Agonist and Antagonist Binding of Muscarinic Acetylcholine Receptors Purified from Porcine Brain: Interconversion of High- and Low-Affinity Sites by Sulfhydryl Reagents

The affinity for muscarinic ligands of a preparation of muscarinic acetylcholine receptors purified from porcine brain was examined by means of competitive binding of [3H]quinuclidinylbenzylate and unlabeled ligands, followed by computer-assisted nonlinear regression analysis. The displacements by antagonists fitted a single-site model. In contrast, the displacements by agonists did not fit the single-site model and could be explained by assuming two populations of binding sites. The proportion of the sites with high affinity for muscarinic agonists (H-sites) ranged from 25 to 35% of the total number of sites. GTP had no effect on the displacements by agonists, a finding indicating that H-sites did not result from interaction between receptors and GTP-binding proteins. In the presence of dithiothreitol, the affinity for muscarinic ligands decreased. The largest effects were observed on the affinity for pirenzepine and that of H-sites for carbachol. Preincubation of the preparation with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in an increase in the proportion of H-sites to 75% of the total number of binding sites. The results of sucrose density gradient centrifugation of the preparation indicated apparent heterogeneity as to molecular size of the receptors, but this heterogeneity did not correlate with that of the affinity for agonists. In addition, the receptors were detected as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation, regardless of the presence or absence of disulfide-reducing reagents. These results suggest that the redox state of thiol groups in the receptor molecules is relevant to their affinities for ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pH on Muscarinic Acetylcholine Receptors from Rat Brainstem

The effect of hydrogen ion concentration on ligand binding to muscarinic acetylcholine receptors was studied in membranes isolated from rat brainstem. The binding of [3H]methylscopolamine was constant between pH 7 and 10. The affinity, but not the number, of [3H]methylscopolamine binding sites decreased below pH 7; at pH 4 little binding was detected. When brainstem membranes were incubated at various pH levels from 3 to 11 for 1 h and then returned to pH 8, [3H]methylscopolamine binding affinity was restored to control levels. Carbamylcholine binding affinity was also depressed in media of low pH. However, this decrease was permanent after a 1-h incubation at pH 4 (i.e. carbamylcholine affinity was not restored on raising the pH to 8). The capacity of a guanine nucleotide to affect carbamylcholine was also abolished by a 1-h incubation at pH 4, and was not restored by raising the pH. The guanine nucleotide-dependent regulatory protein may be irreversibly inactivated or dissociated from the receptor at low pH. The receptor's binding subunit, on the other hand, appears to be much less sensitive to hydrogen ion concentration.     

Muscarinic Acetylcholine Receptors in Torpedo Electric Organ: Effect of Guanine Nucleotides

Abstract: The effect of guanine nucleotides on the binding properties of presynaptic muscarinic receptors has been studied in a membrane preparation from the electric organ of Torpedo marmorata by measuring the competitive displacement of the radiolabelled antagonist, [³H]quinuclidinyl benzilate, by nonradioactive muscarinic ligands. The binding of the antagonists, atropine, scopolamine and pirenzepine was to a single class of sites [slope factors (pseudo Hill coefficients) close to 1] and was unaffected by 0.1 m M GTP. The binding of the N -methylated antagonists, N -methylatropine and N -methyl-scopolamine was more complex (slope factors <1) but also insensitive ( N- methylatropine) to 0.1 m M GTP. Agonist binding was complex and could be resolved into two binding sites with relatively high and low affinities. The proportion of high-affinity sites varied with the nature of the agonist (15–80%). Agonist binding was depressed by 0.1 m M GTP, and the order of sensitivity was oxotremorine-M > carbamoylcholine > muscarine > acetylcholine > arecoline > oxotremorine. The binding of pilocarpine, a partial agonist, was unaffected by GTP. With carbamoylcholine as a test ligand the GTP effect on agonist binding was half-maximal at 12 μM. GDP and guanylylimidodiphosphate produced comparable inhibition of carbamoylcholine binding, but GMP and cyclic GMP were ineffective, as were various adenine nucleotides. Analysis of agonist binding in terms of a two-site model indicates that the predominant effect of guanine nucleotides is to reduce the number of sites of higher affinity.     

Muscarinic Acetylcholine Receptors in Neuroblastoma Cells: Lack of Effect of Veratrum Alkaloids on Receptor Number

The effect of compounds that activate sodium channels on the number of muscarinic acetylcholine receptors in neuroblastoma NIE 115 cells has been investigated. The cells were used in electrically unexcitable ("control" cells) and excitable ("differentiated" cells) states. Although receptor assays using a single concentration of the radioligand [3H]scopolamine methyl chloride indicated a loss of receptors after a 6-h incubation of cells with veratrine, no true loss of receptors was seen with any of the compounds tested (veratridine, veratrine, aconitine) when full saturation analyses were performed in either control or differentiated cells. The apparent receptor loss seen with veratrine was due to a muscarinic receptor-active component of veratrine (not veratridine) occluded by the cells and released into the binding assays upon cell breakage. Veratridine and aconitine have a very low affinity for muscarinic acetylcholine receptors, and the binding of carbamoylcholine to the receptors is unaffected by tetrodotoxin, so that there is no evidence in this system for interaction between muscarinic receptors and sodium channels.     

Muscarinic and Nicotinic Cholinergic Binding Sites in the Terminal Abdominal Ganglion of the Cricket (Acheta domesticus)

Cricket (Acheta domesticus) terminal abdominal ganglia (TG) contain high concentrations (approximately 2 pmol/mg protein) of muscarinic and nicotinic cholinergic binding sites, based on the capacity of TG to bind specifically the labelled ligands L-[3H]quinuclidinyl benzilate ([3H]QNB) and [125I]alpha-bungarotoxin ([125I]alpha-BGT) with high affinity. For both ligands, binding is saturable and reversible. Competitive displacement experiments indicate that the [3H]QNB and [125I]alpha-BGT binding sites probably represent pharmacologically distinct classes of putative TG acetylcholine receptors (AChRs). Results from physiological recording and autoradiographic localization experiments demonstrate that a portion of the putative nicotinic AChRs is localized in synaptic regions of the well-characterized cercal sensory-giant interneuron pathway in the TG, where they are likely to serve as functional synaptic AChRs. Unlike nicotinic ligands, muscarinic agents do not appear to be pharmacologically active in this pathway. Therefore, in the insect CNS, putative muscarinic and nicotinic AChRs coexist at high density, but can be pharmacologically distinguished from one another on the basis of criteria derived from both ligand binding and physiological methods.     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Mechanism of Agonist-Induced Down-Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

Cooperative Interactions at M2 Muscarinic Acetylcholine Receptors: Structure/Activity Relationships in Stepwise Shortened Bispyridinium- and Bis(Ammonio)Alkane-Type Allosteric Modulators

Muscarinic M₂-receptors allow for divergent modes of allosteric action, depending on the structure of the allosteric modulator. Phthalimido-substituted bis(ammonio)alkane-type modulators belong to the common mode allosteric agents, whereas a physicochemically closely related bispyridinium-oxime with dichlorobenzyl-substituents at both ends is an atypical agent. Here, we compared the actions of stepwise shortened compounds composed of the phthalimido moiety and middle chains of either the bispyridinium- or the bis(ammonio)alkane-type. Allosteric interactions were measured in pig M₂ receptors with the orthosteric probe [³H]N-methylscopolamine ([³H]NMS) to label the acetylcholine binding site of the receptors. Dissociation and equilibrium binding experiments revealed parallel structure/activity-relationships in both series of compounds with regard to the cooperativity of interaction with [³H]NMS and to the underlying binding affinities in radioligand-occupied and free receptors. In conclusion, the findings are in line with the hypothesis that the phthalimido-moiety, but not the middle chain, is pivotal for the topology of interaction with the M₂-receptor protein.     

Long-Term Treatment with Glucocorticoids Increases Synthesis and Stability of Junctional Acetylcholine Receptors on Innervated Cultured Human Muscle

Abstract: We have studied the effect of long-term treatment with hydrocortisone on the expression of acetylcholine receptors (AChRs) at the neuromuscular junctions of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord motoneurons. Hydrocortisone increased accumulation of junctional AChRs in a dose-and time-dependent fashion. This increase was due to both decreased degradation and increased synthesis of AChRs. Other glucocorticoids, dexamethasone and prednisolone, exerted similar effects. Our study demonstrates a novel action of glucocorticoids on human junctional AChRs.     

Muscarinic Receptors and Hydrolysis of Inositol Phospholipids in Rat Cerebral Cortex and Parotid Gland

Abstract: Exposure of rat brain or parotid gland slices to muscarinic receptor agonists stimulates a phospholipase C that degrades inositol phospholipids. When tissue slices were labelled in vitro with [³H]inositol, this response could be monitored by measuring the formation of [³H]inositol phosphates. Accumulation of inositol 1,4-biphosphate in stimulated brain slices suggests that polyphosphonositides are the primary targets for phospholipase C activity. Li⁺ (10 m M ) in the medium completely blocked the hydrolysis of inositol 1-phosphate, partially inhibited inositol 1,4bisphosphate hydrolysis, but had no effect on the hydrolysis of inositol 1,4,5-trisphosphate by endogenous phosphatases. Muscarinic receptor pharmacology was studied by measuring the accumulation of [³H]inositol 1-phosphate in the presence of 10 m M Li⁺. In experiments on brain slices, the response to carbachol was antagonised by atropine with an affinity constant of approximately 8.79 ± 0.12. Dose-response curves to several muscarinic agonists were constructed using brain and parotid gland slices. The results are consistent with relatively direct coupling of low-affinity muscarinic receptors to inositol phospholipid breakdown in brain slices; full agonists were relatively more potent in the parotid gland compared with the brain. Explanations for these differences are suggested.     

Effect of G Protein-Coupled Receptor Kinase 2 on the Sensitivity of M4 Muscarinic Acetylcholine Receptors to Agonist-Induced Internalization and Desensitization in NG108-15 Cells

NG108-15 cells express predominantly the M4 subtype of the muscarinic receptor for acetylcholine. Stimulation of these receptors by the agonist carbachol causes an inhibition of cellular adenylyl cyclase and a consequent fall in the intracellular cyclic AMP concentration. Pretreatment of the cells with carbachol caused both internalization and desensitization of the M4 receptor. Overexpression of G protein-coupled receptor kinase (GRK) 2 caused an increase in the rate constant for receptor endocytosis (from 0.06 to 0.18 min(-1)) and a decrease in the EC50 for carbachol stimulation of internalization (from 15 to 3 microM). Overexpression of a dominant negative form of GRK2 had more modest effects, reducing the rate constant for endocytosis (from 0.11 to 0.07 min(-1)) and increasing the EC50 for carbachol stimulation of internalization (from 8 to 17 microM). Neither GRK2 nor dominant negative GRK2 overexpression had any effect on the rate constant for receptor recycling following agonist removal. The time course and extent of receptor desensitization in control cells were identical to the corresponding values for receptor internalization, and the rate and extent of desensitization were again increased by GRK2 overexpression. Exposure of the cells to hyperosmolar sucrose (0.6 M) almost completely blocked agonist-induced receptor internalization in both control and GRK2-overexpressing cells. Sucrose treatment also blocked agonist-induced desensitization. We conclude that the internalization and desensitization of the M4 muscarinic receptor in NG108-15 cells can be modulated in response to changes in GRK2 activity and also that internalization plays a key role in desensitization.     

Immunohistochemical Localization of Glycogen Phosphorylase Isozymes in the Rat Gastrointestinal Muscle Layers and Enteric Nervous System

Glycogen represents the major brain energy reserve though its precise functions are still under debate. Glycogen has also been found in different cell types of the enteric nervous system (ENS), the largest and most complex component of the peripheral nervous system. In the present work we have demonstrated, by application of isozyme-specific antibodies, the presence of isozymes of glycogen phosphorylase (GP), one of the major control sites in glycogen metabolism, in the rat ENS. Immunohistochemistry revealed that isoform BB (brain) is the predominant isozyme expressed in enteric glial cells (EGC) and rare neurons of the myenteric and submucosal plexuses. Isoform MM (muscle) appears in cells which are, according to their location and morphology, probably interstitial cells of Cajal (ICC). In addition, both GP isoforms are expressed in longitudinal and circular intestinal smooth muscle layers. As GP BB is mainly regulated by the cellular AMP level, a special function of glycogen in the energy supply of neural gut functions is suggested.     

Protection by Alcuronium of Muscarinic Receptors Against Chemical Inactivation and Location of the Allosteric Binding Site for Alcuronium

Abstract: We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [³H]methyl- N -scopolamine ([³H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [³H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues ( p -nitrobenzenesulfonyl fluoride and 4-chloro-7-nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N' -dicyclohexylcarbodiimide, and N -ethyl-5-phenylisoxazolium-3'-sulfonate] blocked the binding of [³H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [³H]NMS binding sites against the inactivation by tyrosine- and carboxyl-directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the "classical" muscarinic site and sterically blocks the access to this site.     

α2-Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK-N-SH Human Neuroblastoma Cells

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The alpha 2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 +/- 2%, with an EC50 of 83 +/- 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the alpha 2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 +/- 0.1, 2.2 +/- 0.3, and 1.4 +/- 0.1 microM, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell alpha 2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, muscarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 +/- 0.2 and 0.3 +/- 0.1 microM, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 +/- 1, 16,857 +/- 757, and 148,043 +/- 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic Activation of Muscarinic and Metabotropic Glutamate Receptors Down-Regulates Type I Inositol 1,4,5-Trisphosphate Receptor Expression in Cerebellar Granule Cells

Abstract: The ability of receptors coupled to phosphoinositide turnover to evoke accumulation of inositol 1,4,5-trisphosphate (InsP₃) over extended incubation periods, and consequently to affect the level of InsP₃ receptor expression, was studied in cultured cerebellar granule cells. The cholinergic agonist carbachol (CCh; 1 m M ) evoked a biphasic accumulation of InsP₃, a rapid three- to fourfold peak increase over control levels at ∼10 s, decreasing within 1 min to a long-lasting plateau elevation. Using an antibody against the type I InsP₃ receptor, it was demonstrated that >50% down-regulation of type I InsP₃ receptor expression in cerebellar granule cells occurred within 1 h of incubation with 1 m M CCh. Over 24 h, 1 m M CCh caused an ∼85% decrease in type I InsP₃ receptor levels, and significant decreases in immunoreactivity were evident at much lower concentrations of CCh. Direct assessment of total InsP₃ receptor expression using a radioligand binding method also detected down-regulation, but to an apparently lesser extent. 1-Aminocyclopentane-1 S ,3 R -dicarboxylic acid (200 µ M ), an agonist of metabotropic glutamate receptors, evoked a marked decrease in type I InsP₃ receptors after 24 h of incubation. These findings demonstrate that a functional consequence of maintained InsP₃ production in cerebellar granule cells is the down-regulation of InsP₃ receptor expression and that this down-regulation may be a common mechanism of action of phosphoinositide-linked receptors during prolonged stimulation.     

Characterization of Glutamate Binding Sites in Receptors Assembled from Transfected NMDA Receptor Subunits

Abstract: Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA-sensitive glutamate binding site. We further characterized the glutamate site assembled from NR1a, NR2A, and NR2B NMDA receptor subunits using l -[³H]glutamate and [³H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg²⁺ can alter [³H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [³H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [³H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.     

Effects of Nucleus Basalis Lesions on the Muscarinic and Nicotinic Modulation of [3H] Acetylcholine Release in the Rat Cerebral Cortex

Presynaptic muscarinic and nicotinic receptors in the cerebral cortex reportedly inhibit and increase acetylcholine (ACh) release, respectively. In this study, we investigated whether these receptors reside on cholinergic nerve terminals projecting to the cerebral cortex from the nucleus basalis magnocellularis (nbm). Adult male rats received unilateral infusions of ibotenic acid (5 micrograms/1 microliter) in the nbm. Two weeks later, cerebral cortical cholinergic markers (choline acetyltransferase activity, high-affinity choline uptake, and coupled ACh synthesis) were significantly reduced in synaptosomes prepared from the lesioned hemispheres compared to contralateral controls. The depolarization-induced release of [3H]ACh from these synaptosomes was also reduced in the lesioned hemispheres, reflecting the reduced synthesis of transmitter. However, the nbm lesions had no effect on the inhibition of release induced by 100 microM oxotremorine. Synaptosomal [3H]ACh release was not altered by nicotine or the nicotinic agonists anabaseine and 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine. Nicotine (10-100 microM) did increase [3H]ACh release in control and lesioned hemispheres in cortical minces, but to a similar extent. These results suggest that neither muscarinic nor nicotinic receptors modulating ACh release reside on nbm-cholinergic terminals.     

Differences and Similarities in Muscarinic Receptors of Rat Heart and Retina: Effects of Agonists, Guanine Nucleotides, and N-Ethylmaleimide

Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N-ethylmaleimide decreases agonist affinity for the high-affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of high- and low-affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low-affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high-affinity binding sites in retina. Third, the "partial agonist" pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high- and low-affinity sites and to effect a biological response.     

Stimulation of Phospholipase D Activity in Human Neuroblastoma (LA-N-2) Cells by Activation of Muscarinic Acetylcholine Receptors or by Phorbol Esters: Relationship to Phosphoinositide Turnover

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.