Investigation of Histamine as a Neurotransmitter in the brain

组胺作为大脑神经递质的研究

• 批准号: 63480120
• 负责人: FUKUI Hiroyuki
• 金额: $ 4.48万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480120/
• 关键词: Histamine; Histamine H_1-receptors; Xenopus laevis oocytes; [ ^3H] mepyramine binding; Debrisoquine 4- hydroxylase; Astrocyte; Fura-2; Circadian rhythm; [ ^3H]メピラミン結合試験; H_1受容体; 初代培養; Fura 2; イノシト-ルリン酸; 精製; チトクロ-ムP450; アストログリア細胞; 受容体; 星状膠

1) Cloning of histamine H_1-receptor cDNA :The expression of histamine H_1-receptors in the membranes from bovine adrenal medulla was examined by [ ^3H] mepyramine binding assay and and larger amount (Bmax = ca, 700 fmol/mg protein) of H_1-receptors was expressed. Injection of mRNA prepared from bovine adrenal medulla into Xenopus laevis oocytes resulted in the expression of functional histamine H_1-receptors in the cells, fractionation of adrenal medullary mRNA revealed the existence of two different sizes (2,5-3,5 Hb and 5-6 Kb) of mRNA that encode histamine H_1-receptors.2) Purification and characterization of [ ^3H] mepyramine binding protein from rat liver :[ ^3H] Mepyramine binding protein was solubilized and purified. The purified binding protein showed a single band on SDS electrophoresis and the molecular weight of the purified protein was determined to be 56K. The purified protein revealed to be a family of debrisoquine 4-hydroxylase (DB), cytochrome P450, from the sequence h … More omology analysis of partial amino acid sequences of 12 tryp-sin peptides from the purified protein with cDNA data bank. The purified [ ^3H] Mepyramine binding protein was positive on Western blotting with anti-DB antibody but the molecular weight of the binding protein was larger than that of DB.3) Histamine H^1-receptors expressed on cultured astrocytes (AC) :The expression of histamine H_1-receptors on cultured astrocytes was demonstrated by [ ^3H] mepyramine binding assay. Histamine H_1-receptors were expressed mainly on type-2 astrocytes and H_2-receptors were expressed on type-1 astrocytes. Fluorescent Ca^<--> signals using fura-2 in type-2 astrocytes induced by histamine through H_1-receptors was much more sensitive in processes than in cell soma and each Ca^<--> signal was independent. Increased concentration of histamine resulted in the propagation of Ca^<--> wave from processes to the cell soma. Each astrocyte was different in sensitivity to various neuro-active substances and the results suggested the heterogeneity of astrocytes.4) Effect of histamine and its related substances on circadian rhythm :Injections of histamine (icv) or thioperamide (ip), an antagonist of presynaptic histamine H_3-receptors, caused two distinct Phsae-Shifting effect on free-running circadian parameters of rats. One effect observed by injections during subjective light period was a transient phase-advanced at the maximum of 4 hrs, and the other effect elicited by histamine injections during the early hours of subjective dark period was a permanent phase delay of 2 hrs. And this effect is speculated to derive from an independent mechanism from that of light from a reentrainment study performed under rapid illumination changes. The results together with the existence of the histaminergic terminals in the superchiasmatic nucleus, the major oscillator of mammalian diurnal rhythms, enabled us to conclude that neuronal histamine is involved in the modulation of the bioligical rhythms, and the interaction of histaminergic neurons with light relaying mechanism when circadian rhythmic phase delaying mechanism in concerned. Less

1)组胺H_1受体cDNA的克隆：用[ ^3H]-甲苯胺结合试验检测了牛肾上腺髓质细胞膜上组胺H_1受体的表达，发现有大量的H_1受体表达（Bmax =约700 fmol/mg蛋白）。将牛肾上腺髓质mRNA注入非洲爪蟾卵母细胞，可使卵母细胞表达功能性组胺H_1受体，肾上腺髓质mRNA的分级显示存在两种不同的大小（2）大鼠肝脏[ ^3H]美托咪胺结合蛋白的纯化和性质：溶解并纯化[ ^3H]美吡胺结合蛋白。纯化的结合蛋白经SDS电泳显示单一条带，分子量为56 K。纯化的蛋白质显示为异喹胍4-羟化酶（DB）家族，细胞色素P450，来自序列h ...更多信息 利用cDNA数据库对纯化蛋白中12个胰蛋白酶肽的部分氨基酸序列进行了同源性分析。纯化的[ ^3H]美吡胺结合蛋白与DB抗体的Western印迹反应呈阳性，但结合蛋白的分子量大于DB。3）培养的星形胶质细胞表达组胺H_1受体（AC）：用[ ^3H]美吡胺结合试验证实培养的星形胶质细胞表达组胺H_1受体。组胺H_1受体主要表达于2型星形胶质细胞，H_2受体主要表达于1型星形胶质细胞。组胺<-->通过H_1受体诱导的2型星形胶质细胞Fura-2荧光Ca ~（2+）信号在细胞突起中比在细胞索马中更敏感，且各Ca ~（2+）<-->信号是独立的。组胺浓度增加，Ca ~（2+）<-->波由突起向细胞索马胞体传播。4）组胺及其相关物质对大鼠昼夜节律的影响：注射组胺（icv）或突触前组胺H_3受体拮抗剂硫代哌丁胺（ip）对大鼠自由活动的昼夜节律参数产生两种明显的移相效应。在主观光照期注射观察到的一种效应是最多4小时的短暂相位提前，在主观黑暗期早期注射组胺引起的另一种效应是2小时的永久相位延迟。这种效应被推测来自一个独立的机制，从光的再夹带研究下进行快速照明变化。结合哺乳动物昼夜节律的主要振荡器--视交叉上核中组胺能终末的存在，我们认为神经元组胺参与了生物节律的调节，并在考虑昼夜节律相位延迟机制时，参与了组胺能神经元与光中继机制的相互作用。少

项目成果

N.Inagaki: "Characterization of histamine H_1ーreceptors on astrocytes in primary culture: ^3H mepyramine binding studies." Eur.J.Pharmacol.,. 173. 43-51 (1989)

N.Inagaki：“原代培养物中星形胶质细胞上组胺 H_1 受体的表征：^3H 美吡拉明结合研究。”Eur.J.Pharmacol., 173. 43-51 (1989)

N.Itowi: "Effect of histsmine depletion on the circadian amplitud of the sleepーwakefulness cycle." Physiol.Behave.,. 49. (1991)

N.Itowi：“组胺消耗对睡眠-觉醒周期的昼夜节律幅度的影响。” Physiol.Behave 49。（1991）

M.Hide: European J.Pharamcology. 148. 161-169 (1988)

M.Hide：欧洲药理学杂志。

H.Fukui: "Dependency of histamine induced phasic and tonic contractions on intracellular and extracellular calcium in guinea pig tracheal smooth muscle." Jpn.J.Pharmacol.50. 125-130 (1989)

H.Fukui：“组胺诱导的豚鼠气管平滑肌的阶段性和强直性收缩对细胞内和细胞外钙的依赖性。”

H.Fukui: "Solubilization,characterization and partial purification of ^3H mepyramineーbinding protein,a possible histamine H_1ーreceptor from rat liver." Jpn.J.Pharmacol.,. 46. 127-139 (1988)

H.Fukui：“^3H 美吡拉明结合蛋白（大鼠肝脏中可能的组胺 H_1 受体）的溶解、表征和部分纯化。”Jpn.J.Pharmacol., 46. 127-139 (1988)