Muscarinic receptor subtypes in bovine adrenal medulla

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of differnt selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60–80%) correspond to M2 muscarinic subtypes, and the rest is constitute by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.Abbreviations [3H]QNB [3H]quinuclidinyl benzylate - HHSiD hexahydro-siladifenidol-hydrochloride - AF-DX 116 11-[[2-(diethylamino)methyl]]-1-piperidinyl]-5,11-dihydro-6H-pyrido[2,3,-b][1,4]benzodiazepin-6-one - 4-DAMP 4-diphenylacetoxy-N-methyl piperidine methobromide     

Evidence for Multiple Muscarinic Receptor Subtypes in Human Brain

Pirenzepine, a compound with selective antimuscarinic activity, was used to distinguish muscarinic acetylcholine receptor subtypes in normal human brain. Hill coefficients and IC50 values derived from the inhibition of specific [3H]L-quinuclidinyl benzilate receptor binding suggest the presence of two muscarinic binding sites, differing both in affinity for pirenzepine and in tissue distribution.     

Effect of Postmortem Factors on Muscarinic Receptor Subtypes in Rat Brain

Although prior studies have supported the validity of measuring total muscarinic receptor binding in postmortem brain, there has not been a study of postmortem effects on muscarinic receptor subtypes, M1 and M2, defined by high and low affinity for pirenzepine, respectively. We have examined in rat brain the effect of postmortem delay at room temperature, storage at 4 degrees C and -20 degrees C, and multiple freeze/thaw cycles on total muscarinic binding, measured with [3H]quinuclidinylbenzilate ([3H]QNB) and on M1 muscarinic binding, measured with [3H]pirenzepine ([3H]Pir). We found that delay at room temperature up to 4 h, or storage at 4 degrees C for 24 h or at -20 degrees C for 4 weeks, or 3 freeze/thaw cycles had no effect on [3H]QNB or [3H]Pir binding. Exposure of brain to room temperature for 15 h, however, led to an increase in [3H]QNB binding, without change in [3H]Pir. Scatchard analysis showed an increase in binding sites without a change in affinity. We conclude that [3H]QNB and [3H]Pir are valid measures of total and M1 muscarinic binding, respectively, under these circumstances, but that caution must be used in the interpretation of indirect measures of M2 binding.     

Regulation of Neuronal Muscarinic Acetylcholine Receptor Number by Protein Glycosylation

Tunicamycin, a potent inhibitor of protein glycosylation, was used to study the role of protein glycosylation in the regulation of muscarinic acetylcholine receptor (mAChR) number in cultures of N1E-115, a murine neuroblastoma cell line. At a concentration of 0.35 microgram/ml, tunicamycin inhibited macromolecular incorporation of [3H]mannose by 75-80%, whereas incorporation of [3H]leucine was reduced by only 10%. Treatment with tunicamycin caused a 30% decrease in total membrane mAChR number within 48 h as determined by a filter-binding assay using [3H]quinuclidinyl benzilate ([3H]QNB), a highly specific muscarinic antagonist. Tunicamycin also inhibited the recovery of total membrane mAChR by 70% following carbachol-induced down-regulation. The rate of mAChR degradation (control t1/2 12-14 h) was unaffected by incubation with tunicamycin. Intact cell binding studies using [3H]QNB (a membrane-permeable ligand) to measure total cellular (internal plus cell surface) mAChR and [3H]N-methylscopolamine ([3H]NMS, a membrane-impermeable ligand) to measure cell surface mAChR were conducted to determine whether tunicamycin selectively depleted cell surface mAChR. With 12 h of treatment with tunicamycin, cell surface mAChR number declined by 35%, whereas total cellular mAChR fell by only 10%. The ratio of cell surface receptor to total receptor decreased by 45% after 24 h. These results indicate that protein glycosylation is required for the maintenance of cell surface mAChR number. Incubation with tunicamycin causes a selective depletion of cell surface mAChR, implying that protein glycosylation plays a critical role in transport and/or incorporation of mAChR into the plasma membrane.     

Developmentally Regulated Secreted Factors Control Expression of Muscarinic Receptor Subtypes in Embryonic Chick Retina

Abstract: Two molecular mass subtypes of muscarinic receptor are expressed by the chick retina (72 and 86 kDa). During development, the ratio of subtypes changes, with the 72-kDa form becoming predominant. We have found that subtype switch can occur in retina cell culture, and have investigated factors that influence this in vitro increase in the 72-kDa receptor. Increases similar to those in vivo occurred when cells were cultured at 10⁵ cells/cm², but not at 10-fold lower density. High-density cultures, maintained on coverslips, showed no receptor development when transferred to large volumes of fresh medium, indicating that cell-cell contact alone was not responsible for induction. However, replacement of fresh medium with conditioned medium (from high-density cultures) resulted in normal induction. There were no morphological differences between cultures with high and low levels of the 72-kDa receptor. Conditioned medium also induced 72-kDa receptors in low-density cultures, consistent with a minimal role for cell-cell contact. Efficacy of conditioned medium was markedly dependent on age. Media from cells cultured 1–4 days had no effect, but media from cells cultured 5–8 and 1–8 days elicited 1.6-fold and fourfold increases in the 72-kDa subtype, respectively. The data indicate that maturing retina cells secrete developmentally regulated factors that are necessary for abundant expression of the 72-kDa muscarinic receptor subtype.     

Regional Differences in the Coupling of Muscarinic Receptors to Inositol Phospholipid Hydrolysis in Guinea Pig Brain

The differential effects of muscarinic agents on inositol phospholipid hydrolysis and the role in this process of putative muscarinic receptor subtypes (M1 and M2) were investigated in three regions of guinea pig brain. Addition of the agonist oxotremorine-M to slices of neostriatum, cerebral cortex, or hippocampus incubated in the presence of myo-[2-3H]inositol and Li+ resulted in a large accumulation of labeled inositol phosphates (733, 376, and 330% of control, respectively). In each tissue, the principal product formed was myo-inositol 1-phosphate (59-86%), with smaller amounts of glycerophosphoinositol and inositol bisphosphate. Only trace amounts of inositol trisphosphate could be detected. Regional differences were observed in the capacity of certain partial agonists to evoke inositol lipid hydrolysis, the most notable being that of bethanechol, which was four times more effective in the neostriatum than in either the cerebral cortex or hippocampus. In addition, the full agonists, oxotremorine-M and carbamoylcholine, were more potent stimulators of inositol phosphate release in the neostriatum than in the cerebral cortex. The putative M1 selective agonist 4-m-chlorophenylcarbamoyloxy-2-butynyl trimethyl ammonium chloride had little stimulatory effect in any brain region, whereas the putative M1 selective antagonist pirenzepine blocked the enhanced release of inositol phosphates with high affinity in the cerebral cortex and hippocampus (Ki = 12.1 and 13.9 nM; "M1") but with a lower affinity in the neostriatum (Ki = 160 nM; "M2"). In contrast to its differential effects on stimulated inositol lipid hydrolysis, no regional differences were observed in the capacity of pirenzepine to displace [3H]quinuclidinyl benzilate, a muscarinic antagonist, bound to membrane fractions. Atropine, an antagonist that does not discriminate between receptor subtypes, inhibited the enhanced release of inositol phosphates with similar affinities in the three regions (Ki = 0.40-0.60 nM). The results indicate that by measurement of inositol lipid hydrolysis, regional differences in muscarinic receptor coupling characteristics become evident. These differences, which are not readily detected by radioligand binding techniques, might be accounted for by either the presence of functionally distinct receptor subtypes, or alternatively, by regional variations in the efficiency of muscarinic receptor coupling to inositol lipid hydrolysis.     

Cholinergic Deafferentation of Dorsal Hippocampus by Fimbria-Fornix Lesioning Differentially Regulates Subtypes (m1-m5) of Muscarinic Receptors

Abstract: Unilateral aspiration lesions of the rostral supracallosal stria/cingulum bundle and fimbria-fornix were performed on adult female rats. Ten and 24 days post lesioning, an elevation (17%; p<0.01) of total muscarinic receptors was observed in lesioned versus control hippocampi. By using antisera selective for each of the five molecularly defined subtypes (m1-m5) of muscarinic receptors, significant changes were observed in the levels of expression for at least four receptor proteins. Three receptor subtypes increased in density: m1 by 14% (from 943 to 1,078 fmol/mg); m3 by 77% (from 150 to 268 fmol/ mg); and m4 by 29% (from 220 to 285 fmol/mg). In contrast, a 22% decrease in the density of m2 receptors was found (from 220 to 173 fmol/mg). Detectable levels of m5 receptors were low in the hippocampus (∼1% of total receptors), and reliable measurements were not obtained. The directions of these changes are likely to be related to the pre- or postsynaptic localization of these receptor subtypes.     

Ontogenesis of Muscarinic Receptors and Acetylcholinesterase Activity in Various Areas of Chick Brain

Abstract: Muscarinic receptors, labeled with [³H]quinuclidinyl benzylate (³H]QNB), and acetylcholinesterase activity were studied in five areas of the developing chick brain: (1) hyperstriatum and neostriatum , (2) paleostriatum, (3) optic lobes, (4) mesodiencephalon and (5) cerebellum. The protein content of these areas, expressed as mg/g tissue and total protein, was determined between day -10 and adulthood. Differences in both determinations were observed among the areas. The binding of [³H]QNB was expressed as density (fmol/mg protein) and total number of receptors (fmol/total protein) in the area. Considerable variations were observed among the areas. The cerebellum showed the lowest receptor density and a large decrease in density and total number of receptors in the adult, which may reflect a change in neuronal population. Acetylcholinesterase, in certain areas, accompanied the changes in receptor concentration, but the timing and rate of increase had special features in each case. The most striking one was the cerebellum, in which the enzyme increased steadily postnatally, while the muscarinic receptors dropped to very low values.     

Increased Inositol 1,4,5-Trisphosphate Accumulation Correlates with an Up-Regulation of Bradykinin Receptors in Alzheimer's Disease

Abstract: An alteration in signal transduction systems in Alzheimer's disease (AD) would likely be of pathophysiological significance, because these processes control normal brain functions. Previously, a diminished β-adrenergic-mediated cyclic AMP response was found in cultured fibroblasts from AD patients. Because cross-talk between the phosphoinositide and cyclic AMP pathways exists, the phosphoinositide cascade was studied under conditions that were similar to those for studying the cyclic AMP response. Cells from AD patients and age-matched controls responded to bradykinin (BK) and released inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] in a time- and dose-dependent manner. The level of Ins(1,4,5)P₃ increased rapidly and transiently in response to BK, peaked at 5 s, but still remained 116–132% above the basal level by 30 s. Although the temporal patterns were similar in both groups, the Ins(1,4,5)P₃ concentrations in AD fibroblasts were 73 and 89% above levels in the age-matched controls at 5 and 10 s, respectively. Prostaglandin E₁ also increased Ins(1,4,5)P₃ formation, but this response was not different between the two groups. Although K _D (affinity) values for the BK receptor were similar in both control and AD cells, the number of BK receptors ( B _max) was significantly elevated in AD fibroblasts (186.8 ± 0.8 fmol/mg of protein) as compared with control fibroblasts (57.2 ± 15.3 fmol/mg of protein). These results indicate that the elevated Ins(1,4,5)P₃ production in response to BK in AD fibroblasts is positively correlated with an increase in the receptor numbers.     

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M₂/M₄ antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M₂/M₄ double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M₂/M₄ double-KO mice. In M₂ single-KO and M₄ single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M₃ single-KO and M₁/M₃ double-KO mice were similar to those in wild-type mice. In M₅ single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M₂ and M₄ subtypes reduces glutamate release from primary afferents. Activation of the M₅ subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs.     

Agonist Regulation of Muscarinic Acetylcholine Receptors in Rat Spinal Cord

Abstract: In vitro studies with cultured cells originating from nervous tissue have shown that chronic exposure to muscarinic agonists results in a loss of muscarinic receptors. To determine whether this type of regulation of muscarinic receptor number also occurs in vivo , we infused carbachol into the spinal cords of rats. A single carbachol injection into the lumbar spinal cord caused a significant increase in the nociceptive threshold. This effect of carbachol diminished to control levels after 12 h of repeated agonist injections every 4 h and was blocked by atropine. The desensitization to the antinociceptive effects of carbachol was associated with a loss of muscarinic receptors as determined by the binding of the muscarinic antagonist [³H]quinuclidinyl benzilate. After a 24-h exposure to carbachol given every 4 h, there was about a 60% loss of binding sites. The loss of muscarinic receptors was also blocked by atropine and was reversible. These results represent direct evidence that a muscarinic agonist can regulate receptor number in the central nervous system and suggest that this loss of receptors is associated with a desensitization to the antinociceptive effects of carbachol injected into the spinal cord.     

Muscarinic Acetylcholine Receptors from Avian Retina and Heart Undergo Different Patterns of Molecular Maturation

Muscarinic acetylcholine receptors (mAChRs) from the avian CNS exist in two molecular weight forms whose concentrations change during development. Here, we have compared the development of mAChRs from embryonic hearts with those of the CNS. Analysis of [3H]-propylbenzilylcholine mustard (PrBCM)-labeled retina and heart mAChRs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two atropine-sensitive peaks for each tissue. Apparent molecular masses of retina mAChRs, 86 +/- 0.7 kilodaltons (kDa) and 72 +/- 0.7 kDa, were different from those of heart mAChRs, 77 +/- 1.0 kDa and 52 +/- 0.9 kDa. During retina development, the major receptor type changed from 86 kDa to 72 kDa. No such change occurred during heart development. Furthermore, the 52-kDa species appeared to be generated by endogenous proteolysis, as prolonged incubation of heart membranes at 37 degrees C increased the amount of 52-kDa peptide with a decrease of 77-kDa peptide. Protease inhibitors blocked this conversion. Incubation of retina membranes at 37 degrees C did not result in a conversion of the 86-kDa peptide into the 72-kDa peptide, but it did cause the appearance of a minor amount of 52-kDa peptide. The proteolysis of retina mAChRs was not enhanced by cohomogenizing them with heart tissue, arguing against the presence of releasable proteases in heart. Membrane-bound retina and heart mAChRs displayed similar sensitivity to exogenous (Staphylococcus aureus V8) protease, indicating that heart receptors were not unusually susceptible to proteolytic attack; analysis of the labeled polypeptides with the V8 protease showed different patterns of digestion for the retina and heart receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological Profiles of the Subtypes of Muscarinic Cholinoceptors That Mediate Aggregation of Pigment in the Melanophores of Two Species of Catfish

Using selective antagonists, including pirenzepine, adiphenine, AF-DX116, gallamine, and 4-DAMP we attempted to characterize the muscarinic cholinoceptors on the melanophores of the translucent glass catfish Kryptopterus bicirrhis and the mailed catfish Corydoras paleatus. The M₃ receptor-selective antagonist, 4-DAMP, potently inhibited the acetylcholine-induced aggregation of pigment in both species. It appeared, therefore, that the receptors that mediated the cholinergically evoked aggregation of melanosomes in these species were of the M₃ muscarinic subtype.     

Characterization of Muscarinic Cholinergic Receptors in the Crab Nervous System

The selective muscarinic antagonist L-[3H]-quinuclidinyl benzilate (L-[3H]QNB) binds reversibly and with high affinity (KD = 0.3 nM) to a single population (Bmax = 105 fmol/mg protein) of specific sites in nervous tissue of the crab Cancer magister. The binding site is stereoselective; (-)QNB is over 200 times more potent than (+)QNB as an inhibitor of specific L-[3H]QNB binding. The muscarinic antagonists scopolamine and atropine are over 10,000 times more potent inhibitors of L-[3H]QNB binding than the nicotinic antagonists decamethonium and d-tubocurarine. The muscarinic agonists oxotremorine, pilocarpine, arecoline, and carbachol also compete effectively for the L-[3H]QNB binding site. This pharmacological profile strongly suggests the presence of classical muscarinic receptors in the crab nervous system. These receptors are localized to nervous tissue containing cell bodies and neuropil, whereas specific L-[3H]QNB binding is low or absent in peripheral nerve, skeletal muscle, and artery.     

Altered Ontogenesis of Muscarinic Receptors in Agranular Cerebellar Cortex

Abstract: The developmental pattern, the agonist binding properties and the cellular origin(s) of muscarinic binding sites were investigated in agranular cerebellum of x-irradiated rats, of Gunn rats with hereditary hyperbilirubinemia, and of staggerer mutant mice. The density of muscarinic binding sites was found to be higher than normal in all of these cerebellar types, indicating that granular neurons do not greatly contribute to binding of acetylcholine in the rodent cerebellum. The total number of muscarinic binding sites as measured by binding of [³H]4NMPB remains unchanged in the agranular cerebellum of x-irradiated rats. However, the number of muscarinic sites is reduced by about 30% in the agranular cerebellum of homozygous Gunn rats (jj), in which fibrous astrocytes and Purkinje cells are also damaged. In the cerebellum of staggerer mice (sg/sg), where a cascade of events leads to massive damage to mossy fibers and Golgi cells in addition to granular neurons and Purkinje cells, the content of muscarinic receptors is reduced by 50%. Thus, the number of muscarinic binding sites in the rodent cerebellum seems to depend on the integrity of the additional cell types and cellular elements, damaged in these agranular models. The ontogenetic variations in the affinity of cerebellar muscarinic sites for binding of carbamylcholine in normal and Gunn rat cerebellum were compared with those observed in x-irradiated and staggerer cerebellum, where elimination of granular neurons induces the formation of ‘heterologous’ synapses. Muscarinic binding affinity increases 10-fold during postnatal development in the cerebellum of normal and Gunn rats. In the immature x-irradiated cerebellum, the affinity of muscarinic binding sites was found to be nearly as high as that detected in the adult normal cerebellum. In contrast, cerebella of 5-month-old staggerer mice display 5-fold lower affinity than their normal counterpart values, as low as that determined in normal immature cerebellum. The characteristic ontogenetic pattern of muscarink binding is therefore indicated to be related to the formation of correct circuitry, but not to the presence of granular neurons, in the developing rat cerebellum.     

Changes in Nicotinic and Muscarinic Cholinergic Receptors in Alzheimer-Type Dementia

Nicotinic and muscarinic cholinergic receptors were studied in autopsied brains from four histologically normal controls and five histopathologically verified cases of Alzheimer-type dementia (ATD), using ligand binding techniques. Nicotinic and muscarinic cholinergic receptors were assessed by (-)-[3H]nicotine and [3H]quinuclidinyl benzilate [( 3H]QNB), respectively. Compared with the controls, (-)-[3H]nicotine binding sites in the ATD brain regions examined were significantly reduced in the putamen and the nucleus basalis of Meynert (NbM). [3H]QNB binding was significantly reduced in the hippocampus and NbM. These findings suggest that there are significant changes of nicotinic and muscarinic cholinergic receptors in selected regions of ATD brains.     

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.     

Differential Alteration of Various Cholinergic Markers in Cortical and Subcortical Regions of Human Brain in Alzheimer''s Disease

The main objective of the present study was to determine whether cholinergic markers (choline acetyltransferase activity and nicotinic and muscarinic receptors) are altered in Alzheimer's disease. Choline acetyltransferase activity in Alzheimer's brains was markedly reduced in various cortical areas, in the hippocampus, and in the nucleus basalis of Meynert. The maximal density of nicotinic sites, measured using the novel nicotinic radioligand N-[3H]methylcarbamylcholine, was decreased in cortical areas and hippocampus but not in subcortical regions. M1 muscarinic cholinergic receptor sites were assessed using [3H]pirenzepine as a selective ligand; [3H]pirenzepine binding parameters were not altered in most cortical and subcortical structures, although the density of sites was modestly increased in the hippocampus and striatum. Finally, M2-like muscarinic sites were studied using [3H]-acetylcholine, under muscarinic conditions. In contrast to M1 muscarinic sites, the maximal density of M2-like muscarinic sites was markedly reduced in all cortical areas and hippocampus but was not altered in subcortical structures. These findings reveal an apparently selective alteration in the densities of putative nicotinic and muscarinic M2, but not M1, receptor sites in cortical areas and in the hippocampus in Alzheimer's disease.     

[3H]N-Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture

Saturation experiments with the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax = 1.85 +/- 0.01 pmol/mg of protein at 10 days in culture; KD = 0.128 +/- 0.01 nM. The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki = 273 +/- 13 nM), whereas the M2/M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H) = 31 +/- 5 nM; Ki(L) = 2,620 +/- 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N-methyl-D-aspartate (100 microM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.     

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.