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Choline Uptake and Metabolism in Affinity-Purified Cholinergic Nerve Terminals from Rat Brain 总被引:5,自引:3,他引:2
Peter J. Richardson 《Journal of neurochemistry》1986,46(4):1251-1255
Cholinergic nerve terminals were affinity purified from rat caudate nucleus. These terminals possessed both high- (KT = 2.7 microM) and low- (KT = 58 microM) affinity uptake mechanisms for exogenous [3H]choline. The proportion of [3H]choline acetylated was reduced from 75 to 30% under conditions of anoxia and hypoglycaemia, whereas the phosphorylation of choline increased from 4 to 52%. Choline phosphorylation was also increased when the terminals were preloaded with choline. The affinity-purified terminals were shown to release acetylcholine in a Ca2+-dependent manner on depolarization. The relationship between choline acetylation and phosphorylation in the cholinergic nerve terminal is discussed. 相似文献
3.
Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum 总被引:2,自引:1,他引:1
Yasushi Ikarashi Akira Takahashi Hirohisa Ishimaru Tadashi Arai Yuji Maruyama 《Journal of neurochemistry》1997,69(3):1246-1251
Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D2 antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D2 agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake. 相似文献
4.
L. Eder-Colli P. A. Briand N. Pellegrinelli Y. Dunant 《Journal of neurochemistry》1989,53(5):1419-1427
Monoclonal antibodies were raised against the synaptosomal plasma membranes (SPMs) purified from the electric organ of the Torpedo. One antibody that reacts preferentially with SPMs rather than with other membrane fractions isolated from this tissue was previously found to inhibit hydrophilic and amphiphilic choline-O-acetyltransferase (ChAT) activity. On immunoblots of SPMs, this antibody recognizes two polypeptides of 135 and 66 kilodaltons that are related; the 66-kilodalton polypeptide appears to exist as a monomer and as a dimer in SPMs. The antibody was also able to inhibit the calcium-dependent release of acetylcholine in Torpedo synaptosomes without affecting the total neurotransmitter content. This inhibition was dependent on the antibody concentration and was observed when the release was elicited by either KCl depolarization or the calcium ionophore A23187; this suggests that inhibition was not mediated by a blockage of the depolarization-activated calcium influx. The inhibition could not be prevented by atropine, a result indicating that the antibody does not block release by mimicking the action of acetylcholine on presynaptic muscarinic autoreceptors. Thus, the antigen recognized by this antibody appeared to be involved in acetylcholine release; this antigen could be membrane-bound ChAT, another protein of the SPMs, or both. 相似文献
5.
Yvette Morot Gaudry-Talarmain Marie-Françoise Diebler Seana O''Regan 《Journal of neurochemistry》1989,52(3):822-829
We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process. 相似文献
6.
Increase in Exogenous Choline Fails to Elevate the Content or Turnover Rate of Cortical, Striatal, or Hippocampal Acetylcholine 总被引:1,自引:1,他引:0
Abstract: The present experiments were designed to test whether increasing the availability of choline to rat brain increases the rate of acetylcholine synthesis in that organ. The content of choline and acetylcholine and the turnover rate of acetylcholine in striatum, hippocampus, and cerebral cortex were measured following changes in dietary choline, intraperitoneal choline, or intravenous infusion of choline. Increasing plasma choline caused some increase in tissue choline but did not increase acetylcholine levels nor acetylcholine turn-over rate in any of the areas of brain studied. Indeed, in hippocampus, choline decreased the turnover rate of acetylcholine. 相似文献
7.
High affinity choline uptake (HACU) was investigated in the hippocampal formation following fetal septal cell suspension transplants into rats with fimbria-fornix lesions. Nine-14 weeks after transplantation, HACU was markedly decreased in hippocampi from animals with fimbria-fornix lesions; this decrease was ameliorated by fetal septal cells transplanted into the host hippocampus. HACU related to septal transplantation was activated in vitro by K+, and in vivo by the administration of scopolamine and picrotoxin. These findings suggest that fetal septal cell transplantation can restore HACU in the host hippocampus following fimbria-fornix lesions, and that HACU related to the graft has pharmacological properties similar to those of the normal adult HACU system. The activation of HACU by picrotoxin, a gamma-aminobutyric acid (GABA) antagonist, suggests that transplanted cholinergic neurons receive either direct or indirect functional input from GABAergic afferents from the transplant and/or host hippocampus. Lesions of the fimbria-fornix also resulted in an increased binding to muscarinic receptors in the dorsal hippocampus. This increase in binding was not significantly ameliorated by intrahippocampal grafts of cholinergic neurons. 相似文献
8.
Koichiro Kawashima Toru Hayakawa Yuko Kashima Takeshi Suzuki Kazuko Fujimoto Hisayo Oohata 《Journal of neurochemistry》1991,57(3):882-887
A vertical-type in vivo microdialysis probe and a sensitive, specific radioimmunoassay (RIA) were used to study the mechanism of acetylcholine (ACh) release in the striatum of anesthetized rats. Without the use of physostigmine, a cholinesterase inhibitor, our RIA could still detect the amount of ACh present in the perfusate (5.6 +/- 0.6 fmol/min, n = 16). Tetrodotoxin (1 microM) produced a significant decrease in the amount of ACh collected in the perfusate, suggesting that basal ACh determined under the present experimental conditions was related to cholinergic neural activity. Atropine (0.1-1 microM) applied topically via the dialysis probe did not affect the amount of ACh recovered in the perfusate in the absence of physostigmine. Addition of physostigmine (10 microM) to the perfusion fluid produced about a 100-fold increase in the amount of ACh collected. In the presence of physostigmine, topical administration of atropine and pirenzepine (0.01-1 microM) through a dialysis probe produced a further three- to fourfold increase in ACh output, whereas a slight increase was produced by AF-DX 116 at the highest concentration (1 microM). These results indicate that presynaptic modulation of ACh release in the striatum does not occur under basal conditions, and that presynaptic M1 muscarinic receptors are involved in the modulation of ACh release when the ACh concentration is raised under certain conditions. 相似文献
9.
Kunio Koshimura Soichi Miwa Ken Lee Yasunori Hayashi Hiroshi Hasegawa Keigo Hamahata Motohatsu Fujiwara Mieko Kimura Yoshinori Itokawa 《Journal of neurochemistry》1990,54(2):533-539
The purpose of the present study is to clarify the effects of the administration of choline on the in vivo release and biosynthesis of acetylcholine (ACh) in the brain. For this purpose, the changes in the extracellular concentration of choline and ACh in the rat striatum following intracerebroventricular administration of choline were determined using brain microdialysis. We also determined changes in the tissue content of choline and ACh. When the striatum was dialyzed with Ringer solution containing 10 microM physostigmine, ACh levels in dialysates rapidly and dose dependently increased following administration of various doses of choline and reached a maximum within 20 min. In contrast, choline levels in dialysates increased after a lag period of 20 min following the administration. When the striatum was dialyzed with physostigmine-free Ringer solution, ACh could not be detected in dialysates both before and even after choline administration. After addition of hemicholinium-3 to the perfusion fluid, the choline-induced increase in ACh levels in dialysates was abolished. Following administration of choline, the tissue content of choline and ACh increased within 20 min. These results suggest that administered choline is rapidly taken up into the intracellular compartment of the cholinergic neurons, where it enhances both the release and the biosynthesis of ACh. 相似文献
10.
Dissociated rat septal nucleus cells cultured in defined medium exhibited twofold increases in the maximal rates of sodium-dependent, high-affinity choline uptake and acetylcholine formation when grown in the presence of phosphoethanolamine. The effect was concentration-dependent (EC50 = 15 microM) and appeared to be associated with in vitro maturation of cholinergic neurons rather than with enhanced survival. Choline acetyltransferase, acetylcholinesterase, and choline kinase activities were unaffected by this treatment. The effect of phosphoethanolamine was specific for cholinergic neurons, because treatment with this compound did not alter the kinetic constants for high-affinity neuronal uptake of gamma-aminobutyric acid or dopamine. The action appeared to be mediated primarily through activation of the sodium-dependent, high-affinity transport mechanism for choline as opposed to alterations in the storage and release of acetylcholine. 相似文献
11.
The extracellular concentration of dopamine (DA) and 3,4-dihydroxyphenylacetic acid in the substantia nigra (SN) and striatum was estimated by microdialysis. The dialysate content of DA from the SN was recorded during infusion of a DA uptake blocker (nomifensine; 5 mumol/L) dissolved in the perfusion fluid. Perfusion of tetrodotoxin (1 mumol/L) produced a virtually complete disappearance of nigral and striatal DA release. Dendritic as well as terminal release of DA was inhibited for several hours when the nerve impulse flow in dopaminergic neurons was blocked by systemic administration of gamma-butyrolactone (750 mg/kg, i.p.). The systemic administration (0.3 mg/kg, i.p.) as well as infusion (1 mumol/L) of the D2 agonist (-)-N-0437 [2-(n-propyl-N-2-thienylethylamino)-5-hydroxytetralin] produced a significant decrease in the release of DA in both the striatum and the SN. DA levels were recorded in the striatum both with and without addition of nomifensine to the perfusion fluid. The decrease in the striatum after (-)-N-0437 was suppressed in the presence of nomifensine. Infusion (1 mumol/L) as well as systemic administration (40 mg/kg) of sulpiride caused a similar increase in the release of striatal DA; this increase was, in both experiments, potentiated by nomifensine coinfusion. Sulpiride administration induced a small increase in the release of nigral DA. Infusion of (-)-N-0437 or (-)-sulpiride into the nigra caused a moderate decrease and increase, respectively, of striatal DA level.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Abstract: The activity of choline acetyltransferase was used as an index of cholinergic structures in regions of rat brain. The activities of ATP citrate lyase and choline kinase correlated poorly with cholinergic activity in whole tissue fractions, contrasting with the good correlation between acetylcholinesterase and choline acetyltransferase. Choline acetyltransferase was preferentially localised in synaptosomes prepared from regions of high (striatum) or intermediate (cortex, medulla oblongata/pons) cholinergic activity. In general, this was not true for either choline kinase or ATP citrate lyase. 相似文献
13.
Identification of Sodium-Dependent, High-Affinity Choline Uptake Sites in Rat Brain with [3 H]Hemicholinium-3 总被引:1,自引:1,他引:0
[3H]Hemicholinium-3 (HC-3) was used to label sodium-dependent, high-affinity choline uptake sites in regions of rat brain. Autoradiography revealed a high density of [3H]HC-3 binding sites in brain regions with a high density of cholinergic terminals, such as the interpeduncular nucleus, caudate-putamen, and olfactory tubercle. This distribution of [3H]HC-3 binding sites was in close agreement with the amounts of choline acetyltransferase in specific nuclei and subregions of rat brain. Destruction of presynaptic cholinergic projections in the cerebral cortex and the basal ganglia by injection of excitotoxins reduced [3H]HC-3 binding by 40-50%. These data indicate that sodium-dependent [3H]HC-3 binding sites are related to the choline transport system present in cholinergic neurons. 相似文献
14.
Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild-type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for approximately 24% of the cell cytosolic proteins as being ChAT. The apparent Km values of the enzyme for choline and acetyl-CoA were 299 and 221 microM, respectively, whereas the respective Vmax values were 10.6 and 11.4 mumol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one-step purification procedure starting with 10(8) infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore-mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level. 相似文献
15.
Abstract: The posterior stomach nerve (PSN) is a crustacean sensory nerve containing about 60 cholinergic neurons, which are devoid of synaptic interactions. Kinetic analysis shows that the PSN takes up [3 H]choline by both low-affinity ( K m = 163 μM) and high-affinity (Na+ -dependent) ( K m = 1 μM) processes. The capacity of the high-affinity system is only about 1% that of the low-affinity system. The high-affinity system is not tightly coupled to acetylcholine (ACh) synthesis, and it appears that both ACh and phosphorylcholine are formed from an intracellular pool of choline, which is fed by both uptake systems. There are differences in the rates of [3 H]choline uptake and 3 H metabolite accumulation between regions of the PSN that contain neuronal cell bodies and those that do not. These differences may arise from differences in the relative proportion of neuronal to nonneuronal tissue in each nerve region. 相似文献
16.
Masato Ando Mitsuyoshi Iwata Kazuya Takahama Yutaka Nagata 《Journal of neurochemistry》1987,48(5):1448-1453
The activities of choline kinase (CK) and choline acetyltransferase (ChAT) were examined in vitro in superior cervical sympathetic ganglia (SCG) excised from rats following aerobic incubation for 1 h in a medium containing various choline concentrations, with and without application of a high KCl level (70 mM). Ganglionic CK activity was strongly inhibited (by approximately 75%) at low extracellular choline concentrations (1-5 microM) but rose as the choline concentration was raised to 10-50 microM in the incubation medium, then fell and rose again with further increases in choline concentration. A similar but moderate accelerative effect on ganglionic CK activity was also observed after addition of acetylcholine (ACh; 1 mM) without eserine. Whereas specific CK activity did not change significantly in axotomized SCG, in which the ratio of glial cells to neurons is greatly increased for a week after the operation., it was remarkably increased after denervation, in which the preganglionic cholinergic nerve terminals had degenerated. When either a high KCl level or hemicholinium-3 (HC-3; 50 microM) was added to the medium in the presence or absence of choline, ganglionic CK activity was markedly inhibited. On the other hand, ChAT activity in the SCG remained at a significantly high level during incubation with low choline concentrations (1-10 microM), but the enhanced enzyme activity became inhibited as the extracellular choline concentration was raised to 50-100 microM in the medium. Addition of HC-3 to the medium did not alter ganglionic ChAT activity at low choline concentrations. However, application of quinacrine (10 microM) considerably reduced ganglionic CK activity and also suppressed ChAT activity induced by high KCl levels.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Abstract: We studied the effect of α-latrotoxin (αLTX) on [14C]acetylcholine ([14C]ACh) release, intracellular Ca2+ concentration ([Ca2+]i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10?10-10?8M) caused an elevation of the [Ca2+]i as detected by Fura 2 fluorescence and evoked [14C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na+ in the external medium and another that did not. Displacement of Na+ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca2+]i and [14C]ACh release by αLTX. The Na+-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca2+]i and [14C]ACh release remained in the absence of Na+. Both the Na+-dependent and the Na+-independent components of the αLTX-evoked [14C]ACh release partly required the presence of either Mg2+ or Ca2+. The nonneurotransmitter [14C]choline was released along with [14C]ACh, but this release did not depend on the presence of either Na+ or Ca2+, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na+-dependent divalent cation entry and (b) Na+-independent divalent cation entry, and (2) nonspecific Na+- and divalent cation-independent leakage. 相似文献
18.
Release of Ethanolamine, but Not of Serine or Choline, in Rat Pontine Nuclei on Stimulation of Afferents from the Cortex, In Vivo 总被引:1,自引:0,他引:1
Henry Perschak Max Wolfensberger Kim Quang Do Yves Dunant Michel Cuénod 《Journal of neurochemistry》1986,46(5):1338-1343
Release of ethanolamine, serine, and choline in rat pontine nuclei on electrical stimulation of afferents from the cortex was investigated using in vivo push-pull cannula techniques. Ethanolamine was determined by using gas chromatographic techniques; serine was measured with a HPLC system; and choline was assayed with a luminescence method. Resting elution rates of ethanolamine, serine, and choline were 50.8 +/- 8.4, 34.8 +/- 12.6, and 1.16 +/- 0.20 pmol/5 min, respectively. Stimulation of the cortico-pontine tract evoked a highly significant 3.4-fold increase in release of ethanolamine, whereas serine and choline release was unaffected. Reactions in membrane phospholipids are most likely involved in the stimulation-dependent release of ethanolamine and special consideration was given to base-exchange reactions. Alternatively, a release from intracellular, possibly synaptic stores cannot be excluded. 相似文献
19.
We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control. 相似文献
20.
Jordi Alberch Esther Pérez-Navarro Ernest Arenas Jordi Marsa 《Journal of neurochemistry》1991,57(5):1483-1487
The role of nerve growth factor (NGF) and its receptor (NGFR) in the regulation of cholinergic activity has been studied during the aging process. NGFRs were quantified in cortical membranes using a radioactive binding assay. NGF levels and choline acetyltransferase (ChAT) activity were determined in cortex, hippocampus, neostriatum, and septum. These assays were performed in both adult (6-month-old) and aged (36-month-old) rats. High- and low-affinity 125I-NGF binding sites were present in cortex of adult and aged rats. Furthermore, we observed a decrease in number and affinity of both NGFRs in aged rats. ChAT activity in these rats was lower (approximately 30%) than in adult rats in all the brain regions examined. NGF levels were not modified in cortex and hippocampus and were decreased in neostriatum (55%) and septum (35%). In conclusion, our results suggest that, during the aging process, the cholinergic impairment is related to a decrease in NGF levels in neostriatum but not in cortex and hippocampus. The reduction in level of NGF protein in septum could be due to a decrease in number of high-affinity 125I-NGF binding sites. 相似文献