STEREOSPECIFIC IN VlTRO BINDING OF [3H]DEXETIMIDE TO BRAIN MUSCARINIC RECEPTORS

A binding assay for muscarinic cholinergic receptors has been developed using labelled dexetimide as ligand and a filtration technique. The main features of this assay are its stereospecific nature, the very high affinity of the ligand for the specific receptors sitcs and its very low affinity for non-specific binding sites. The latter point was further investigated using labelled levitimide, the inactive enantiomer. The binding was found to be neither stereospecific nor saturable and displacement by both enantiomers revealed a particular curve with a very flattened course. Kinetic experiments with [³H]dexetimide suggest the occurrence of a heterogenous population of muscarinic receptors in the rat striatum. A study of the regional distribution of muscarinic receptors in rat brain showed a high concentration in the dopaminergic areas, the cortex and the hippocampus, but practically none in the cerebellum. The subcellular distribution pattern revealed a marked enrichment of [³H]dexetimide stereospecific binding sites in the microsomal fraction of rat striatum and hippocampus. Such a distribution was not found with [³H]levitimide. All the characteristics of this binding assay make dexetimide a very appropriate ligand for labelling muscarinic receptors in vitro as well as in vivo.     

Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors

The present study shows that N-[3H]methylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD = 5.0 nM; Bmax = 8.2 fmol/mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high-affinity binding sites (KD = 4.8 nM; Bmax = 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]pirenzepine. In contrast, the M2 ligand, [3H]AF-DX 116, appears to bind to two classes of binding sites, i.e., a high-affinity (KD = 3 nM)/low-capacity (Bmax = 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD = 28.4 nM) and higher capacity (Bmax = 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4-diphenylacetoxy-N-methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD = 0.5 nM)/low capacity (Bmax = 19.5 fmol/mg of protein) and one of low affinity (KD = 57.5 nM)/high capacity (Bmax = 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration-dependent manner, and the maximal increase in acetylcholine release (59.0-68.0%) occurred at 10(-7) M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and tetrodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-beta-erythroidine, d-tubocurarine, and kappa-bungarotoxin, but was insensitive to either atropine or alpha-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic cholinergic receptors on rat pancreatic acini by N-[3H]methylscopolamine binding. Their relationship with calcium 45 efflux and amylase secretion

N-[3H]Methylscopolamine (NMS) binding, amylase secretion, and 45Ca efflux from dispersed rat pancreatic acini were investigated in parallel, in the presence or absence of 4 muscarinic agonists and 3 muscarinic antagonists. Scatchard analysis of [3H]NMS saturation isotherms gave a KD of 0.9 nM and an average binding capacity of 24,000 sites per cell. Binding competition curves with the antagonists atropine, dexetimide, and NMS gave KD values of 3.5, 3.5, and 0.5 nM, respectively. With the 3 full agonists oxotremorine, muscarine, and carbamylcholine, the receptor population could be divided into two classes of binding sites: a minor one (15%) with high affinity (KD = 20-35 nM) and a major one (85%) with low affinity (KD = 3-65 microM). There was a receptor reserve of about 50% with respect to carbamylcholine-stimulated amylase secretion. Further analysis of dose-effect curves suggests that low affinity binding sites were involved in the secretory response to muscarinic stimulation. Pilocarpine, like muscarinic antagonists, recognized all binding sites with the same affinity but acted as a partial agonist on amylase secretion and 45Ca efflux.     

Autoradiographic distribution of high affinity muscarinic and nicotinic cholinergic receptors labeled with [3H]acetylcholine in rat brain

The relative distribution of muscarinic and nicotinic cholinergic receptors labeled with [3H]acetylcholine was determined using autoradiography. [3H]Acetylcholine binding to high affinity muscarinic receptors was similar to what has been described for an M-2 distribution: highest levels of binding occurred in the pontine and brainstem nuclei, anterior pretectal area and anteroventral thalamic nucleus, while lower levels occurred in the caudate-putamen, accumbens nucleus and primary olfactory cortex. Nicotinic receptors were labeled with [3H]acetylcholine to the greatest extent in the interpeduncular nucleus, several thalamic nuclei, medial habenula, presubiculum and superior colliculus, and to the least extent in the hippocampus and inferior colliculus. By using autoradiography to localize cholinergic binding sites throughout the brain it was observed that the distributions of high affinity muscarinic and nicotinic sites labeled with the endogenous ligand, [3H]acetylcholine are different from each other and are different from distributions of muscarinic and nicotinic sites labeled with muscarinic and nicotinic antagonists.     

Corticosteroid modulation of muscarinic receptors in rat myocardial membranes

Rat ventricular myocardial membanes contain muscarinic acetylcholine receptors which can be identified by binding of the muscarinic antagonist (-)-[³H]quinuclidinyl benzilate. Scatchard analysis of saturation binding data revealed binding to a single class of non-cooperative sites (0.693 pmol/mg protein) with high affinity (i.e. with an equilibrium dissociation constant of 0.24 nM). Competition binding curves of the agonist carbamylholine were shallow (with a Hill coefficient, n_H of 0.71) for membranes of untreated rats, suggesting the presence of two receptor subpopulations with different agonist affinity. These curves were steeper (n_H = 0.86) for adrenalectomized animals and more shallow (n_H = 0.62) for hydrocortisone-treated animals. In contrast, both treatments did not affect the total receptor number. This suggests that corticosteroids are required for the myocardial muscarinic receptors to adopt high agonist affinity. However, the inhibition of adenylate cyclase by muscarinic agonists disappeared after both corticosteroid treatment and adrenalectomy. But agonist receptor binding could still be modulated by guanine nucleotides. This indicates that both high and low affinity froms of muscarinic receptors induced by altered corticosteroid states retain functional coupling with the inhibitory nucleotide binding site, but are uncoupled from the adenylate cyclase catalytic subunit, C.     

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.     

Variations in binding affinities of the peripheral nicotinic acetylcholine receptor can be attributed to endogenous acetylcholine

Equilibrium binding studies performed with fresh membrane fragments from Torpedo marmorata reveal a low affinity for [3H]acetylcholine with an equilibrium dissociation constant in the micromolar range and no indication of cooperative interactions. The low binding affinity is an artifact caused by the presence of endogenous acetylcholine and is not related to the active conformation of the receptor. Endogenous acetylcholine is identified by its interaction with acetylcholine esterase and choline kinase. It is present in presynaptic vesicles as shown in electron micrographs. Leakage of these synaptosomes is of the order of 300 pmol acetylcholine per g tissue as determined by means of binding studies performed with [3H]acetylcholine. In the absence of endogenous acetylcholine, equilibrium binding studies show a high affinity for [3H]acetylcholine and a slight cooperativity of sites (K1D = 30nM; K2D = 10nM). The addition of detergents, local anesthetics or alcohols to a further increase in affinity and to a decrease in cooperativity (K1D = 11nM; K2D = 5nM). No low-affinity binding can be detected in the micromolar range.     

Differentiation between ligand trapping into intact cells and binding on muscarinic receptors

Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells.     

Solubilization of muscarinic acetylcholine receptor by zwitterionic detergent from rat brain cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [³H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with K_D value of 0.7 nM and a B_max value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors.     

[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.     

Identification of Muscarinic Receptors in Rat Cerebral Cortical Microvessels

Microvessels isolated from rat cerebral cortex consist mainly of capillaries (greater than 85%). Fresh, intact microvessel preparations have been analyzed by radioligand binding techniques for muscarinic receptors. Scatchard analysis of specific quinuclidinyl benzilate (QNB) binding indicates that microvessels possess a large number of muscarinic sites (914 fmol/mg protein) of high affinity (KD = 0.034 nM). The association and dissociation rate constants (0.37 min-1 nM-1 and 0.0067 min-1, respectively) yield an equilibrium KD of 0.018 nM. Displacement of [3H]QNB by muscarinic ligands and control substances is typical of muscarinic receptors. The results indicate that cerebral microvessels possess a large population of muscarinic receptors.     

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.     

Solubilization of muscarinic acetylcholine receptors of bovine brain

The effectiveness of several detergents and salts in solubilizing the muscarinic acetylcholine receptor (identified by its atropine-sensitive [³H]3-quinuclidinyl benzilate (QNB) binding) from bovine striatal membranes is reported. The highest density of receptor is obtained by extraction with 1% digitonin-0.1 mM EDTA. Although the total solubilized muscarinic receptors (sites/ml) are increased and the nonspecific binding is decreased when 1 M NaCl is included in this extraction medium, the receptor density (sites/mg protein) is lower. The solubilized receptors have the same specific QNB binding affinity, and sensitivity to a variety of drugs, as the membrane-bound muscarinic receptors.     

A cholinergic receptor site on murine lymphocytes with novel binding characteristics

To further analyze functionally important cholinergic receptors on lymphocytes, we studied the binding of the muscarinic antagonist Quinuclidinyl benzilate (QNB) to murine splenic lymphocytes. Studies of displacement of [³H]QNB by unlabelled QNB on lymphocytes revealed at least two binding sites. Scatchard analysis of equilibrium binding isotherms also distinguished two sites with apparent Kds of 480 nM and 16 μM. There was greater specific QNB binding to B cell-enriched lymphocyte fractions than to T cell fractions. Lymphocyte binding demonstrated temperature-dependent dissociability, and specific binding occurred on isolated lymphocyte membranes as well. Both muscarinic and nicotinic ligands competed for QNB binding to lymphocytes with low and nearly equal affinity. Therefore, QNB binding sites on lymphocytes appear to be of low affinity and of mixed muscarinic and nicotinic character.     

The effect of sodium channel activators on muscarinic receptors of neuroblastoma cells

Incubation of neuroblastoma NIE 115 cells with veratrine leads to an apparent reduction in the number of muscarinic acetylcholine receptors assayed by [3H]scopolamine methyl chloride binding. No true down-regulation of the receptors occurs but a component of veratrine with muscarinic receptor affinity, which is not veratridine, enters the intracellular water space during the incubation period and competes with [3H]scopolamine methyl chloride for the muscarinic binding sites in subsequent ligand binding assays unless it is carefully washed away. Treatment of cells with the agonist carbamoylcholine does, however, lead to a true downregulation of muscarinic receptors.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

α2-Adrenergic Receptors in Neuronal and Glial Cultures: Characterization and Comparison

Membranes prepared from either neuronal or glial cultures contain alpha 2-adrenergic receptors as determined by the characteristics of [3H]yohimbine [( 3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 +/- 1.35 nM (n = 10) for neuronal cultures and 18.42 +/- 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a Bmax of 1.6 +/- 0.33 pmol/mg protein (n = 10) compared with 0.143 +/- 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for alpha 2-adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the alpha 2-adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynanthine, or propranolol. The agonists showed the same pattern with the alpha 2-selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog. 5'-guanylyl-imidodiphosphate, were able to lower the affinity of the alpha 2-receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of alpha 2-adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain alpha 2-adrenoceptors.     

Autoradiographic localization of nicotinic receptor binding in rat brain using [3H]methylcarbamylcholine, a novel radioligand

Light microscopic autoradiography was used to visualize the neuroanatomical distribution of nicotinic receptors in rat brain using a novel radioligand, [3H]methylcarbamylcholine (MCC). Specific [3H]MCC binding to slide-mounted tissue sections of rat brain was saturable, reversible and of high affinity. Data analysis revealed a single population of [3H]MCC binding sites with a Kd value of 1.8 nM and Bmax of 20.1 fmol/mg protein. Nicotinic agonists and antagonists competed for [3H]MCC binding sites in slide-mounted brain sections with much greater potency than muscarinic drugs. The rat brain areas containing the highest densities of [3H]MCC binding were in thalamic regions, the medial habenular nucleus and the superior colliculus. Moderate densities of [3H]MCC binding were seen over the anterior cingulate cortex, the nucleus accumbens, the zona compacta of substantia nigra and ventral tegmental area. Low densities of [3H]MCC binding were found in most other brain regions. These data suggest that [3H]MCC selectively labels central nicotinic receptors and that these receptors are concentrated in the thalamus, the medial habenular nucleus and the superior colliculus of the rat brain.     

Neosurugatoxin, a Specific Antagonist of Nicotinic Acetylcholine Receptors

Neosurugatoxin (NSTX) (3 nM-30 nM), recently isolated from the Japanese ivory mollusc (Babylonia japonica) exerted a potent antinicotinic action in the isolated guinea pig ileum. Specific [3H]nicotine binding to rat forebrain membranes was saturable, reversible, and of high affinity. Nicotinic cholinergic agonists exhibited a markedly greater affinity for [3H]nicotine binding sites than a muscarinic agonist, oxotremorine. Although alpha-bungarotoxin had no effect on [3H]nicotine binding, low concentrations (1 nM-1 microM) of NSTX inhibited [3H]nicotine binding in the forebrain membranes and its IC50 value was 69 +/- 6 nM. On the other hand, NSTX did not affect muscarinic receptor binding in the brain. These data indicate that NSTX may be of appreciable interest as a neurotoxin with a selective affinity for ganglionic nicotinic receptors.     

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.