8-Br-cyclic GMP mimics activation of muscarinic autoreceptor and inhibits acetylcholine release from rat hippocampal slices
8-Br-cyclic GMP mimics activation of muscarinic autoreceptor and inhibits acetylcholine release from rat hippocampal slices
复制标题
8-Br-环状 GMP 模拟毒蕈碱自身受体的激活并抑制大鼠海马切片中乙酰胆碱的释放
DOI：
10.1016/0006-8993(81)90255-9
复制
发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
T. Bártfai
中科院分区：
文献类型：
--
作者：
Ö. Nordström;T. Bártfai
在大鼠海马乙酰胆碱通过刺激毒蕈碱乙酰胆碱受体抑制其自身的释放12，13。在河豚毒素的存在下，对来自海马的分离的神经末梢的悬浮液进行的实验表明，在没有激活神经元环路的情况下施加毒蕈碱反馈调节1。胆碱能神经末梢上的毒蕈碱型自身受体可能介导[~ 3 H]乙酰胆碱（[~ 3 H] ACh）I释放的抑制。刺激周围和中枢神经系统中的毒蕈碱受体导致环GMP水平增加2，6，9。本研究解决的问题，是否胆碱能激动剂的影响，在毒蕈碱自身受体，介导的环GMP。用二丁酰环鸟苷酸作初步实验表明可能是这种情况。1.用Mcllllovian组织切碎器将大鼠右冠沿矢状方向切成0.4mm厚的薄片。在Krebs-Ringer缓冲液中，将切片与1 μ M胆碱和50 nM作为示踪剂1的[3 H]胆碱（[3 H] Ch）孵育（NaCl，138 mM; KCl，5 mM; MgCl 2，1 mM; NaH-PO 4，1 mM; NaHCO 3，11 mM;葡萄糖，10 mM NaOOCCH 3，1 mM;和CaCl 2，2 mM）在37 ℃下用95 ℃ O2和5 ℃ CO2连续鼓泡40分钟。当使用含有25 mM KBr的Krebs-Ringer缓冲液（而不是5 mM KBr）时，Na+浓度降低20 mM以维持等渗透压。在37 ℃下研究了[aH] ACh和[aH] Ch的诱发（K = 25 mM）释放以及药物对其的影响。[3H]用庚酮萃取[aH] ACh分离ACh和[3 H] Ch。后者在Lumagel的Beckman闪烁光谱仪（来自Lumac System AG，巴塞尔）中除去溶剂后计数。根据Folbergrov~ iS方法从[~ 3 H] ACh释放实验中所用的切片中提取环GMP。将含有EDTA（lmM）的lml高氯酸（0.3M）加入到每个切片（< 10 mg蛋白质）中，并将样品在干冰上快速冷冻。解冻样品后，加入0.1 ml HCl（0.1 M）的甲醇溶液，并对样品进行超声处理。将超声处理物离心，并将蛋白质沉淀，preeipita-
In the rat hippocampus acetylcholine depresses its own release via stimulation of muscarinic acetylcholine receptors12, 13. Experiments carried out, in the presence of tetrodotoxin, on a suspension of isolated nerve-endings from hippocampus indicated that the muscarinic feedback regulation is exerted without activation of neuronal loops 1. It was concluded that muscarinic autoreceptors localized on cholinergic nerveendings may mediate inhibition of the release of [aH] acetylcholine ([3H] ACh) I. Stimulation of muscarinic receptors on the periphery and in the central nervous system leads to increases in cyclic GMP levels2, 6, 9. The present study addresses the question of whether the effects of cholinergic agonists, at the muscarinic autoreceptor, are mediated by cyclic GMP. Preliminary experiments with dibutyryl cyclic GMP indicated that this may be the case 1.Rat hippocampi were cut into 0, 4 mm thick slices by a Mcllwain tissue chopper in sagittal direction. The slices were incubated with 1/~ M choline and with [3H] choline ([3H] Ch) 50 nM as a tracer 1, in Krebs-Ringer's buffer (NaC1, 138 mM; KC1, 5 raM; MgC12, 1 raM; NaH~ PO4, 1 mM; NaHCO3, 11 mM; glucose, 10 mM NaOOCCHa, 1 mM; and CaC12, 2 mM) for 40 mio at 37 C under continuous bubbling with 95~ 02 and 5~ COz. When Krebs-Ringer's buffer with 25 mM K÷ was used (instead of 5 mM K÷) the Na+ concentration was lowered by 20 mM to maintain iso-osmolarity. The evoked (by K÷ 25 mM) release of [aH] ACh and [aH] Ch and the effect of drugs on it was studied for 5 min at 37 C.[3H] ACh and [3H] Ch were separated by heptanon extraction s of [aH] ACh. The latter was counted after removal of the solvent in a Beckman scintillation spectrometer in Lumagel, from Lumac System AG, Basel. Cyclic GMP was extracted from the slices used in the experiments on [3H] ACh release according to Folbergrov~ iS. One ml perchloric acid (0.3 M) containing EDTA (1 raM) was added to each slice (< 10 mg protein) and the samples were rapidly frozen on dryice. After thawing the samples, 0.1 ml HCI (0.1 M), in methanol was added and the samples were sonicated. The sonicate was centrifuged and the protein pellet, preeipita-