Investigation of Histamine as a Neurotransmitter in the brain

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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