大鼠脑室内注射氨甲酰胆碱对肾钠,钾,水排出的影响

在麻醉大鼠侧脑室注射胆碱能激动剂氨甲酰胆碱（ＣＢＣ）引起显著的促钠排泄、促钾排泄和利尿反应（Ｐ＜０．０５）,其中促钠排泄反应与剂量之间呈量效关系（ｒ＝０．９９９７,Ｐ＜０．０５）。由脑室注射ＣＢＣ（２．７４×１０－３μｍｏｌ）引起的上述反应可以被胆碱能Ｍ受体阻断剂阿托品或Ｎ受体阻断剂六甲双胺预处理完全阻断（Ｐ＜０．０５）。同样,ＣＢＣ的肾脏效应也可被肾上腺素能α受体阻断剂酚妥拉明预处理所部分阻断（Ｐ＜０．０５）。上述结果表明脑室注射ＣＢＣ引起的促钠排泄、促钾排泄和利尿反应是刺激了脑胆碱能Ｍ或Ｎ受体,有部分效应可能继发刺激去甲肾上腺素能α受体。     

Influence of a genetic variant of CHAT gene over the profile of plasma soluble ChAT in Alzheimer disease

The choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are fundamental to neurophysiological functions of the central cholinergic system. We confirmed and quantified the presence of extracellular ChAT protein in human plasma and also characterized ChAT and VAChT polymorphisms, protein and activity levels in plasma of Alzheimer''s disease patients (AD; N = 112) and in cognitively healthy controls (EC; N = 118). We found no significant differences in plasma levels of ChAT activity and protein between AD and EC groups. Although no differences were observed in plasma ChAT activity and protein concentration among ChEI-treated and untreated AD patients, ChAT activity and protein levels variance in plasma were higher among the rivastigmine-treated group (ChAT protein: p = 0.005; ChAT activity: p = 0.0002). Moreover, AD patients homozygous for SNP rs1880676 A allele exhibited higher levels of ChAT activity. Considering this is the first study to report the influence of genetic variability of CHAT locus over ChAT activity in AD patients plasma, it opens a new set of important questions on peripheral cholinergic signaling in AD.     

Effects of MK-771, A TRH analog,on pentobarbital-induced alterations of cholinergic parameters in discrete regions of rat brain

MK-771 (l-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide) was administered intraventricularly to conscious and pentobarbital-narcotized rats. In the conscious rats MK-771 did not affect the regional levels of acetylcholine (ACh) or the rate of sodium-dependent high-affinity choline uptake (HACU). MK-771 was found to antagonize pentobarbital-induced elevations of ACh levels in the cortex, hippocampus and striatum. MK-771 also reversed the depressant effects of pentobarbital on the HACU of the cortex and hippocampus. Striatal HACU was unaltered by the administration of pentobarbital or the combination of pentobarbital and MK-771.     

非神经元型胆碱能系统在（前）脂肪细胞的表达及nAChRα7功能状态对前脂肪细胞Visfatin基因表达的影响

原核生物、真核生物、植物体的非神经细胞和组织中,尤其是多种免疫活性细胞中,均证实乙酰胆碱酯酶（acetylcholinesterase,AChE）、胆碱乙酰转移酶（choline acetyltransferase,ChAT）和乙酰胆碱受体（acetylcholine receptor, AChR）各亚型在内的胆碱能系统组分的存在,其中烟碱样乙酰胆碱受体α7（nicotinic acetylcholine receptor α7,nAChRα7）是烟碱样胆碱能抗炎通路（nicotinic anti-inflammatory pathway）中重要的分子核心机制,同时也是机体限制宿主防御反应扩大的内源性抗炎机制之一. 本文旨在探讨（前）脂肪细胞上非神经元型胆碱能系统是否存在及初步揭示烟碱样胆碱能受体α7对前脂肪细胞功能的影响. 以体外培养的3T3-L1前脂肪细胞为研究对象,采用免疫组化和蛋白质免疫印迹技术,分别检测前脂肪细胞和成熟脂肪细胞中乙酰胆碱酯酶、胆碱乙酰转移酶和烟碱样乙酰胆碱受体α7的3种胆碱能系统主要组分的蛋白表达. 另将前脂肪细胞分为给予广谱烟碱样乙酰胆碱受体激动剂尼古丁、特异性烟碱样乙酰胆碱受体α7激动剂氯化胆碱及特异性烟碱样乙酰胆碱受体α7拮抗剂甲基牛扁亭碱干预12 h、24 h、36 h,并设立相应处理时间的空白对照组,逆转录聚合酶链反应检测前脂肪细胞visfatin mRNA表达情况. 免疫组化染色可见前脂肪细胞中AChE、ChAT及AChRα7均有阳性表达;蛋白免疫印迹检测进一步半定量证实了前脂肪细胞和成熟脂肪细胞中AChE、ChAT及AChRα7的蛋白表达;拮抗剂甲基牛扁亭碱(10⁶～10⁴mol/L)时间、剂量依赖性上调前脂肪细胞visfatin mRNA表达(1.3～1.55fold,P<0.01),与对应空白对照组相比,存在显著性统计学差异; 加入不同剂量的尼古丁和氯化胆碱,则前脂肪细胞中visfatin mRNA表达水平与对应空白对照组相比,均不同程度地下降,其中以氯化胆碱的抑制效应更为显著. 前脂肪细胞与成熟脂肪细胞中均存在有独立的胆碱能体系,其中AChRα7很可能在调节脂肪细胞因子分泌及肥胖相关的病理生理过程中发挥重要作用.     

EDITOR SPOTLIGHT: Interview with Cholinergic Mechanisms Special Issue Guest Editor Hermona Soreq

Hermona Soreq holds a Hebrew University Slesinger Chair in Molecular Neuroscience and is among the founding members of the Edmond and Lily Safra Center of Brain Sciences (ELSC). Soreq's research (H-impact: 98) focuses on acetylcholine (ACh)-related pathways and combines RNA-sequencing technologies, transgenic engineering, and molecular biology tests with in-depth analysis approaches. Her work addresses microRNAs (miRs) and transfer RNA fragments (tRFs) which have rapidly acquired wide recognition as global controllers of regulatory processes in healthy and diseased brain and body, including anxiety, inflammation, and cognition. Altogether, Soreq's work leads to molecular neuroscience-driven prevention and/or intervention with diseases involving impaired ACh signaling, including schizophrenia, bipolar disorder, Alzheimer's disease, and stress. Hermona led this Special Issue based on the 17th Symposium on Cholinergic Mechanisms (ISCM2022). We interviewed her on the progress in the field, what she wants to achieve as Senior Editor for the Gene Regulation and Genetics category at the Journal of Neurochemistry, key moments, and future directions.     

Rabbit blastocysts accumulate [3H]quinuclidinyl benzilate in vitro

Day-6 rabbit blastocysts were able to accumulate [3H]quinuclidinyl benzilate (QNB) from their environment. This accumulation was reduced approximately 50% in the presence of 1.5 x 10(-4) M atropine (an accepted antagonist for ligands which bind to muscarinic cholinergic receptors). The accumulation of QNB was sensitive to temperature and was apparently saturable. In the presence of 2 nM QNB, Day-6 blastocysts accumulated 30.3 +/- 2.0 fmoles per blastocyst. When the cellular elements alone were examined, lesser amounts of specific binding were detected. Owing to the complexity of this multicompartmental system, Scatchard analysis did not provide meaningful results. This accumulation appears higher than that reported for other tissues such as rabbit heart homogenates or rabbit uterine endometrial cells. This muscarinic cholinergic accumulation may have some roll in blastocyst-maternal recognition.     

Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [³H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [³H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [³H]inositol-l-phosphate. Incubation of slices withN-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [³H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4,5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.     

淋巴细胞上的非神经性乙酰胆碱系统

尽管乙酰胆碱作为神经递质存在于哺乳动物神经系统中的事实已广为人知,但近年来在淋巴细胞等非神经性组织和细胞中也发现了乙酰胆碱。淋巴细胞具备一个独立的非神经性乙酰胆碱系统,包括:乙酰胆碱、胆碱酯酶、胆碱乙酰转移酶、毒蕈碱乙酰胆碱受体和烟碱能乙酰胆碱受体等组分。免疫系统与淋巴细胞胆碱能系统之间可以相互作用。免疫刺激后的淋巴细胞可增强胆碱能系统的表达;激活后的乙酰胆碱受体参与淋巴细胞的免疫调节。淋巴细胞上胆碱能系统的这些发现将为相关疾病的研究和寻找有效的防治药物提供新的研究思路。     

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.     

Novel,testis‐specific mRNA transcripts encoding N‐terminally truncated choline acetyltransferase

Previous studies reported the presence of choline acetyltransferase (ChAT) mRNA and protein in the mammalian testis. We have now found that none of the ChAT mRNAs produced in the testis is capable of encoding a full‐length ChAT protein. Two ChAT cDNAs were isolated from an adult rat testis cDNA library encoding N‐terminally truncated ChAT proteins of 450 and 414 amino acids (aa), respectively, the former containing a novel N‐terminal extension of 69 residues. Rapid Amplification of cDNA Ends (RACE) analysis revealed a complex pattern of 5′ untranslated mRNA termini generated from the ChAT gene locus in the testis, all representing truncated versions of the ChAT enzyme. Two of these proteins were produced in transfected fibroblasts and found to lack ChAT activity. Neither did they show binding to the ChAT substrates, acetyl CoA and choline, in a competition assay. These results indicate that mammalian testis lacks a bona fide ChAT enzyme but expresses truncated ChAT proteins with a possible unique function to the testis. Mol. Reprod. Dev. 53:274–281, 1999. © 1999 Wiley‐Liss, Inc.     

Lack of an association between SNPs within the cholinergic receptor genes and smoking behavior in a Czech post-MONICA study

Smoking has a significant heritable component of approximately 30–60%. Recent genome wide association studies have identified single nucleotide polymorphisms (SNPs) within the nicotinic cholinergic receptor subunits 3 (rs578776), 5 (rs16969968) and β3 (rs6474412), which are associated with nicotine dependence in Western European populations. To analyze the association in a Czech population, we genotyped 1,191 males and 1,368 females (post-MONICA study). The WHO protocol was used to examine smoking status and the number of cigarettes smoked per day. There were 32.1% current and 27.6% past smokers among the males and 22.5% current and 13.8% past smokers among the females. We have not confirmed the original results: the SNPs rs16969968 (p = 0.07), rs578776 (p = 0.16) and rs6474412 (p = 0.76) were not associated with smoking status (never-smokers vs. ever-smokers) in the entire population, if a codominant model of analysis was used. This result was valid for both the male and female subpopulations if analyzed separately and adjusted for age. Finally, in ever-smokers, the number of cigarettes smoked per day was also independent of different genotypes, regardless of which polymorphism (and gender) was analyzed (the lowest p value was 0.49). The association between the cholinergic receptors–nicotinic subunits (-3, -5 and -ß3), and smoking behavior may be population-dependent.     

Intra- and extraganglionic peripheral cholinergic neurons in the urogenital organs of the cat

Summary The distribution of cholinergic neurons in the urinary tract and male genital organs of the cat was studied by a histochemical method for acetylcholinesterase. In addition to cell clusters in autonomic ganglia (intraganglionic cells), isolated extraganglionic cholinergic cells were found within the innervated tissues, usually in association with nerve trunks and blood vessels. Smaller neural cells with multiple axonal processes, identical to Cajal's interstitial cells, were found in the meshes of the terminal nerve plexus in smooth muscle, lamina propria and vascular wall.It is concluded that peripheral cholinergic neurons, like their adrenergic analogues, are arranged as a short intraganglionic, a shorter extraganglionic, and a terminal system of neurons.Supported in part by grants 10465 and 11285 from the USPHS and the Henry C. Buswell Urology Research Fund.     

Distribution and characteristics of nerve growth factor binding on cholinergic neurons of rat and monkey forebrain

In sections of rat forebrain, perikarya labeled radioautographically with¹²⁵I-NGF resembled cholinesterase-positive neurons in their distribution within striatum and basal forebrain. Neurons with NGF receptors were also visualized in radioautographs prepared from the basal forebrain of a cerebrus monkey. Present techniques fail to detect axons projecting from basal forebrain to hippocampus or cortex which have been shown to take up NGF selectively in retrograde transport studies. In studies with membrane-enriched preparations from rat, high-affinity binding of¹²⁵I-NGF (half maximal saturation in the 15–30 pM range) was detected in basal forebrain and striatum; lower levels of high-affinity binding were seen in hippocampus and neocortex. The binding and molecular properties of these receptors are similar to those described in other NGF-responsive tissues. These observations are further evidence supporting a biological role for NGF on some forebrain cholinergic neurons in adult rat.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Structural models of the nicotinic acetylcholine receptor and its toxin-binding sites

Models of the protein structure of agonist-, competitive antagonist-, and snake neurotoxin-binding sites were designed using the sequence of the first 54 residues of the acetylcholine receptor (AChR) subunit from Torpedo californica. These models are based on the premise that the N-terminal portions of the subunits form the outermost extracellular surface of the AChR and that agonists bind to this portion. The models were developed by predicting the secondary strucutre of the-subunit N-terminal segment from its sequence, then using these predictions to fold the segment into tertiary structures that should bind snake neurotoxins, agonists, and antagonists. Possible gating mechanisms and quaternary structures are suggested by the proposed tertiary structures of the subunits. Experiments are suggested to test aspects of the models.Supported by Armed Forces Radiobiology Research Institute, Defense Nuclear Agency, under Research Work Unit MJ 00032. The views presented in this paper are those of the author. No endorsement by the Defense Nuclear Agency has been given or should be inferred.     

Effect of atropine and gammahydroxybutyrate on inschemically induced changes in the level of radioactivity in [3H]inositol phosphates in gerbil brain in vivo

Brain ischemia in gerbils was induced by ligation of both common carotid arteries for 1 min or 10 min. Sham-operated animals served as controls. Intracerebral injection of [³H]inositol into gerbil brain 16 hr before ischemic insult resulted in equilibration of the label between inositol lipids and water-soluble inositol phosphate.A short ischemic period (1 min) resulted in a statistically significant increase in the radioactivity of inositol triphosphate (IP₃) and inositol monophosphate (IP), by about 48% and 79%, respectively, with little change in that of the intermediate inositol biphosphate (IP₂), which increased by about 16%. When the ischemic period was prolonged (10 min), an increase in the radioactivity of inositol monophosphate exclusively, by about 84%, was observed. The level of radioactivity in inositol phosphates IP₂ and IP₃ decreased by about 50%, probably as a consequence of phosphatase activation by the ischemic insult.The agonist of the cholinergic receptor, carbachol, injected intracerebrally (40 g per animal) increased accumulation of radioactivity in all inositol phosphates. The level of radioactivity in IP₃, IP₂, and IP was elevated by about 40, 23, and 147%, respectively.The muscarinic cholinergic antagonist, atropine, injected intraperitoneally in doses of 100 mg/kg body wt. depressed phosphoinositide metabolism in control animals. The level of radioactivity in water-soluble inositol metabolites in the brain of animals pretreated with atropine was evidently about 32% lower than in untreated animals.Pretreatment with atropine decreased the radioactivity of all inositol phosphates in the brain of animals subjected to 1-min ischemia and the radioactivity of IP in the case of 10-min brain ischemia. Gammabutyrolactone (GBL) administered intraperitoneally in the anesthetic dose 300 mg/kg body wt. diminished inositol monophosphate accumulation induced by either ischemic condition.Results from these in vivo studies are evidence that the blockage of cholinergic receptors by atropine depresses the response of phosphoinositides to physiological and particularly pathological stimuli.The results suggest that stimulation of the cholinergic receptor system is involved in the degradation of polyphosphoinositides during ischemia.     

Cholinergic-specific glycoconjugates

Cholinergic nerve terminals utilize glycoconjugates in several ways, as surface markers and as structural components of the synaptic vesicles present within them. The surface markers have been discovered immunochemically: antibodies raised against them are able specifically to sensitize the cholinergic subpopulation of mammalian brain synaptosomes to complement-mediated lysis. One such group of antigens (Chol-1) have been identified as a novel series of minor gangliosides having in common a sialylatedN-acetylgalactosamine residue. These gangliosides may constitute the major gangliosides at cholinergic terminals. A second surface antigen (Chol-2) is thought to be a protein with an epitope in common with aTorpedo electric organ ganglioside. Cholinergic synaptic vesicles are rich in a proteoglycan which appears to assist in the sequestration of acetylcholine within the vesicle and to stabilize the vesicle membrane during cycles of exocytosis and recovery. It may be the cholinergic equivalent of the chromogranins.Abbreviations AP affinity purified - ATPase adenosine 5-triphosphate phosphohydrolase - cer ceramido - ChAT choline acetyltransferase - Chol-1, –2 cholinergic-specific antigens - DA dopamine - DOG deoxyglucose - ELISA enzyme-linked immunosorption assay - EOD electric organ discharge - FAB fast atomic bombardment - GABA -aminobutyrate - GAG glycosaminoglycan - gal galactosyl - gaINAc N-acetylgalactosaminyl - glc glucosyl - Glu glutamate - 5-HT 5-hydroxytryptamine - LDH lactate dehydrogenase - NA noradrenaline - NGF nerve growth factor - S, S-S mono-, disialyl - SPM synaptosomal plasma membrane - TH tyrosine hydroxylase - TLC thin-layer chromatography - TSM Torpedo electromotor synaptosomal membrane - VIP vasoactive intestinal polypeptide - VPG vesicle proteoglycan Special issue dedicated to Dr. Leon Wolfe.     

Antisense inhibition of neuronal nicotinic receptors in the tobacco-feeding insect,Manduca sexta

Acetylcholine is the predominant excitatory transmitter in the insect central nervous system with many of its effects mediated by nicotinic acetylcholine receptors. These receptors are present at very high density and are structurally heterogeneous, although little is known about functional distinctions between them. An interesting system for examining these receptors is the larval stage of Manduca sexta, a nicotine-resistant tobacco-feeding insect. The nicotinic responses of cultured neurons were found to be blocked by mecamylamine and curare but highly resistant to alpha-bungarotoxin. The responses were also unaffected by the reducing agent dithiothreitol and the alkylating agent bromoacetylcholine suggesting that the alpha-subunit dicysteine agonist binding site is protected. To begin determining the functional roles of different subunits in these receptors, cultured neurons were treated with oligonucleotides based on the gene sequence of the alpha subunit, MARA1. Antisense DNA caused a significant downward shift in the amplitude distribution of nicotinic responses compared to sense or reverse antisense treatments. These treatments did not affect currents mediated by the application of GABA. The reduction in the nicotinic depolarization and inward currents did not affect the rate of current onset or recovery, suggesting that antisense MARA1 causes a partial block of all nicotinic responses in these neurons. These results demonstrate that receptor gene expression in insect neurons can be manipulated in a sequence-specific manner by antisense treatment and they provide evidence that MARA1 is important for normal nicotinic responses in Manduca.     

Acetylcholine synthesis in human CSF: Implications for study of central cholinergic metabolism

Investigation of neurological diseases involving central cholinergic dysfunction has led to numerous studies seeking a peripheral marker of cholinergic activity in brain. The main objective of these studies was to determine whether the ACh synthesizing activity present in human CSF was due to the presence of the enzyme choline acetyltransferase (ChAT; 68kDa). When CSF was fractionated into low and high molecular weight (M_r) components, 80% of the ACh synthesizing activity (AChSA) was found to be associated with the fraction <10 kDa. The remaining 20% was evenly distributed among fractions in the 5–30, 30–50, 50–300, and 300 kDa fractions. Although boiling destroyed all activity >10 kDa, the ChAT inhibitor NVP, at concentrations equal to or greater than that required to inhibit ChAT in human cortical tissue, did not alter the ACh-SA in either fraction. Results indicate that normal human CSF does not contain ChAT and all ACh-SA in CSF reflects non-enzymatic imidazole/histidine-like catalyzed synthesis.     

Sexually dimorphic responses to neonatal basal forebrain lesions in mice: II. Cortical morphology

Previous studies in the mouse have shown that neonatal lesions to the cholinergic basal forebrain (nBM) areas result in transient cholinergic depletion of neocortex and precipitate altered cortical morphogenesis. Lesion-induced morphological alterations in cortex persist into adulthood and are accompanied by behavioral changes, including spatial memory deficits. The current study investigated whether neonatal nBM lesions affect male and female mice differently in adulthood. Quantitative morphometry of cortical layer width was employed to assess alterations in cytoarchitecture in neonatally nBM-lesioned and littermate control mice of both sexes following behavioral testing. Our results showed significant decreases in cortical layer IV and V widths across somato/motor cortex in neonatally nBM lesioned mice of both sexes. Sexually dimorphic responses were observed in cortical layer II/III and total cortical width, limited to the area containing the “barrel cortex” representation of the whisker hairs. In lesioned females, layer II/III and total cortical width were decreased relative to female controls, and in lesioned males, layer II/III was increased relative to controls, whereas total cortical width was unchanged. In male but not female mice we observed significant correlations between decreased widths in layer IV and V and impaired performance on a spatial memory task. The current data further support a role of developing cholinergic cortical afferents in the modulation of cortical morphogenesis and cortical circuits involved in cognitive behaviors. In addition, our observations provide further evidence for sexually dimorphic development and function in cognitive centers of the rodent brain. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 595–606, 1998