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Localization of glycine receptor α1-subunit mRNA-containing neurons in the rat brain
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LOCALIZATION OF GLYCINE RECEPTOR a 1-SUBUNIT mRNA-CONTAINING NEURONS IN THE RAT BRAlN. KOUJI SATOH~ TAKANORI S A I K A ~ , J - H ZHANG~-~ MAKOTO SATOH,KEIYA TADA. AND MASAYA TOHYAMA.. D e p a r t m e n t of Anatomy II, Osaka University Medical School, Yamadaoka, Suita, Osaka 565, Japan. The localization of g l y c i n e r e e e t o r s in the r a t b r a i n w a s e x a m i n e d b y m e a n s of in situ hybridization histochemistry using an olig0nucle0tide probe to the sequence of the ~ I s u b u n i t . Stronglyor m o d e r a t e l y labeled neurons were m a i n l y f o u n d in the l o w e r b r a i n s t e m . Our data are almost consistent with the data of previous labeled ligand binding experiments. In a d d i t i o n , we compare this d a t a w i t h l o c a l i z a t i o n of o t h e r s u b u n i t s ( ~ a n d 9 3 K D p r t e i n ) of s t r y c i n i n e sensitive glycine receptor. We find that discrepancy exists between 10calizations of t h e s e subunits. For example, although in the l o w e r b r a i n s t e m , we c a n f i n d all t h r e e s u b u n i t s , o n l y ~ s u b u n i t w a s d e t e c t e d a n d w e c a n ' t find other two subunits in the c e r e b r a l c o r t e x . T h e s e r e s u l t s s u g g e s t that glycine receptor has heterogeneity in its c o m p o s i t i o n .
MECHANISM OF POLYAMINE-INDUCED MODULATION OF THE NMDA RECEPTOR COMPLEX. YUKIO YONEDA, KIYOKAZU OGITA, AND RIYO ENOMOTO*, Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-O1, Japan. Polyamine spermidine (SPD) markedly potentiated the binding of [3H](+)-5-methyl-lO, lldihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) to open cationic channels associated with the N-methyl-D-aspartate (NMDA)-sensitive subclass of excitatory amino acid receptors in the presence of a maximally effective concentration of both L-glutamic acid (Glu) and glycine (Gly) in brain synaptic membranes treated with Triton X-100. Among several natural and synthetic polyamines tested, spermine was most effective in potentiating the binding followed by bis-(3-aminopropyl)amine, SPD and 1,3-diaminopropane in that order. The potentiation by SPD was noncompetitively prevented by a competitive type of antagonists at the Gly as well as NMDA sites. The densities of [3H]MK-801 binding sites were unevenly distributed in the rat brain, while the affinities were not significantly different from each other. The hippocampus had the highest density in the presence of Glu/Gly with progressively lower densities in the cerebral cortex, striatum, hypothalamus, midbrain, cerebellum and medulla-pons. Further addition of SPD significantly increased the affinities in structures with a high density without altering the density itself, without changes in either the affinity or density in the cerebellum. The positive polyamines were all ineffective in potentiating the binding in the cerebellum with a concomitant potentiation of that in the hippocampus and cerebral cortex. These results suggest that heterogeneous NMDA receptor complex is present in the cerebellum in terms of differential sensitivity to potentiation by polyamines.
SOLUBILIZATION OF THE NMDA RECEPTOR COMPLEX FROM THE RAT BRAIN. KIYOKAZU OGITA, TAKEO SUZUKI*, AND YUKIO YONEDA, Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-01, Japan. The detergent deoxycholic acid (DOC) was most effective in solubilizing the binding activity of [3H](+)-5-methyl-lO,ll-dihydro-5H-dibenzo[a,d]cyclohepten-5,10_imine (MK-801) to open cationic channels associated with the N-methyl-D-aspartate (NMDA)-sensitive subclass of excitatory amino acid receptors from rat brain menforanes among various detergents tested. Neither L-glutamic acid (Glu) nor glycine (Gly) potentiated the binding at concentrations below 1 ram in solubilized supernatants, while the binding was significantly inhibited by the addition of competitive NVLDA and Gly antagonists. In contrast, both amino acids were effective in potentiating the binding in a concentration-dependent manner in preparations after gel filtration on a Sephadex G-25 column of solubilized supernatants. Polyamine spermidine (SPD) markedly potentiated the binding in preparations both before and after gel filtration. In preparations after gel filtration, either Glu alone or Glu/Gly markedly accelerated the initial association rate of [3H]MK-801 binding without significantly affecting the binding at equilibrium, whereas SPD did not additionally facilitate the initial association rate found in the presence of both Glu and Gly with a concomitant elevation of the steady-state level. ~ o n g a variety of NF~A channel blockers, (+)-MK-801 exhibited the most potent displacement of the binding in the presence of all three different stimulants in preparations after gel f i l t r a t i o n followed by (-)-MK-801, N - [ l - ( 2 - t h i e n y l ) c y c l o h e x y l ] p i p e r i d i n e , phencyclidine, N-allylnormetazocine and ketamine in that order. These results suggest that the whole supermacromolecular NMDA receptor complex is co-solubilized under the experimental conditions employed.
LOCALIZATION OF GLYCINE RECEPTOR a 1-SUBUNIT mRNA-CONTAINING NEURONS IN THE RAT BRAlN. KOUJI SATOH~ TAKANORI S A I K A ~ , J - H ZHANG~-~ MAKOTO SATOH,KEIYA TADA. AND MASAYA TOHYAMA.. D e p a r t m e n t of Anatomy II, Osaka University Medical School, Yamadaoka, Suita, Osaka 565, Japan. The localization of g l y c i n e r e e e t o r s in the r a t b r a i n w a s e x a m i n e d b y m e a n s of in situ hybridization histochemistry using an olig0nucle0tide probe to the sequence of the ~ I s u b u n i t . Stronglyor m o d e r a t e l y labeled neurons were m a i n l y f o u n d in the l o w e r b r a i n s t e m . Our data are almost consistent with the data of previous labeled ligand binding experiments. In a d d i t i o n , we compare this d a t a w i t h l o c a l i z a t i o n of o t h e r s u b u n i t s ( ~ a n d 9 3 K D p r t e i n ) of s t r y c i n i n e sensitive glycine receptor. We find that discrepancy exists between 10calizations of t h e s e subunits. For example, although in the l o w e r b r a i n s t e m , we c a n f i n d all t h r e e s u b u n i t s , o n l y ~ s u b u n i t w a s d e t e c t e d a n d w e c a n ' t find other two subunits in the c e r e b r a l c o r t e x . T h e s e r e s u l t s s u g g e s t that glycine receptor has heterogeneity in its c o m p o s i t i o n .
MECHANISM OF POLYAMINE-INDUCED MODULATION OF THE NMDA RECEPTOR COMPLEX. YUKIO YONEDA, KIYOKAZU OGITA, AND RIYO ENOMOTO*, Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-O1, Japan. Polyamine spermidine (SPD) markedly potentiated the binding of [3H](+)-5-methyl-lO, lldihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) to open cationic channels associated with the N-methyl-D-aspartate (NMDA)-sensitive subclass of excitatory amino acid receptors in the presence of a maximally effective concentration of both L-glutamic acid (Glu) and glycine (Gly) in brain synaptic membranes treated with Triton X-100. Among several natural and synthetic polyamines tested, spermine was most effective in potentiating the binding followed by bis-(3-aminopropyl)amine, SPD and 1,3-diaminopropane in that order. The potentiation by SPD was noncompetitively prevented by a competitive type of antagonists at the Gly as well as NMDA sites. The densities of [3H]MK-801 binding sites were unevenly distributed in the rat brain, while the affinities were not significantly different from each other. The hippocampus had the highest density in the presence of Glu/Gly with progressively lower densities in the cerebral cortex, striatum, hypothalamus, midbrain, cerebellum and medulla-pons. Further addition of SPD significantly increased the affinities in structures with a high density without altering the density itself, without changes in either the affinity or density in the cerebellum. The positive polyamines were all ineffective in potentiating the binding in the cerebellum with a concomitant potentiation of that in the hippocampus and cerebral cortex. These results suggest that heterogeneous NMDA receptor complex is present in the cerebellum in terms of differential sensitivity to potentiation by polyamines.
SOLUBILIZATION OF THE NMDA RECEPTOR COMPLEX FROM THE RAT BRAIN. KIYOKAZU OGITA, TAKEO SUZUKI*, AND YUKIO YONEDA, Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-01, Japan. The detergent deoxycholic acid (DOC) was most effective in solubilizing the binding activity of [3H](+)-5-methyl-lO,ll-dihydro-5H-dibenzo[a,d]cyclohepten-5,10_imine (MK-801) to open cationic channels associated with the N-methyl-D-aspartate (NMDA)-sensitive subclass of excitatory amino acid receptors from rat brain menforanes among various detergents tested. Neither L-glutamic acid (Glu) nor glycine (Gly) potentiated the binding at concentrations below 1 ram in solubilized supernatants, while the binding was significantly inhibited by the addition of competitive NVLDA and Gly antagonists. In contrast, both amino acids were effective in potentiating the binding in a concentration-dependent manner in preparations after gel filtration on a Sephadex G-25 column of solubilized supernatants. Polyamine spermidine (SPD) markedly potentiated the binding in preparations both before and after gel filtration. In preparations after gel filtration, either Glu alone or Glu/Gly markedly accelerated the initial association rate of [3H]MK-801 binding without significantly affecting the binding at equilibrium, whereas SPD did not additionally facilitate the initial association rate found in the presence of both Glu and Gly with a concomitant elevation of the steady-state level. ~ o n g a variety of NF~A channel blockers, (+)-MK-801 exhibited the most potent displacement of the binding in the presence of all three different stimulants in preparations after gel f i l t r a t i o n followed by (-)-MK-801, N - [ l - ( 2 - t h i e n y l ) c y c l o h e x y l ] p i p e r i d i n e , phencyclidine, N-allylnormetazocine and ketamine in that order. These results suggest that the whole supermacromolecular NMDA receptor complex is co-solubilized under the experimental conditions employed.