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219 Characterization of glycine release mediated by glycine transporter 1 stably expressed in HEK-293 cells
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Characterization of glycine release mediated by glycine transporter 1 stably expressed in HEK-293 cells Department of Neuroanatomy, Biomedical Research Center, Osaka University Medical School l, Department of Anatomy and Neuroscience, Osaka University Medical School 2 Kohji Sate’ , Kazuko Sakata2, Shoichi Shimada2, Masaya Tohyama2 We constructed a cell line which stably expresses glycine transporter 1 (GlyTl) proteins. The cell line showed significant 142C-giycine uptake and could keep steep glycine concentration gradient between intracellular and extracellular space (in>out). Using this cell line, we investigated glycine release mediated by this transporter. The 142C-Glycine release was enhanced by extracellular glycine and sarcosine, a selective inhibitor of the transporter, in a dose-dependent manner. In addition, the replacement of extracellular Na+ with Lis’ or extracellular Cl- with acetate- markedly increased the release. Furthermore, we investigated the effects of extracellular Ca2+ and K+. The removal of these ions also showed enhancement of the release. These results suggest that glycine transporter 1 protein, which might be involved in the NMDA receptor neurotransmission, can release glycine into the extracellular space in the vicinity of synpases, and that the release might be influenced by the extrace
220
MOTOR NERVE AXOTOMY INDUCES DIFFERENTIAL EXPRESSION OF TWO CHOLINERGIC MARKERS: CHOLINE ACETYLTRANSFERASE AND VESICULAR ACETYLCHOLINE TRASPORTER Dept. Neurology, Tokyo Metropolitan Inst. Neurosci., 2-6, Musashidai, Fuchu City, Tokyo 183, Japan’, Dept. Anatomy & Embryology, Tokyo Metropolitan Inst. Neurosci., 2-6, Musashidai, Fuchu City, Tokyo 183, Japan2 JUNK0
MATSUURAl,
KYOKO
AJIK12, TOMOYUKI
ICHIKAWA2,
HIDEMI
MISAWA’
Acetylcholme is synthesized in the cytoplasm of choline&c neurons by choline acetyltransferase (ChAT) and then packaged in synaptic vesicles by vesicular acetylcholine transporter (VAChT). Recently VAChT gene was shown to be located within the first intron of the ChAT gene. In order to analyze their expression in vivo, we compared the expression of respective mRNA and protein by in situ hybridization and immunohistochemistry following transection of the hypoglossal nerve in adult rats. Unilateral transection resulted in. a dramatic loss of ChAT and VAChT mRNA expression one week following surgery in the ipsilateral hypoglossal motor nucleus. Although ChAT protein was virtually undetectable parallel to ChAT mRNA in the injured side, VAChT protein showed no obvious change between the injured and control sides. These results suggest the possibility that transcription of the two genes are tightly linked, but their translation products are differentially regulated in the cell.
221
INVERSION OF AN INWARD CURRENT INDUCED BY DOPAMINE AFTER THE APPLICATION OF GTP-ANALOG OBSERVED IN THE NEURONS OF APLYSIA Department of Physiology, School of Medicine, Iwate Medical University, Morioka, 020, Japan’, Laboratory of Physics, School of Allied Medical Sciences, Hirosaki University, Hirosaki, 036, Japan2 SATOSHI KAWASAKI’, KAZUHIKO SASAKI’, MITSUHIKO MATSUMOT02, REIKO FUJITA’,
SHINGO KIMURA’, MAKOTO SATO’
KOICHIRO.
TAK.ASHIMA’,
Dopamine (DA) induces a Na+- (inward) current response in identified neurons of Aplysia under voltage clamp. This response is produced by activation of Di-type receptor, and blocked by both guanosinethiodiphosphate and cholera toxin (CTX). Intracellular injection of guanosinethiotriphosphate (GTPgS) into the same type of cell produced an irreversible Kf- (outward) current, as it did in other cells. Surprisingly, the DA-induced inward current reversed to an outward (K+) current when examined during the response to GTPgS. Both the DA- and GTPgS-induced K+-current were significantly depressed by 10 mM Ba2+ and 4aminopyridine (4AP), but not by 10 mM tetraethylammonium (TEA). The K+-current response to DA disappeared after the application of CTX, although the response to GTPgS was still produced normally. The results suggest that the Di-receptor may couple with K-+-channel via CTX-sensitive G-protein during the activation of other type of G-protein.
219
Characterization of glycine release mediated by glycine transporter 1 stably expressed in HEK-293 cells Department of Neuroanatomy, Biomedical Research Center, Osaka University Medical School l, Department of Anatomy and Neuroscience, Osaka University Medical School 2 Kohji Sate’ , Kazuko Sakata2, Shoichi Shimada2, Masaya Tohyama2 We constructed a cell line which stably expresses glycine transporter 1 (GlyTl) proteins. The cell line showed significant 142C-giycine uptake and could keep steep glycine concentration gradient between intracellular and extracellular space (in>out). Using this cell line, we investigated glycine release mediated by this transporter. The 142C-Glycine release was enhanced by extracellular glycine and sarcosine, a selective inhibitor of the transporter, in a dose-dependent manner. In addition, the replacement of extracellular Na+ with Lis’ or extracellular Cl- with acetate- markedly increased the release. Furthermore, we investigated the effects of extracellular Ca2+ and K+. The removal of these ions also showed enhancement of the release. These results suggest that glycine transporter 1 protein, which might be involved in the NMDA receptor neurotransmission, can release glycine into the extracellular space in the vicinity of synpases, and that the release might be influenced by the extrace
220
MOTOR NERVE AXOTOMY INDUCES DIFFERENTIAL EXPRESSION OF TWO CHOLINERGIC MARKERS: CHOLINE ACETYLTRANSFERASE AND VESICULAR ACETYLCHOLINE TRASPORTER Dept. Neurology, Tokyo Metropolitan Inst. Neurosci., 2-6, Musashidai, Fuchu City, Tokyo 183, Japan’, Dept. Anatomy & Embryology, Tokyo Metropolitan Inst. Neurosci., 2-6, Musashidai, Fuchu City, Tokyo 183, Japan2 JUNK0
MATSUURAl,
KYOKO
AJIK12, TOMOYUKI
ICHIKAWA2,
HIDEMI
MISAWA’
Acetylcholme is synthesized in the cytoplasm of choline&c neurons by choline acetyltransferase (ChAT) and then packaged in synaptic vesicles by vesicular acetylcholine transporter (VAChT). Recently VAChT gene was shown to be located within the first intron of the ChAT gene. In order to analyze their expression in vivo, we compared the expression of respective mRNA and protein by in situ hybridization and immunohistochemistry following transection of the hypoglossal nerve in adult rats. Unilateral transection resulted in. a dramatic loss of ChAT and VAChT mRNA expression one week following surgery in the ipsilateral hypoglossal motor nucleus. Although ChAT protein was virtually undetectable parallel to ChAT mRNA in the injured side, VAChT protein showed no obvious change between the injured and control sides. These results suggest the possibility that transcription of the two genes are tightly linked, but their translation products are differentially regulated in the cell.
221
INVERSION OF AN INWARD CURRENT INDUCED BY DOPAMINE AFTER THE APPLICATION OF GTP-ANALOG OBSERVED IN THE NEURONS OF APLYSIA Department of Physiology, School of Medicine, Iwate Medical University, Morioka, 020, Japan’, Laboratory of Physics, School of Allied Medical Sciences, Hirosaki University, Hirosaki, 036, Japan2 SATOSHI KAWASAKI’, KAZUHIKO SASAKI’, MITSUHIKO MATSUMOT02, REIKO FUJITA’,
SHINGO KIMURA’, MAKOTO SATO’
KOICHIRO.
TAK.ASHIMA’,
Dopamine (DA) induces a Na+- (inward) current response in identified neurons of Aplysia under voltage clamp. This response is produced by activation of Di-type receptor, and blocked by both guanosinethiodiphosphate and cholera toxin (CTX). Intracellular injection of guanosinethiotriphosphate (GTPgS) into the same type of cell produced an irreversible Kf- (outward) current, as it did in other cells. Surprisingly, the DA-induced inward current reversed to an outward (K+) current when examined during the response to GTPgS. Both the DA- and GTPgS-induced K+-current were significantly depressed by 10 mM Ba2+ and 4aminopyridine (4AP), but not by 10 mM tetraethylammonium (TEA). The K+-current response to DA disappeared after the application of CTX, although the response to GTPgS was still produced normally. The results suggest that the Di-receptor may couple with K-+-channel via CTX-sensitive G-protein during the activation of other type of G-protein.