Different agonist binding properties of M1 and M2 muscarinic receptors in calf brain cortex membranes

The muscarinic antagonist 1-[benzilic 4,4'-3H]-quinuclidinyl benzilate [3H]-QNB) bound to a single class of non-cooperative sites in calf cerebral cortex membranes (KD = 0.29 nM and Bmax = 1.06 pM/mg protein). Computer-assisted analysis of the shallow pirenzepine/[3H]-QNB competition binding curves indicated that 68% of these sites were of the M1-subtype and the remaining 32% of the M2 subtype. Respective Ki-values for pirenzepine were 27 nM and 1.14 microM. Binding characteristics of the antagonist atropine and of the agonist carbachol for M2 were evaluated by performing competition binding with 0.5 nM [3H]-QNB in the presence of 2 microM pirenzepine. The binding characteristics for the M1 receptors were obtained indirectly by subtracting the curve for M2 from the total curve, or directly by competition binding with 0.3 nM [3H]-pirenzepine. Atropine competition curves were steep for M1 and M2 and were not affected by 1 mM GTP nor by 1 mM N-ethylmaleimide. The carbachol competition curve was shallow for M2. The steep curves for M1 indicate that this receptor subclass was only composed of low agonist affinity sites. GTP, which caused a rightward shift and a steepening of the carbachol competition curve for M2, did not affect the curves for M1. N-ethylmaleimide provoked a leftward shift and a steepening of the carbachol competition curve for M2 and abolished GTP modulation. A leftward shift was also observed for M1, but of a smaller magnitude (i.e. 3-4-fold for M1 compared to 17-fold for M2). These data suggest that, in calf brain cortex, M1 and M2 receptors show different susceptibility towards GTP and N-ethylmaleimide modulation.     

Muscarinic M3 receptors are coupled to two signal transduction pathways in rat submandibular cells

The receptor subtypes involved in muscarinic-induced phosphoinositide hydrolysis and adenylate cyclase inhibition in rat submandibular acinar cells were characterized by comparing the inhibitory potencies of four muscarinic antagonists on the two signal transduction responses. Carbachol-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was inhibited by all antagonists with a potency rank order of 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP) = atropine much greater than pirenzepine much greater than AF-DX 116 (P less than 0.01). The same rank order was observed in antagonist-reversal of the reduction of cAMP caused by carbachol in the model. These findings suggest that muscarinic effects are mediated by M3 receptors in both the phosphoinositide and adenylate cyclase pathways in the submandibular gland.     

Determination of muscarinic agonist potencies at M1 and M2 muscarinic receptors in a modified pithed rat preparation

Summary The agonistic potencies of (±)muscarine, (±)cis - 2 - methyl - 5 - [(dimethylamino)methyl] - 1,3 -oxathiolane methiodide (cis-oxathiolane) and its two enantiomers were determined at muscarinic M₁ and M₂ receptors in the pithed rat. In non-pretreated animals, i.v. administration of these agents produced bradycardic effects mediated by cardiac M₂ receptors followed by increases in heart rate mediated by M1 receptors in sympathetic ganglia. As these responses have been shown to partly overlap, “true” M₁ and M₂ potencies were determined after selective blockade of M₁ and M₂ receptors by pirenzepine and methoctramine, respectively. A similar rank order of agonist potencies was obtained at M₁ and M₂ receptors: (+)cis-oxathiolane > (±)cis-oxathiolane > (±)muscarine > (-)cis-oxathiolane. At both receptor subtypes, (+)cis-oxathiolane was considerably more potent (ca. 30-fold) than its corresponding (−) enantiomer indicating that the agonist binding sites of the two receptor subtypes may have similar stereochemical properties. While (±)muscarine showed similar potencies at M₁ and M₂ receptors, racemic cis-oxathiolane and its two enantiomers showed a slight selectivity (3–7 fold) for M₁ receptors indicating the potential usefulness of these compounds in the development of selective M₁ receptor agonists. Send offprint requests to F. Cantalamessa at the above address     

Tetrahydropyrimidine derivatives display functional selectivity for M1 muscarinic receptors in brain

Selective muscarinic agonists might be useful in the treatment of Alzheimer's disease. Previous studies identified several amidine derivatives as selective and efficacious m1 agonists, using muscarinic receptor subtypes expressed in cell lines. In the present studies, the functional selectivities, side effect profiles and memory-enhancing properties of these ligands were examined through a series of in vitro and in vivo experiments. CDD-0078, CDD-0097, and CDD-0102 behaved as partial agonists by stimulating PI turnover in rat cortical slices to roughly 100% above basal levels. Pirenzepine was more potent than either AF-DX 116 or p-F-hexahydrosiladifenidol (p-F-HHSiD) in blocking the PI responses of each ligand, suggesting that the responses were due to activation of M₁ receptors. The time course of pharmacological responses was examined following i.p. injections of muscarinic agonists. Low does (0.1 and 1.0 mg/kg) of CDD-0078, CDD-0097, and CDD-0102 did not elicit signs of cholinergic activity during the 2-hr testing period. The highest dose tested (10 mg/kg) produced a modest degree of salivation and lacrimation during the first 30-min period. Core body temperature remained unaffected. Central nervous system (CNS) penetration was evaluated through ex vivo binding studies. CDD-0097 inhibited 1.0 nM [³H]pirenzepine binding in a dose-dependent manner 30 min following i.p. injections. In behavioral studies, CDD-0097 (1.0 mg/kg) completely reversed the memory deficits induced by hemicholinium-3 or by IgG-192 saporin in two types of memory tasks. It did not impair the performance of control animals in either task. In summary, CDD-0097 displayed a limited side-effect profile and the ability to penetrate into the central nervous system and stimulate M₁ receptors. The amidine derivatives should be useful in further exploring the functional consequences of activating M₁ muscarinic receptors in the CNS. The beneficial effects of CDD-0097 on memory function warrant further examination of the compound as a selective M₁ agonist for the treatment of Alzheimer's disease. Drug Dev. Res. 40:171–184, 1997. © 1997 Wiley-Liss, Inc.     

Selective antagonists provide evidence that M1 muscarinic receptors may mediate carbachol-induced drinking in the rat

The present study served to investigate the ability of seven selective muscarinic antagonists to inhibit carbachol-induced drinking in the rat. The muscarinic antagonists were given by intracerebroventricular (i.c.v.) injection 1 min before the i.c.v. injection of carbachol (1 microgram/rat). The M2 antagonist, methoctramine, was inactive up to 80.3 nmol/rat. The M3 antagonist, p-fluoro-hexahydro-sila-difenidol, elicited a modest (42%) but statistically significant inhibition of drinking only at 80 nmol/rat. On the other hand, the selective M1 antagonists, (R)-trihexphenidyl, o-methoxy-sila-hexocyclium and pirenzepine, produced a marked and dose-dependent inhibition of carbachol-induced drinking, their ID50 values being 0.51, 7.36 and 9.31 nmol/rat. Also the M1/M3 antagonists, 4-diphenylacetoxy-N-methylpiperidine methiodide and hexahydro-sila-difenidol, were potent inhibitors of carbachol-induced drinking, their ID50 values (0.28 and 11.09 nmol/rat) being related to their pA2 values for M1 receptors in rabbit vas deferens. These data suggest that carbachol-induced drinking may be mediated by activation of muscarinic M1 receptors.     

Neuroleptic binding to muscarinic M2 receptors of normal human heart in vitro and comparison with binding to M1 and dopamine D2 receptors of brain.

We determined by radioligand binding the equilibrium dissociation constants (Kd's) for seventeen neuroleptics at muscarinic M2 receptors of human heart atrium and compared these data with our previous data for binding to muscarinic M1 and dopamine D2 receptors of human brain. At the M2 receptor, the most potent compound was thioridazine; the least, molindone. If selectivity is defined as Kd at one receptor less than or equal to 0.1 Kd at the other receptor, no compound was selective for the M2 subtype. Two compounds, clozapine and triflupromazine, were selective for the M1 subtype. Thus, few neuroleptics have the assumed preferred property of M1 over M2 subtype selectivity. Such a feature could reduce extrapyramidal side effects, while reducing the likelihood of certain cardiac side effects.     

Quantitative autoradiographic localization of the M1 and M2 subtypes of muscarinic acetylcholine receptors in the monkey brain

The distribution of muscarinic acetylcholine receptors (mAChR) was investigated in the monkey brain by means of quantitative in vitro autoradiography. 3H-QNB, 3H-pirenzepine (PZ) and 3H-AF-DX 116 were used for labelling total mAChR, M1 and M2 receptors, respectively. 3H-PZ and 3H-AF-DX 116 showed specificity to each receptor subtype in the monkey brain. On sections containing the putamen and globus pallidus, the sum of Bmax values of 3H-PZ and 3H-AF-DX 116 binding sites was almost close to that of 3H-QNB binding sites. Autoradiographic distributions of muscarinic subtype receptors in the monkey brain were similar to those reported in the rat brain; that is, M1 receptors were dominant in most areas of the telencephalon, while M2 receptors were richly distributed in the brainstem and cerebellum. However, some nuclei of the brainstem such as the central gray matter, superior colliculus, substantia nigra, nucleus of the oculomotor nerve, pontine nucleus and inferior olivary nucleus, had relatively high ratios of M1 receptors in the monkey brain. In addition, the cortical lamminar distribution of M2 receptors noticed in the rat was not observed in the monkey brain. Knowledge about the localizations of M1 and M2 receptors in various brain regions in the monkey brain will increase our understanding of the functions of the brain cholinergic system in the primate.     

Distribution of m2 muscarinic receptors in rat brain using antisera selective for m2 receptors.

The DNA fragment encoding the third intracellular loop of rat m2 muscarinic receptor was fused to the gene for staphylococcal Protein A. The resultant fusion protein, expressed in bacteria, was purified via IgG affinity chromatography and used as an antigen to raise a polyclonal antiserum. Chinese hamster ovary cells transfected with cDNA coding for a single muscarinic receptor subtype were used as tissue sources to screen antisera. The antiserum was shown to immunoprecipitate quantitatively (greater than 90%) m2 receptors but not to precipitate m1, m3, m4, or m5 receptors. Additionally, immunoprecipitation by m2 antiserum could be inhibited by protein containing the third intracellular loop of the m2 receptor. This selective m2 antiserum was then used to study the distribution and density of m2 receptors in rat brain and heart. In agreement with previous studies, m2 receptors were found to be abundant in heart and comprise at least 92% of the total muscarinic receptor density. Hindbrain, brain stem, and midbrain regions such as cerebellum (75%), pons/medulla (70%), and thalamus/hypothalamus (43%) are also enriched in m2 receptors. In contrast, forebrain regions contain markedly lower percentages of m2 receptors, with cortex expressing 20%, hippocampus 19%, striatum 12%, and olfactory tubercle 20% of the total receptor density. Although the density of m2 receptors expressed as a percentage of total varied considerably from brain region to brain region, the absolute density of these receptors appeared relatively uniform throughout the brain. This study demonstrates that a gene fusion system can be used for efficient antibody production. The use of similar fusion protein antisera directed against other subtypes of muscarinic receptors should prove useful in future studies on regulation, function, and structure of muscarinic receptors.     

Binding of selective antagonists to four muscarinic receptors (M1 to M4) in rat forebrain

To compare the proportions of four muscarinic receptors in different rat brain regions, we used competition curves with four selective antagonists, at 1-[N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) binding equilibrium and after allowing [3H]NMS dissociation for 35 min. Himbacine and methoctramine were shown to discriminate two muscarinic receptor subtypes having a high affinity for 4-diphenylacetoxy-N-methylpiperidine methiodide and hexahydrosiladifenidol, intermediate affinity for pirenzepine, and low affinity for AF-DX 116. One M4 subtype had a high affinity for himbacine and methoctramine; it was found predominantly in homogenates from rat striatum (46% of total [3H]NMS receptors) and in lower proportion in cortex (33% of [3H]NMS receptors) and hippocampus (16% of [3H]NMS receptors). Its binding properties were identical to those of muscarinic receptors in the neuroblastoma x glioma NG 108-15 hybrid, suggesting that it was encoded by m4 mRNA. The M3 subtype (typically found in rat pancreas, a tissue expressing the m3 mRNA) had a low affinity for himbacine and methoctramine and represented about 10% of all [3H]NMS receptors in rat brain cortex, hippocampus, striatum, and cerebellum. M1 and M2 receptors were identified in rat brain by their high affinity for pirenzepine and AF-DX 116, respectively.     

Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M2 receptors to certain OPs.     

M1 and M3 muscarinic receptors are involved in the release of urinary bladder-derived relaxant factor

We have investigated the muscarinic receptor subtype(s) mediating the release of urinary bladder-derived relaxant factor that is demonstrated by a coaxial bioassay system. Acetylcholine-induced relaxation of a precontracted anococcygeus muscle mounted within the bladder was considered as an evidence for the release of this factor. M₁-muscarinic agonist McN-A-343 and the cholinesterase inhibitor physostigmine also elicited relaxation responses in the coaxial bioassay besides acetylcholine. Acetylcholine-induced relaxation was antagonized by the subtype-selective muscarinic antagonists (pK_B): M₃-antagonist darifenacin (9.36 ± 0.11), M₃/M₁-antagonist 4-DAMP (9.30 ± 0.11), M₁-antagonist telenzepine (8.56 ± 0.21), M₄-antagonist tropicamide (6.63 ± 0.17) and M₂-antagonist AF-DX 116 (6.01 ± 0.21). The pK_B values of these antagonists have suggested that stimulation of M₁- and M₃-muscarinic receptors in the bladder wall mediates the release of urinary bladder-derived relaxant factor. In addition, McN-A-343, by activating the facilitatory M₁ receptors and physostigmine by inhibiting the acetylcholinesterase may induce the release of this factor through endogenous acetylcholine in the coaxial bioassay system.     

Gallamine binding to muscarinic M1 and M2 receptors, studied by inhibition of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to rat brain membranes

Whereas classic muscarinic antagonist ligands appear to recognize only a single class of muscarinic receptor sites, the recently discovered antagonist pirenzepine appears to distinguish at least two classes of sites. Its unique binding properties, demonstrated in both indirect and direct binding studies, have led to an emerging concept of high affinity (M1) and low affinity (M2) sites. This concept has been supported by pharmacologic studies of functional muscarinic responses, as well as by data suggesting different effector relationships for the two sites. Gallamine possesses muscarinic antagonist properties, and it also recognizes heterogeneity among muscarinic receptors. The purpose of this study was to define gallamine-recognized heterogeneity in terms of the pirenzepine-defined M1, M2 concept. This has been done by studying the ability of gallamine to inhibit [3H]pirenzepine binding to the M1 site, and to inhibit [3H]quinuclidinylbenzilate ([3H]QNB) binding in cerebellar membrane preparations, which contain almost exclusively the M2 site. The results show that gallamine binds with high affinity to the M2 site, with Ki = 2.4 nM, and lower affinity to the M1 site with Ki = 24 nM. Within these classes gallamine does not recognize heterogeneity. The ability of gallamine to inhibit [3H]QNB binding to cortex is best described by a two-site model comprised of 77% low affinity gallamine sites (M1) and 23% high affinity gallamine sites (M2). Thus, the heterogeneity among muscarinic receptors which is recognized by gallamine within the receptor binding paradigms of this study can be attributed to the M1, M2 subtypes as defined by pirenzepine binding. In addition, gallamine at low concentrations appears to bind as a pure competitive antagonist at these two sites, indicated by linear Schild plots with slopes of 1.0, the lack of an effect on dissociation of radioligands, and the ability to protect [3H]pirenzepine and [3H]QNB-binding sites from alkylation by propylbenzylcholine mustard. These studies do not exclude the possibility of a non-competitive interaction of gallamine with the muscarinic receptor observed by other investigators at high gallamine concentrations, and postulated to occur at a site adjacent to the primary muscarinic site. It is proposed that gallamine is capable of interacting with both the primary muscarinic site and an allosteric site. These results support the emerging concept of M1 and M2 muscarinic subclasses and suggest that gallamine and related compounds may be useful in defining muscarinic receptor subclasses, given their higher affinity for the M2 site.     

Allosteric site on muscarinic acetylcholine receptors: identification of two amino acids in the muscarinic M2 receptor that account entirely for the M2/M5 subtype selectivities of some structurally diverse allosteric ligands in N-methylscopolamine-occupied receptors

Two epitopes have been identified recently to be responsible for the high-affinity binding of alkane-bisammonium and caracurine V type allosteric ligands to N-methylscopolamine (NMS)-occupied M2 muscarinic acetylcholine receptors, relative to M5 receptors: the amino acid M2-Thr423 at the top of transmembrane region (TM) 7 and an epitope comprising the second extracellular loop (o2) of the M2 receptor including the flanking regions of TM4 and TM5. We aimed to find out whether a single amino acid could account for the contribution of this epitope to binding affinity. Allosteric interactions were investigated in wild-type and mutant receptors in which the orthosteric binding site was occupied by [3H]NMS (5 mM Na,K,Pi buffer, pH 7.4, 23 degrees C). Using M2/M5 chimeric and point-mutated receptors, the relevant epitope was narrowed down to M2-Tyr177. A double point-mutated M2 receptor in which both M2-Tyr177 and M2-Thr423 were replaced by the corresponding amino acids of M5 revealed that these two amino acids account entirely for the (approximately 100-fold) M2/M5 selectivity of the alkane-bisammonium and the caracurine V type allosteric ligands. At NMS-free M2 receptors, the caracurine V derivative also displayed approximately 100-fold M2/M5 selectivity, but the double point mutation reduced the M2 affinity by only approximately 10-fold; thus, additional epitopes may influence selectivity for the free receptors. A three-dimensional model of the M2 receptor was used to simulate allosteric agent docking to NMS-occupied receptors. M2-Tyr177 and M2-Thr423 seem to be located near the junction of the allosteric and the orthosteric areas of the M2 receptor ligand binding cavity.     

Rat hippocampal muscarinic autoreceptors are similar to the M2 (cardiac) subtype: comparison with hippocampal M1, atrial M2 and ileal M3 receptors.

1. Affinity constants for 15 non-selective or putatively selective muscarinic antagonists were determined at muscarinic autoreceptors and postsynaptic receptors (linked to phosphatidylinositol (PI) hydrolysis) in rat hippocampal slices, at muscarinic receptors mediating contractility in guinea-pig atria or ileal smooth muscle and at binding sites in rat cerebral cortical membranes labelled with [3H]-1-quinuclidinyl benzilate or [3H]-pirenzepine. 2. Comparison of the affinities of these antagonists at central M1 receptors (inositol-monophosphate formation in rat hippocampal slices) with their affinities at peripheral M1 receptors (inhibition by McN-A-343 of electrically stimulated twitches in rabbit vas deferens) provides support for the suggestion that these receptors may differ pharmacologically. 3. Comparison of affinity constants obtained by displacement of specifically bound [3H]-pirenzepine from rat cerebral cortical membranes with those obtained in functional tests showed poor correlations between affinities for binding sites and for functional atrial receptors or for hippocampal autoreceptors. A significant correlation was found between affinities for [3H]-pirenzepine binding and those determined at muscarinic receptors linked to PI turnover in rat hippocampus. A significant correlation was also obtained between the affinities for specific [3H]-pirenzepine binding sites in cortical membranes and the affinities at ileal receptors. 4. Comparison of the affinity values for muscarinic autoreceptors in rat hippocampus with affinity values obtained from in vitro models of muscarinic receptor subtypes showed no significant correlations between these autoreceptors and either M1 or M3 receptors. A significant correlation was found between antagonist affinities for hippocampal autoreceptors and muscarinic receptors in the heart. Therefore, muscarinic autoreceptors in rat hippocampus are pharmacologically similar to the M2 (cardiac) muscarinic receptor subtype.     

星形胶质细胞毒蕈碱样乙酰胆碱受体M2、M4亚型基因克隆及序列分析

目的：克隆胶质细胞M_2、M_4受体亚型基因序列，并比较胶质细胞M_2、M_4受体亚型基因序列和蛋白质序列与神经元细胞M_2、M_4受体基因序列和蛋白质序列间的差异。方法：根据神经元细胞M_2、M_4受体基因序列设计出针对M_2、M_4受体基因序列全长的特异性探针，采用RT-PCR方法扩增胶质细胞M_2、M_4受体亚型基因序列，并对其进行克隆测序。结果：通过RT-PCR方法扩增胶质细胞M_2、M_4受体亚型基因序列，与神经元细胞M_2、M_4受体比较，M_2受体差异碱基17个，发生氨基酸改变的有8个；M_4受体差异碱基3个，发生氨基酸改变的有2个。结论：胶质细胞M_2、M_4受体与神经细胞M_2、M_4受体亚型在基因序列和氨基酸序列上具有明显差异。     

Evidence for prejunctional M2 muscarinic receptors in pulmonary cholinergic nerves in the rat.

1. The effects of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine and methoctramine) were used to investigate the existence of prejunctional muscarinic receptors on cholinergic nerves in the rat lung. The tracheal tube preparation was used in vitro, and contraction of the trachealis muscle was induced by electrical field stimulation (EFS) and by application of an exogenous muscarinic agonist (pilocarpine), and measured as an increase in intraluminal pressure in the tube. 2. The muscarinic antagonists, gallamine and methoctramine, enhanced the contractions induced by nerve stimulation, while contractions elicited by exogenous application of pilocarpine were inhibited by the antagonists. 3. In contrast, pirenzepine blocked contractions induced by both EFS and pilocarpine in a dose-dependent manner (EC50 0.1 microM) due to blockade of the postjunctional muscarinic receptors on airway smooth muscle. Potentiation of the response to EFS was never seen with this antagonist. 4. The muscarinic agonist, pilocarpine, caused a slow maintained increase in tone of the tracheal tube and at the same time reduced the contractions induced by EFS. This inhibitory effect was blocked by gallamine and methoctramine. 5. The results suggest that prejunctional inhibitory muscarinic receptors may be localised on the parasympathetic cholinergic nerve terminals innervating tracheal smooth muscle in the rat. This confirms previous findings obtained by measuring transmitter release in this species. The present results suggest that these receptors are of the M2 subtype. Blockade of these autoreceptors with gallamine or methoctramine would increase the output of acetylcholine (ACh) and thereby enhance the nerve-induced contraction of tracheal smooth muscle.     

星形胶质细胞毒蕈碱样乙酰胆碱受体M1、M3、M5亚型基因克隆及序列分析

目的克隆胶质细胞M1、M3、M5受体亚型基因序列,比较胶质细胞和神经细胞M1、M3、M5受体亚型基因序列、蛋白质序列的差异。方法根据神经细胞M1、M3、M5受体基因序列全长设计特异性探针,采用RT-PCR方法扩增胶质细胞M1、M3、M5受体亚型基因序列,并对其进行克隆测序。结果测序得到胶质细胞M1、M3、M5受体亚型基因序列,与神经细胞比较,M1受体差异碱基4个,发生氨基酸改变的有1个;M3受体差异碱基有8个,发生氨基酸改变的位点有4个;M5受体差异碱基1个,发生氨基酸改变的有1个。结论胶质细胞与神经细胞M1、M3、M5受体亚型在基因和蛋白序列上具有明显差异。     

Adaptive changes in M1 muscarinic receptors localized to specific rostral brain regions during and after morphine withdrawal

Morphine-dependent rats were allowed to undergo withdrawal by abrupt discontinuation of the drug. The regional expression of brain M1 muscarinic receptors was measured directly by autoradiographic determination with [(3)H] pirenzepine, and indirectly by quantifying the relative levels of M1 mRNA encoding the receptor protein. Patterns of receptor changes after morphine treatment were in general agreement using the two methods. Frontal cortical samples derived from morphine-dependent rats exhibited a 28% increase in M1 receptor mRNA measured at the end of the infusion. At the peak of the withdrawal, M1 mRNA levels for dependent rats were much lower (33.4%) than those for control rats. Hippocampal samples derived from morphine-dependent rats exhibited no changes in M1 mRNA levels after the morphine infusion. During the peak of withdrawal, however, hippocampal M1 mRNA levels were reduced (57%) compared with levels for controls. The M1 mRNA levels remained at this reduced degree of expression even after withdrawal symptoms had subsided. Addition of diisopropylflurophophate (DFP) to the morphine infusion schedule inhibited the adaptive changes in M1 mRNA levels induced by morphine. During the peak period of withdrawal, M1 mRNA levels in the hippocampus declined by only 18% as compared with 57% for the morphine control group. The adaptive decrease in hippocampal M1 receptors after withdrawal subsided may reflect prolonged heightened cholinergic activity in an area where such cholinergic innervation plays an important role in memory.