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328 Localization of prostaglandin I2 receptor in the brain stem
S46 3',,97
IMMUNOHISTOCHEMICAL LOCALIZATION OF ENDOTHELIN TYPE A RECEPTOR IN RAT BRAIN.
KIYOSHI KUROKAWA r HISAO YAMADA AND JUNZO OCHI, Department of the Anatomy, Shiga University of Medical Science, Otsu 520-21, Japan. Endothelin- 1, which was first biochemically discovered as a potent vasoconstrictor peptide, has been detected immunohistochemically in central nervous system(CNS). The binding sites are found in CNS, suggesting that endothelin may act as a neuromodulater. Using the anti-endothelin type A receptor antibody we produced, immunohistochemical analysis of the receptor was performed in rat brain. The intensely immunostaining neurons were found in substantia nigra (panes compacta et lateralis), locus ceruleus, nucleus tractus solitarii, nucleus pefiventicularis, nucleus arcuatus, zona incerta and lamina glomerulosa bulbi olfactorii. The moderately immunostaining neurons were found in nuclei paraventricularis et supraopticus. These nuclei correspond to the locations of catecholaminenergic neurons, especially of dopmaninergic neurons. Some of these nuclei belong to cardiovascular modulatory centers in CNS. These result may indicate that endothelin acts on the catecholamininergic neurons and influcences the blood pressure control in CNS.
328
LOCALIZATION OF P R O S T A G L A N D I N I2 RECEPTOR IN T H E BRAIN STEM KIYOSHI M A T S U M U R A , YUMIKO W A T A N A B E , HAJIME TAKECHI, H I R O T A K A ONOE A N D .YASUYOSHI W A T A N A B E . Subfemtomole BioreqoFlnition Proiect. Research Development Corr~oration of laoan and Det~artment of Neuroscience, Osaka Rio.science Institute. Suita. Osaka 565. lat3an.
An in vitro receptor autoradiography for PGI2 was performed in rat brain using [3tt]iloprost, a stable agonist for PGI2"receptors. Highest density of iloprost binding sites was found in the medial and commissural parts of the nucleus tractus solitarius (NTS) (111 fmol/mg tissue) and the area postrema (48 fmol/mg). Superficial layers of the spinal trigeminal nucleus (TRIG) (22 fmol/mg) and dorsal horn (DID (15 fmol/mg) of the spinal cord were also high in the density, bloderate density (5-10 fmol/mg) was found in the thalamus, hippocampus, cerebral cortex and dorsal cochlea nucleus, lloprost binding pattern was different from those of other prostaglandins so far studied (i.e. PGE1, PGE2, PGD2, and PGF2a), except for those of PGE1 and PGE2 in the NTS, TRIG and DIt. Precise comparison of binding sites for iloprost, PGE1 and PGE2 within the NTS in serial sections revealed difference in the distribution pattern between iloprost and PGEs. In the developmental course, iloprost binding sites in these three regions have already been expressed in the prenatal stage (at late embryonic day 20), whereas PGE2 binding sites were unclear in the background level. Finally, addition of high concentration (10/JM) of unlabelled iloprost to the incubation mixture almost completely abolished [3tt]iloprost binding, whereas the same treatment with 10/AM PGE2 only partially reduced [3tt]iloprost binding. These results indicate that iloprost bound to a specific receptor, that are distinct from those for other prostaglandins.
329
ANTIBODIES AGAINST A METABOTROPIC GLUTAMATE RECEPTOR, mGluR1, INHIBIT LONGTERM DEPRESSION IN CEREBELLAR CULTURE. RYUICHI SHIGEMOTO I SHI(~ETADA NAKANISHI2. ,AND TOMOO HIRANO 3, IDeot. of Morphol. Brain Sci., 2Inst. of Immunol., 3Dept. of Physiol., Fac. of Med. Kyoto Univ., Kyoto 606-01, _Japan. Two trpE-fusion proteins containing distinct extracellular sequences of the rat metabotropic glutamate receptor, mGluRl, were used to produce antibodies. On immunoblo~-, each of the antibodies specifically reacted with mGluRl in the rat cerebellum. The antibodies suppressed glutamate-induced increase of inositol phosphates in CHO cells transfected with mGluRl cDNA, but not in those transfected with mGluR5 cDNA. We applied these antibodies to cultured Purkinje cells (PC) to test their effects on long-term depression (LTD). A cultured PC was whole cell voltage-clamped at -80 mV and glutamate was applied by iontophoresis on a dendrite of PC once every 20 seconds. Following stable recordings of glutamate-induced current, PC was depolarized to 0 mV for 4 rain. This procedure depressed the amplitude of glutamateinduced current to about 70 % for more than 30 min. In the presence of each mGluR1 antibody, depression of the glutamate-induced current was completely inhibited. Tfiese results indicate that activation of mGluR1 is necessary for the induction of LTD in cultured PC and the mGluR 1 antibodies can be used as selective antagonists.
IMMUNOHISTOCHEMICAL LOCALIZATION OF ENDOTHELIN TYPE A RECEPTOR IN RAT BRAIN.
KIYOSHI KUROKAWA r HISAO YAMADA AND JUNZO OCHI, Department of the Anatomy, Shiga University of Medical Science, Otsu 520-21, Japan. Endothelin- 1, which was first biochemically discovered as a potent vasoconstrictor peptide, has been detected immunohistochemically in central nervous system(CNS). The binding sites are found in CNS, suggesting that endothelin may act as a neuromodulater. Using the anti-endothelin type A receptor antibody we produced, immunohistochemical analysis of the receptor was performed in rat brain. The intensely immunostaining neurons were found in substantia nigra (panes compacta et lateralis), locus ceruleus, nucleus tractus solitarii, nucleus pefiventicularis, nucleus arcuatus, zona incerta and lamina glomerulosa bulbi olfactorii. The moderately immunostaining neurons were found in nuclei paraventricularis et supraopticus. These nuclei correspond to the locations of catecholaminenergic neurons, especially of dopmaninergic neurons. Some of these nuclei belong to cardiovascular modulatory centers in CNS. These result may indicate that endothelin acts on the catecholamininergic neurons and influcences the blood pressure control in CNS.
328
LOCALIZATION OF P R O S T A G L A N D I N I2 RECEPTOR IN T H E BRAIN STEM KIYOSHI M A T S U M U R A , YUMIKO W A T A N A B E , HAJIME TAKECHI, H I R O T A K A ONOE A N D .YASUYOSHI W A T A N A B E . Subfemtomole BioreqoFlnition Proiect. Research Development Corr~oration of laoan and Det~artment of Neuroscience, Osaka Rio.science Institute. Suita. Osaka 565. lat3an.
An in vitro receptor autoradiography for PGI2 was performed in rat brain using [3tt]iloprost, a stable agonist for PGI2"receptors. Highest density of iloprost binding sites was found in the medial and commissural parts of the nucleus tractus solitarius (NTS) (111 fmol/mg tissue) and the area postrema (48 fmol/mg). Superficial layers of the spinal trigeminal nucleus (TRIG) (22 fmol/mg) and dorsal horn (DID (15 fmol/mg) of the spinal cord were also high in the density, bloderate density (5-10 fmol/mg) was found in the thalamus, hippocampus, cerebral cortex and dorsal cochlea nucleus, lloprost binding pattern was different from those of other prostaglandins so far studied (i.e. PGE1, PGE2, PGD2, and PGF2a), except for those of PGE1 and PGE2 in the NTS, TRIG and DIt. Precise comparison of binding sites for iloprost, PGE1 and PGE2 within the NTS in serial sections revealed difference in the distribution pattern between iloprost and PGEs. In the developmental course, iloprost binding sites in these three regions have already been expressed in the prenatal stage (at late embryonic day 20), whereas PGE2 binding sites were unclear in the background level. Finally, addition of high concentration (10/JM) of unlabelled iloprost to the incubation mixture almost completely abolished [3tt]iloprost binding, whereas the same treatment with 10/AM PGE2 only partially reduced [3tt]iloprost binding. These results indicate that iloprost bound to a specific receptor, that are distinct from those for other prostaglandins.
329
ANTIBODIES AGAINST A METABOTROPIC GLUTAMATE RECEPTOR, mGluR1, INHIBIT LONGTERM DEPRESSION IN CEREBELLAR CULTURE. RYUICHI SHIGEMOTO I SHI(~ETADA NAKANISHI2. ,AND TOMOO HIRANO 3, IDeot. of Morphol. Brain Sci., 2Inst. of Immunol., 3Dept. of Physiol., Fac. of Med. Kyoto Univ., Kyoto 606-01, _Japan. Two trpE-fusion proteins containing distinct extracellular sequences of the rat metabotropic glutamate receptor, mGluRl, were used to produce antibodies. On immunoblo~-, each of the antibodies specifically reacted with mGluRl in the rat cerebellum. The antibodies suppressed glutamate-induced increase of inositol phosphates in CHO cells transfected with mGluRl cDNA, but not in those transfected with mGluR5 cDNA. We applied these antibodies to cultured Purkinje cells (PC) to test their effects on long-term depression (LTD). A cultured PC was whole cell voltage-clamped at -80 mV and glutamate was applied by iontophoresis on a dendrite of PC once every 20 seconds. Following stable recordings of glutamate-induced current, PC was depolarized to 0 mV for 4 rain. This procedure depressed the amplitude of glutamateinduced current to about 70 % for more than 30 min. In the presence of each mGluR1 antibody, depression of the glutamate-induced current was completely inhibited. Tfiese results indicate that activation of mGluR1 is necessary for the induction of LTD in cultured PC and the mGluR 1 antibodies can be used as selective antagonists.