates nerve terminal neurotransmitter release in the CNS.     

Continuous Determination by a Chemiluminescent Method of Acetylcholine Release and Compartmentation in Torpedo Electric Organ Synaptosomes

Abstract: The detection of acetylcholine (ACh) with a chemiluminescent procedure enables one to follow continuously the release of transmitter from stimulated synaptosomes and to study the compartmentation of ACh in resting and active nerve terminals. A compartment of ACh liberated almost entirely by a single freezing and thawing could be directly measured and compared with a compartment of ACh resistant to several cycles of freezing and thawing but liberated by a detergent (60–70% of the total). It is the compartment liberated by freezing and thawing that is reduced when synaptosomes are stimulated. Up to half the total synaptosomal ACh content is readily releasable provided the calcium entry is maintained, or if a strong releasing agent such as the venom of Glycera convoluta is used. In addition, it is shown that synaptosomes contain only negligible amounts of choline, and that the proportion of the two ACh compartments is not influenced by changing extracellular calcium just before their determination.     

Calcium-Independent Release of Acetylcholine from Electric Organ Synaptosomes and Its Changes by Depolarization and Cholinergic Drugs

Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+-independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2-(4-phenylpiperidino)cyclohexanol (10 microM), ouabain (104 microM), atropine (10 microM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+-independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35-fold at 203 mM K+). Carbamoylcholine (104 microM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 microM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1-3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ectonucleotidase Activities Associated with Cholinergic Synaptosomes Isolated from Torpedo Electric Organ

Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Simultaneous Release of Acetylcholine and ATP from Stimulated Cholinergic Synaptosomes

Abstract: The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromatin cells in the adrenal medulla.     

Energy Metabolism and Quantal Acetylcholine Release: Effects of Botulinum Toxin, l-Fluoro-2,4-Dinitrobenzene, and Diamide in the Torpedo Electric Organ

In the Torpedo electric organ, a modified nerve-muscle system, type A botulinum toxin blocked the release of acetylcholine (ACh) quanta, both neurally evoked and spontaneous. At the same time, the toxin increased the release of a class of small miniature potentials (the subminiature potentials), reduced the ATP and more the creatine phosphate content of the tissue, and impaired the activity of creatine kinase (CK). Thus, we compared this pattern of changes with those provoked by 1-fluoro-2,4-dinitrobenzene (FDNB), an efficient inhibitor of CK. As expected, FDNB rapidly inactivated CK, which resulted in a profound depletion of ATP whereas the stores of creatine phosphate were preserved. In addition, FDNB caused conspicuous morphological alterations of nerve endings and ACh depletion. This agent also suppressed evoked and spontaneous quantal release whereas the occurrence of subminature potentials was markedly increased. Diamide, a penetrating thiol oxidizing substance, provoked first a transient rise in quantal ACh release and then blockade of transmission with, again, production of a large number of subminiature potentials. Creatine phosphate was depleted in the tissue by diamide, the ATP content reduced, and CK activity partly inhibited. The morphology of nerve terminals did not show obvious changes with either diamide or botulinum toxin at the stage of transmission failure. Although the three poisons acted by different mechanisms, this resulted in a rather similar pattern of physiological changes: failure of quantal release and enhancement of subquantal release. These results and experiments on synaptosomes indicated that CK inhibition was probably a crucial mechanism for FDNB but not for diamide or botulinum intoxication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Newly Synthesized and Preformed Acetylcholine Are Released from Torpedo Synaptosomes by Different Pathways

In this study, we investigated the mechanisms underlying the release of preformed and of newly synthesized acetylcholine (ACh) from isolated Torpedo nerve terminals (synaptosomes). This was pursued by examining and comparing the effects of anticytoskeletal and anticalmodulin drugs and of activating the presynaptic muscarinic ACh receptors on the release of preformed endogenous ACh and of newly synthesized radiolabeled ACh. The anticytoskeletal drugs vinblastine, cytochalasin B, and colchicine inhibit the Ca2+-dependent K+-mediated release of newly synthesized radiolabeled ACh, but have no effect on the release of preformed ACh. By contrast, the muscarinic agonist oxotremorine markedly inhibits the release of preformed ACh, but has little effect on the release of newly formed ACh. Treatment of the synaptosomes with the calmodulin antagonist trifluoperazine inhibits the release of both ACh pools concomitantly. These findings show that preformed and newly synthesized ACh are released by different routes and suggest that their secretion is mediated by converging pathways. The significance of these results in view of the previously demonstrated preferential release of newly synthesized ACh is discussed.     

Compared Effects of Two Vesicular Acetylcholine Uptake Blockers, AH5183 and Cetiedil, on Cholinergic Functions in Torpedo Synaptosomes: Acetylcholine Synthesis, Choline Transport, Vesicular Uptake, and Evoked Acetylcholine Release

We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.     

Thiamine and Cholinergic Transmission in the Electric Organ of Torpedo

The electric organ of Torpedo marmorata was found to contain as much as 120 +/- 24 nmol of thiamine per g of fresh tissue. The vitamin was distributed as nonesterified thiamine (32%), thiamine monophosphate (22%), thiamine diphosphate (8%), and an important proportion of thiamine triphosphate (38%). A high level of thiamine triphosphate was found in synaptosomes isolated from the electric organ. In contrast, the synaptic vesicles did not show any enrichment in thiamine, whereas they contained a marked peak of acetylcholine (ACh) and ATP. Thus thiamine seems to be very abundant in cholinergic nerve terminals; its localization is apparently extravesicular, either in the axoplasm or in association with plasma membrane. When calcium was reduced and magnesium increased in the external medium, the efficiency of transmission was diminished, owing to inhibition of ACh release; in a parallel manner the degree of thiamine phosphorylation was found to increase--this condition is known to modify the repartition of ACh between vesicular and extravesicular compartments. Electrical stimulation, which causes periodic variations of the level of ACh and ATP, also caused significant changes in thiamine esters. In addition, related changes of the vitamin and the transmitter were observed under other conditions, suggesting a functional link between the metabolism of thiamine and that of ACh in cholinergic nerve terminals.     

Diacylglycerol-Induced Stimulation of Neurotransmitter Release from Rat Brain Striatal Synaptosomes

These studies were undertaken to test the hypothesis that alterations in phosphatidylinositol metabolism can modulate neurotransmitter release in the central nervous system. The effects of 1,2-diacylglycerols (DAGs) on dopamine release in the rat central nervous system were determined by measuring dopamine release from rat striatal synaptosomes in response to two DAGs (sn-1,2-dioctanoylglycerol and 1-oleoyl-2-acetylglycerol) that can activate protein kinase C and one DAG (deoxydioctanoylglycerol) that does not activate this kinase. Dioctanoylglycerol and 1-oleoyl-2-acetylglycerol, at a concentration of 50 micrograms/ml, stimulated the release of labeled dopamine from striatal synaptosomes by 35-50 and 17%, respectively. Dioctanoylglycerol-induced release was also demonstrated for endogenous dopamine. In contrast, deoxydioctanoylglycerol (50 micrograms/ml) did not stimulate dopamine release. Dioctanoylglycerol-induced dopamine release was independent of external calcium concentration, indicating a utilization of internal calcium stores. Dioctanoylglycerol (50 micrograms/ml) also produced a 38% increase in labeled serotonin release from striatal synaptosomes. The addition of dioctanoylglycerol to the striatal supernatant fraction increased protein kinase C activity. These results are consistent with the concept that an increase in phosphatidylinositol metabolism can stimulate neurotransmitter release in the central nervous system via an increase in DAG concentration. The data suggest an involvement of protein kinase C in the DAG-induced release, but other sites for DAG action are also possible.     

Acetylcholine Incorporation by Cholinergic Synaptic Vesicles from Torpedo marmorata

[3H]Acetylcholine efflux and Na+-K+ ATPase ion pump activity were measured concomitantly in rat cortical synaptosomes. Ouabain (500 microM), strophanthidin (500 microM), and parachloromercuribenzene sulfonate (500 microM) each inhibited ouabain-sensitive 86Rb uptake and elevated [3H]acetylcholine release independently of the external calcium concentration. Veratridine (10 microM), electrical field stimulation (60 V, 60 Hz, 5-ms pulse duration), or the calcium ionophore A23187 (10 micrograms/ml) also inhibited ouabain-sensitive 86Rb uptake and released [3H]acetylcholine, but via a calcium-dependent process. Veratridine-induced [3H]acetylcholine release and ion pump inhibition were correlated over a wide range of drug concentrations and both effects were blocked by pre-treatment with tetrodotoxin (1 microM). The rate of [3H]acetylcholine efflux from superfused synaptosomes was increased within 15 s of exposure to ouabain, strophanthidin, veratridine, A23187, or field stimulation, while ouabain-sensitive 86Rb uptake was significantly decreased within a similar interval. These results suggest that [3H]acetylcholine release is due at least in part to inhibition of Na+-K+ ATPase.     

Cholinergic Modulation of the Release of [3H]Acetylcholine from Synaptosomes of the Myenteric Plexus

Abstract: The purpose of these experiments was to determine if cholinergic agents affected the release of acetylcholine (ACh) from a synaptosomal preparation of the guinea pig ileum myenteric plexus. The synaptosomal preparation was first incubated with the precursor [³H]choline; subsequently, release of the stored [³H]ACh was measured. The release was decreased by oxotremorine or exogenous ACh plus hexamethonium and increased by exogenous ACh plus atropine. The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) evoked release that was inhibited by nicotinic antagonists or muscarinic agonists. Release was stimulated half-maximally by approximately 2 μ m - and maximally by 10 μ m -DMPP. Either in the absence of calcium or at 0°C, DMPP was without effect. The effect of 10 μ m -DMPP was brief, a significant stimulation occurring only within the first 2 min at 37°C. Tetrodotoxin also inhibited excitation by DMPP but not completely. Thus, the release of [³H]ACh appears to be presynaptically modulated, negatively by muscarinic agonists and positively by nicotinic agonists.     

Cetiedil, a Drug That Inhibits Acetylcholine Release in Torpedo Electric Organ

The effects of cetiedil, a vasodilatator substance with reported anticholinergic properties, were examined on cholinergic presynaptic functions at the nerve electroplaque junction of Torpedo marmorata using either synaptosomes or slices of intact tissue. Cetiedil abolished the calcium-dependent release of acetylcholine (ACh) triggered by depolarization or by addition of A23187 ionophore, a finding localizing the site of action downstream from the calcium entry step. In addition, a direct effect on the release process itself was indicated by the observation that cetiedil blocks the release of ACh mediated by a recently isolated presynaptic membrane protein, the mediatophore, reconstituted into ACh-containing proteoliposomes. In all three preparations, ACh release was inhibited by cetiedil with a Ki of 5-8 microM. Under the conditions used in these release experiments, the synthesis of ACh and its compartmentation within the nerve terminals were not modified. However, the drug was able to reduce high-affinity choline uptake and vesicular ACh incorporation when it was given together with the radioactive precursor, a result showing that cetiedil has a broad inhibitory action on cholinergic uptake processes.     

Release of Acetylcholine from Rat Brain Synaptosomes Stimulated with Leptinotarsin, A New Neurotoxin

Abstract: Leptinotarsin is a neurotoxic protein found in the hemolymph of potato beetles of the genus Leptinotarsa. In order to study the action of leptinotarsin from two species, L. haldemani and L. decemlineata , synaptosomes were prelabeled with [³H]choline in order to synthesize [³H] acetylcholine (ACh). These synaptosomes were then immobilized on Millipore filters and used for assay. Toxins from both species induce the release of radioactivity in this system. Fractionation of the released radioactivity indicated that ACh was released in preference to choline. The toxin that caused release was heat-labile and was partially dependent on Ca²⁺ in the perfusing medium. Release followed apparent first order kinetics when stimulation was effected with leptinotarsin from L. haldemani (leptinotarsin-h), but was more complex when using leptinotarsin from L. decemlineata (leptinotarsin-d). Increasing the concentration of toxin increased the rate of release, but the shapes of the dose-release curves elicited by the leptinotarsins from the two species were different. While leptinotarsin-h exhibited a simple, saturating dose-release curve, leptinotarsin-d was characterized by a sigmoid function, which was well described, with a Hill coefficient of 1.8. Antibodies directed toward black widow spider venom glands had no effect upon the releasing activity of leptinotarsin-h but could partially neutralize that of leptinotarsin-d. Toxins from both species have been partially purified and do not appear to be identical. The purified toxins should be useful tools with which to study the release of acetylcholine.     

Effect of Taurine on Neurotransmitter Release from Insect Synaptosomes

The effect of taurine on the release of [3H]acetylcholine ([3H]ACH) and [3H]gamma-aminobutyric acid ([3H]GABA) from preloaded locust synaptosomes has been studied. Veratridine (100 microM) and K+ (100 mM) both evoked [3H]ACh release and this was reduced in a concentration-dependent manner by taurine (5, 10, and 20 mM). In contrast to this, veratridine induced no observable release of [3H]GABA, and the response to K+ was slight. In the presence of taurine, however, a concentration-dependent enhancement of [3H]GABA release was observed. Since nipecotic acid (1 mM), an inhibitor of neuronal GABA uptake, also revealed [3H]GABA release induced by veratridine, it is suggested that both this effect and that of taurine are due to prevention of GABA reuptake. These results suggest that taurine may act as a neuromodulator in insects.     

The Synthesis, Storage, and Release of Propionylcholine by the Electric Organ of Torpedo marmorata

Abstract: Little is known about the specificity of the mechanisms involved in the synthesis and release of acetylcholine for the acetyl moiety. To test this, blocks of tissue from the electric organ of Torpedo were incubated with either [1-¹⁴C]acetate or [1-¹⁴C]propionate, and the synthesis, storage, and release of [1-¹⁴C]acetylcholine and [¹⁴C]propionylcholine were compared. To obtain equivalent amounts of the two labeled choline esters, a 50-fold higher concentration of propionate than of acetate was needed. Following subcellular fractionation, similar proportions of [¹⁴C]acetylcholine and [¹⁴C]propionylcholine were recovered with synaptosomes and with synaptic vesicles. Furthermore, both labeled choline esters were protected to a similar extent from degradation during homogenization of tissue in physiological medium, indicating that the two choline esters were equally well incorporated into synaptic vesicles. Yet depolarization of tissue blocks by 50 m M KCI released much less [¹⁴C]propionylcholinc than [¹⁴C]acetylcholine. During field stimulation of the tissue blocks, the difference between the releasibility of the two choline esters was less marked, but acetylcholine was still released in preference to propionylcholine. Evidence for specificity of the release mechanism was also obtained when the release of the two choline esters in response to field stimulation was compared in tissue blocks preincubated with both [³H]choline and [¹⁴C]propionate.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)