| Co-Investigator(Kenkyū-buntansha) |
HAYCOCK John W. Professor, Medical Center, Biochemistry Dep. Lousiana State University, 教授
JOH Tong H. Professor, Medical College, Cornell University, 教授
FISCHER Edmond H. Professor, Biochemistry Dep., Univ. of Washington, 教授
HOUSER Carolyne Assistant of Professor, School of Medicine, UCLA, 助教授
JENDEN Donald Chairman and Professor, School of Medicine, UCLA, 教授
OLSEN Richard W. Professor, School of Medicine, UCLA, 教授
MIYOSHI Rie Research Associate, Tokyo Women's Medical college, 助手 (80209965)
YONEDA Yukio Associate Professor, Faculty of Medicine, Setsunan University, 薬学部, 助教授 (50094454)
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| Research Abstract |
The present study is to characterize excitatory amino acid receptors in the brain, focusing on the hippocampus and cerebellum. The used methods were binding experiments, in vitro receptor autoradiography and chemical analysis of receptor proteins. Emphasis was also put on age-related changes of binding characters and distributions of each subcomponent of NMDA receptor complex.
NMDA receptor is forming a complex with complicated structure consisting of numerous subcomponents such as glutamate binding site, glycine binding site, polyamine binding site and ion channel.
We found out that specific binding of NMDA recognition site was markedly enhanced' by pretreatment of Triton X-100. . This method made it possible to elucidate binding characteristics of NMDA receptor complex. More concretely, inhibition experiments of NMDA binding were done by glycine, D-serine, 6-7-di-chloroquinoxaline-2, 3-dione (DCQX), 7-chlorokynurenate (7-CIKYNA) and mechanisms of these inhibitions were analysed.
It has
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been an established fact that glycine potent iates NMDA binding. We performed experiments on recognition of glycine binding sites by 7-chlorokynurena (7-CIKYNA) and pointed out that this substance is useful for labelling strychnine-insensitive glycine binding sites. In relation with polyamine binding site, we performed H-spermidine 3binding experimens in the hippocampus and noticed that polyamine potentiated H-MK 801 binding.
Further experiments were done on how bivalent cations. influence on these inhibition experiments. Studies were also extended to comparison of binding characteristics of NMDA receptor between the hippocampus and cerebellum.
As for the part of receptor protein chemistry, NMDA-sensitive Hglutamate binding protein was solubilized and partially purified. The purified protein showed excellent binding ability.
Age-related changes of excitatory amino acid receptors were investigaed in the rat brain through quantitative in vitro autoradiography. Receptors for L-glutamate have been classified into three subtypes, termed the N-methyl-D-aspartate (NMDA), quisqualate and kainate types. Among them, it is demonstrated that the NMDA subtype is particularly important in learning and memory and in neuronal call death. In this study, we. focused on the NMDA receptor complex. Fischer 344 strain male rats aged 2 (young adult) and 21 (old) months were used in these experiments. The NMDA receptors showed the highest density in the CAl region of the hippocampus, especially in the stratum radiatum and stratum oriens. The adjacent stratum pyramidale had a relatively low concentration of binding. The cerebral cortex also contained high concentrations of the NMDA receptors and exhibited both regional and laminar variations. Moderate levels of receptors were found in the caudate-putamen, olfactory bulb, nucleus accumbens, septum, thalamus and amygdala. Midbrain regions and the cerebellar cortex showed low levels of receptors. Among the NMDA receptor complex, strychnine-insensitive glycine receptors which were labeled with H-glycine were severely reduced in tylencephalic regions in the aged rat brain. In midbrain regions, decreases of 'H-glycine binding 3s ites in parallel with age remained less than those in the telencephalon. ii-glycine binding sites in the cereyellum were not significantly changed. On the -other hand, binding sites of H-CPP (an anta 2onist for an NMDA recognition site) declined slightly in the forebrain area. H-CPP binging sites in the hippocampus did not change significantly in aged animals. Our results indicate that abnormalities of NMDA receptor regulation by glycine recognition sites play an important role in degeneration of neuroni during aging process. Less
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