Investigation of Histamine as a Neurotransmitter in the brain

1)组胺H_1受体基因的克隆：用[~3H]甲吡胺结合法检测组胺H_1受体在牛肾上腺髓质细胞膜上的表达，并获得大量的组胺H_1受体表达(Bmax=ca，700fmol/mg蛋白)。将牛肾上腺髓质提取的信使核糖核酸注入非洲爪哇卵母细胞后，细胞内有功能性组胺H_1受体的表达，肾上腺髓质信使核糖核酸的分级显示存在两种不同大小(2，5-3，5HB和5-6kb)的编码组胺H_1受体的信使核糖核酸。2)大鼠肝脏[~(3 H)]甲吡胺结合蛋白的纯化和性质：[~(3 H)]甲吡胺结合蛋白的溶解和纯化。纯化的结合蛋白经SDS-PAGE分析显示为单一条带，其相对分子质量为56K。纯化的蛋白是来自h…序列的脱氢异喹4-羟基酶家族，细胞色素P450利用cDNA库对纯化蛋白中12个Tryp-sin多肽的部分氨基酸序列进行进一步的基因组学分析。(3)培养的星形胶质细胞(AC)表达组胺H_1受体：用[~3H]甲吡胺结合实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，组胺H_2受体主要表达于1型星形胶质细胞。在组胺通过H_1受体诱导的2型星形胶质细胞中，使用Fura-2的钙荧光信号在过程中比在细胞胞体中更敏感，并且每个钙信号是独立的。组胺浓度的增加导致钙波从突起传播到细胞胞体。不同星形胶质细胞对不同神经活性物质的敏感性不同，提示星形胶质细胞的异质性。4)组胺及其相关物质对昼夜节律的影响：注射组胺(Icv)或突触前组胺H_3受体拮抗剂硫代巴比妥胺(IP)对大鼠自由跑昼夜节律参数有两种明显的相移效应。在主观暗期注射观察到的一个效应是最多4小时的暂时性提前，而在主观暗期早期注射组胺所引起的另一个效应是2小时的永久性相位延迟。据推测，这种效应来自一种独立的机制，不同于在快速光照变化下进行的再卷吸研究中的光的机制。结合哺乳动物昼夜节律的主要振荡器--交叉上核中组胺能终末的存在，我们得出结论：在昼夜节律延迟机制中，神经元组胺参与了生物节律的调节，组胺能神经元与光传递机制的相互作用。较少

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