Himbacine recognizes a high affinity subtype of M2 muscarinic cholinergic receptors in the rat cerebral cortex

Wang, Jian xin; Roeske, William R.; Wang, Wan; Yamamura, Henry I.

doi:10.1016/0006-8993(88)91306-6

Cited by 19 publications

(6 citation statements)

References 13 publications

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“…A number of recent pharmacological classifications have described three subtypes of muscarinic receptor, but it is not clear whether the classifications refer to the same three subtypes and, in particular, whether the binding site in glands is the same as the functional receptor in smooth muscle (Mitchelson, 1988). In confirmation of previous reports (Barlow et al, 1976;Mutschler & Lambrecht, 1984;Giachetti et al, 1986;Hammer et al, 1986;de Jonge et al, 1986;Anwar-ul et al, 1986;Melchiorre et al, 1987;Michel & Whiting, 1988a;Wang et al, 1988) Doods et al, 1987), where M2 receptors predominate in heart, be applied to muscarinic receptor subtypes studied in both binding and functional studies. These receptors probably correspond to the ml, m2 and m3 genes of Bonner et al (1987) (HM1, HM2 and HM4 genes of Peralta et al, 1987).…”

Section: Discussionsupporting

confidence: 74%

“…A number of recent pharmacological classifications have described three subtypes of muscarinic receptor, but it is not clear whether the classifications refer to the same three subtypes and, in particular, whether the binding site in glands is the same as the functional receptor in smooth muscle (Mitchelson, 1988). In confirmation of previous reports (Barlow et al, 1976;Mutschler & Lambrecht, 1984;Giachetti et al, 1986;Hammer et al, 1986;de Jonge et al, 1986;Anwar-ul et al, 1986;Melchiorre et al, 1987;Michel & Whiting, 1988a;Wang et al, 1988), we found 4-DAMP, HSD, AF-DX 116, himbacine and methoctramine to discriminate between functional muscarinic responses in atria and ileum, and between apparently homogeneous populations of [3H]-NMS binding sites in heart and submandibular gland. The correlations we have found between these measures support suggestions that the binding sites in heart (mainly ventricle) are equivalent to the receptors studied in the functional atrial preparation, and that the binding sites in gland are equivalent to the receptor subtype mediating the functional response recorded from ileum.…”

Section: Discussionmentioning

confidence: 91%

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Functional and binding studies with muscarinic M₂‐subtype selective antagonists

Lazareno¹,

Roberts²

1989

British J Pharmacology

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1 The potency of a series of selective muscarinic antagonists has been measured on two functional isolated tissue preparations (rat ileum and atria) and these compared with their potency on a range of binding preparations in order to determine whether the subtypes of M2 receptor measured functionally are the same as those measured in binding studies. 2 On the functional preparations pirenzepine, hexahydrosiladiphenidol (HSD) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were more potent on the ileum than on the atrium (3 fold, 29 fold and 5 fold respectively), whereas himbacine, AF-DX 116 and methoctramine showed the opposite selectivity (5 fold, 3 fold and 56 fold respectively). Atropine had a similar potency on the ileum and atrium. 3 [3H]-N-methyl scopolamine was used to study M2 binding sites on membranes from rat heart and rat submandibular gland. Each preparation appeared to contain a homogeneous binding site population. The potencies of the five M2 selective antagonists (and pirenzepine) in binding studies to heart membranes were very similar to those observed in functional studies of rat atria (correlation coefficient = 0.98). Similarly the binding to submandibular gland membranes was very similar to that observed in functional studies on rat ileum (correlation coefficient = 0.97). 4 [3H]-pirenzepine was used to examine the binding of these antagonists to M1 binding sites on membranes from rat cerebral cortex. The affinities of 4-DAMP, HSD, AF,-DX1 16 and himbacine at M1 sites were similar to their affinities on the gland. Only pirenzepine and methoctramine had higher affinity on Ml sites than on the gland.5 Himbacine had a 20 fold lower affinity at M1 binding sites than at heart sites, and it should therefore be an important tool in identifying M1 sites. 6 Inhibition of [3H]-N-methyl scopolamine binding to rat ileum and rat brainstem by M2-selective antagonists was best described by a two-site model. In both cases the major population of sites (70-90%) appeared to be similar to sites found on the heart (correlation coefficients = 0.95 and 0.97). The other site appeared to be similar to that on the submandibular gland (correlation coefficients = 0.96 and 1.00). 7 The correlations observed in these studies in which a range of selective muscarinic antagonists was used lend weight to previous studies indicating the presence of three functionally important muscarinic receptor subtypes, typified by the binding sites studied in the cerebral cortex, submandibular gland and heart. 8 We propose that the sub-classification of the M2 muscarinic receptor into M2 and M3 subtypes on the basis of ligand binding studies should be extended to cover functionally-defined receptors as well.

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Section: Discussionsupporting

confidence: 74%

“…A number of recent pharmacological classifications have described three subtypes of muscarinic receptor, but it is not clear whether the classifications refer to the same three subtypes and, in particular, whether the binding site in glands is the same as the functional receptor in smooth muscle (Mitchelson, 1988). In confirmation of previous reports (Barlow et al, 1976;Mutschler & Lambrecht, 1984;Giachetti et al, 1986;Hammer et al, 1986;de Jonge et al, 1986;Anwar-ul et al, 1986;Melchiorre et al, 1987;Michel & Whiting, 1988a;Wang et al, 1988), we found 4-DAMP, HSD, AF-DX 116, himbacine and methoctramine to discriminate between functional muscarinic responses in atria and ileum, and between apparently homogeneous populations of [3H]-NMS binding sites in heart and submandibular gland. The correlations we have found between these measures support suggestions that the binding sites in heart (mainly ventricle) are equivalent to the receptors studied in the functional atrial preparation, and that the binding sites in gland are equivalent to the receptor subtype mediating the functional response recorded from ileum.…”

Section: Discussionmentioning

confidence: 91%

Functional and binding studies with muscarinic M₂‐subtype selective antagonists

Lazareno¹,

Roberts²

1989

British J Pharmacology

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“…Moreover, our results provide direct evidence that in the rat brain, muscarinic-M2 receptors consist of a heterogeneous population of sites. This hypothesis is supported by indirect evidence from studies of antagonist competition of [3Hloxotremorine-M binding and himbacine inhibition of r3HIAF'-DX 116 binding (Wang et al, 1988) to muscarinic sites in the rat brain. In contrast, previous direct data obtained from saturation analysis of [3H]AF-DX 116 binding to rat brain homogenates have revealed binding of this radioligand to a single class of relatively high affinity (I(d = 21-57 nM) muscarinic sites (Wang et al, 1987b,c).…”

Section: Discussionmentioning

confidence: 84%

Characterization of [³H]AF‐DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic‐M₂ receptor sites

Araujo

Lapchak

Regenold

et al. 1989

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This study shows that [3H]AF-DX 116 binds specifically, saturably, and with high affinity to putative muscarinic-M2 receptor sites in the rat brain. In homogenates of the hippocampus, cerebral cortex, striatum, thalamus, and cerebellum, [3H]AF-DX 116 appears to bind two subpopulations of muscarinic sites: one class of higher affinity sites (Kd less than 4.0 nM) and one class of lower affinity sites (Kd greater than 50 nM, except in the cerebellum). The apparent maximal capacities (Bmax) of [3H]AF-DX 116 sites in forebrain tissues ranged between 34 and 69 fmol/mg protein for the higher affinity site, and between 197 and 451 fmol/mg protein for the lower affinity site. In cerebellar homogenates, the maximal capacity of [3H]AF-DX 116 binding sites was 10.4 +/- 0.4 (Kd = 1.9 +/- 0.2 nM) and 39.1 +/- 2.6 (Kd = 26 +/- 7 nM) fmol/mg protein for the higher and the lower affinity site, respectively. Determination of the Kd for the higher and lower affinity [3H]AF-DX 116 sites from association and dissociation constants yielded similar values to those obtained from the saturation data. The ligand selectivity pattern reveals that AF-DX 116 is more potent than (-)QNB greater than atropine greater than methoctramine greater than 4-DAMP greater than gallamine greater than NMS greater than carbamylcholine greater than oxotremorine greater than pirenzepine much greater than nicotine in competing for the higher affinity [3H]AF-DX 116 sites. With few exceptions, the pattern was similar for the lower affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Recently, it has been proposed that there is muscarinic receptor heterogeneity within the CNS, including the cerebellum (Hammeret al, 1980(Hammeret al, , 1986Lapchak et al, 1989~). Different subtypes of muscarinic receptor may be selectively labeled using specific ligands, such as [3H]pirenzepine (M,) (Hammer et al, 1980;Watson et al, 1984;Lapchak et al, 1989a,b), [3H]AF-DX 116 (M2) (Hammer et al, 1986;Wang et al, 1987Wang et al, , 1988Lapchak et al, 1989b), or [3H]ACh (M2) (Kellar et al, 1985;Lapchak et al, 1989ab). We have also used the antagonist [3H]4-DAMP to selectively label cerebellar M3 muscarinic binding sites, because Doods et al ( 1987) have suggested that the antagonist 4-DAMP competes for a population of muscarinic binding sites (designated M3) in CNS tissue which is different from either MI or M2 binding sites.…”

Section: Discussionmentioning

confidence: 99%

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

Lapchak

Araujo

Quirion

et al. 1989

Journal of Neurochemistry

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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)

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Himbacine recognizes a high affinity subtype of M2 muscarinic cholinergic receptors in the rat cerebral cortex

Cited by 19 publications

References 13 publications

Functional and binding studies with muscarinic M₂‐subtype selective antagonists

Functional and binding studies with muscarinic M₂‐subtype selective antagonists

Characterization of [³H]AF‐DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic‐M₂ receptor sites

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

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Himbacine recognizes a high affinity subtype of M2 muscarinic cholinergic receptors in the rat cerebral cortex

Cited by 19 publications

References 13 publications

Functional and binding studies with muscarinic M2‐subtype selective antagonists

Functional and binding studies with muscarinic M2‐subtype selective antagonists

Characterization of [3H]AF‐DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic‐M2 receptor sites

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

Contact Info

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Resources

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Functional and binding studies with muscarinic M₂‐subtype selective antagonists

Functional and binding studies with muscarinic M₂‐subtype selective antagonists

Characterization of [³H]AF‐DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic‐M₂ receptor sites