- Email: [email protected]
Identification and characterization of a vesicular nucleotide transporter
Abstracts
S77
P1-a26 Functional expression of Transient Receptor Potential V2 (TRPV2) in mouse myenteric plexus Hiroshi Mihara 1 , Ammar Boudaka 1 , Koji Shibasaki 1 , Akihiro Yamanaka 1 , Toshiro Sugiyama 2 , Makoto Tominaga 1
P1-a29 Characterization of transport properties of purified vesicular inhibitory amino acid transporter (VIAAT) Narinobu Juge, Akiko Muroyama, Miki Hiasa, Hiroshi Omote, Yoshinori Moriyama
1
Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
Okazaki Institute for Integrative Bioscience, Section of Cell Signaling, Japan; Department of Gastroenterology and Hematology, University of Toyama, Japan 2
Background/aim: TRPV2 was shown to receive noxious thermalstimuli, mechanical stimuli. Previous study showed that it was expressed in fine myelinated afferent neurons and several peripheral tissues. However, its distribution and function in enteric nervous system remain unknown. In present study, we examined the functional expression of TRPV2 in the mouse intestine. Methods/results: An analysis by RT-PCR demonstrated the gene expression in the myenteric neural cells. The immunoreactivity was observed in 90% of nNOSexpressing neurons, 19% of calbindin-expressing IPAN and some others. An analysis by Ca2+ imaging of primary myenteric culture cells demonstrated that some high potassium-responders also responded to specific TRPV2 agonist. Conclusion: These results suggest that TRPV2 is functionally expressed mainly in myenteric inhibitory motor neurons.
The vesicular inhibitory amino acid transporter (VIAAT) is responsible for vesicular storage of GABA and glycine in the GABAergic and glycinergic neurons that is the major pathway for inhibitory signal transmission in the central nervous system. In spite of the well-accepted significance of VIAAT in inhibitory neurotransmission, elucidation of the molecular mechanism of the transporter is largely unknown due to the lack of an in vitro assay procedure to assess recombinant VIAAT activity. Here, we purified VIAAT from overexpressed insect cells and reconstituted into liposomes. Upon the formation of membrane potential, the proteoliposomes actively took up GABA and glycine. E213A mutant lost uptake activity for all these substrates. The transport properties of GABA and glycine, in particular, driving force and requirement of anions were characterized and discussed with special reference to those of vesicular glutamate transporter. doi:10.1016/j.neures.2009.09.281
doi:10.1016/j.neures.2009.09.278
P1-a27 Vesicular nucleotide transporter is responsible for vesicular storage and exocytosis of ATP from astrocyte Miki Hiasa 1 , Keisuke Sawada 1 , Kayoko Fujishita 2 , Yoshinori Moriyama 1 , Syuichi Koizumi 2 1
Okayama Univ., Japan;
2
Univ. of Yamanashi, Japan
ATP is released from astrocytes and plays an important role(s) as a gliotransmitter in neural–astrocytes and astrocyte–astrocyte interactions. The issues where and how ATP is stored before secretion in astrocytes are less understood. Recently, we have identified vesicular nucleotide transporter (VNUT) that is responsible for vesicular storage of nucleotides in secretory vesicles in ATP-secreting cells (ref. Sawada et al., 2008). Here we show that VNUT is higher expressed in astrocytes in rat hippocampus and associated with secretogranine 2- or chromogranine A-containing granules. Upon stimulation with glutamate, astrocytes secrete ATP in a Ca2+ and temperature dependent fashion. Suppression of VNUT gene expression decreased the glutamate-evoked ATP secretion. These results demonstrated that ATP is stored in the dense-cored vesicles through VNUT-mediated active transport and then secreted through exocytosis.
Reference Sawada, K., et al., 2008. PNAS. doi:10.1016/j.neures.2009.09.279
P1-a28 Identification and characterization of a vesicular nucleotide transporter Keisuke Sawada, Miki Hiasa, Noriko Echigo, Narinobu Juge, Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama Okayama University Graduate School, Okayama, Japan ATP is stored in secretory vesicles, exocytozed, and acts as an intercellular transmitter upon binding to the purinoceptors. The mechanism of vesicular storage and subsequent exocytosis of ATP was far less understood. Here we identified vesicular nucleotide transporter (VNUT) that is responsible for vesicular storage of nucleotides in neurons and endocrine cells (Ref. Sawada et al., 2008). VNUT is a novel member of the SLC17 anion transporter family. The proteoliposomes containing purified VNUT actively took up ATP, ADP and GTP using membrane potential (Ψ ) as driving force. VNUT is expressed in neurons and PC12 cells. Upon stimulation with membrane depolarization, ATP is secreted in a Ca2+ -dependent fashion from these cells. Suppression of VNUT gene expression decreased ATP secretion. We concluded that VNUT plays an essential role in ATP secretion in these cells.
P1-a30 Gene expression profiling of mouse cerebellar Purkinje cells Kiyotaka Akiyama, Setsuko Nakanishi, Takayuki Naito Okinawa Institute of Science Technology, Japan Neural cells in the brain are classified into highly diverse cell types. For understanding of neural system, it is important to know precise gene expression in each of the diverse cell types. We analyzed gene expression of mouse cerebellar Purkinje cell (PC). PCs were collected by laser capture microdissection and RNAs were isolated from them. Following amplification of mRNAs, genome-wide expression profile was constructed using microarray analysis. However it is possible that these samples contain mRNAs derived from surrounding cells other than PC. In order to eliminate the effect of surrounding cells on the expression profile, a correction has been done by comparison of PC data with PC layer data. Among 377 genes which are thought to have important roles in neuronal activities (including neurotransmitter synthesizing enzyme, neuropeptide, ligand-gated channel, and G-protein coupled receptor), expression of 60 genes was detected in PC. Copy number of these genes was determined by quantitative PCR. This precise gene expression profile is important for understanding the function of PC. doi:10.1016/j.neures.2009.09.282
P1-a31 Selective depressing effects of octanol on the ionotropic receptor responses of the ganglion cells of Aplysia Shingo Kimura, Satoshi Kawasaki, Reiko Fujita, Misato Harata, Noriyuki Watanabe, Kazuhiko Sasaki Dept. Physiol., Iwate Med. Univ., Morioka, Japan To compare the effects of long-chain n-alcohols on various receptor-induced responses of neurons, we examined effect of 1-octanol on different ionotropic and metabotropic types of receptors in Aplysia ganglion cells under voltage-clamp. Application of 1-octanol significantly and reversibly decreased fast Na+ -current responses of a nicotinic- and a dopamine-receptor, fast Cl− -current responses of GABAA receptor and another type of nicotinic-receptor. In contrast, the same concentration of octanol did not affect K+ -current responses of M2-, D2-, H2-, and FMRFamidereceptors, and slow Na+ -currrent responses of D1- and 5HT-receptors. The former 4 types of receptors are known to be ionotropic and the latter 6 types are metabotropic. Therefore, all types of ionotropic receptor responses examined so far were selectively inhibited by octanol irrespective of types of receptor and ionophore, indicating that the effect is not due to competition at their receptors. doi:10.1016/j.neures.2009.09.283
P1-a32 Analysis of locomotor control using transgenic mice having controllable D1R expression Asako Sato 1 , Toshikuni Sasaoka 1,2 , Satoko Arakawa 1,3 , Tomoko Morita 1,4 , Kuniko Katsuki 1 , Motoya Katsuki 1,2 1
Reference Sawada, K., et al., 2008. PNAS 105, 5683–5686. doi:10.1016/j.neures.2009.09.280
Natl. Inst. for Basic Biol., Okazaki, Japan; 2 SOKENDAI, Japan; Med. Dent. Univ., Tokyo, Japan; 4 Univ. Tokyo, Tokyo, Japan
3
Tokyo
Dual deficiency of dopamine D1 receptor (D1R) and D2 receptor (D2R) in mice leads to impairment of locomotion and eating behavior, and the mice die prematurely. To investigate the regulation in locomotion and eating behavior, we generated transgenic mice harboring tetracycline-regulated expression of the D1R gene on the
S77
P1-a26 Functional expression of Transient Receptor Potential V2 (TRPV2) in mouse myenteric plexus Hiroshi Mihara 1 , Ammar Boudaka 1 , Koji Shibasaki 1 , Akihiro Yamanaka 1 , Toshiro Sugiyama 2 , Makoto Tominaga 1
P1-a29 Characterization of transport properties of purified vesicular inhibitory amino acid transporter (VIAAT) Narinobu Juge, Akiko Muroyama, Miki Hiasa, Hiroshi Omote, Yoshinori Moriyama
1
Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
Okazaki Institute for Integrative Bioscience, Section of Cell Signaling, Japan; Department of Gastroenterology and Hematology, University of Toyama, Japan 2
Background/aim: TRPV2 was shown to receive noxious thermalstimuli, mechanical stimuli. Previous study showed that it was expressed in fine myelinated afferent neurons and several peripheral tissues. However, its distribution and function in enteric nervous system remain unknown. In present study, we examined the functional expression of TRPV2 in the mouse intestine. Methods/results: An analysis by RT-PCR demonstrated the gene expression in the myenteric neural cells. The immunoreactivity was observed in 90% of nNOSexpressing neurons, 19% of calbindin-expressing IPAN and some others. An analysis by Ca2+ imaging of primary myenteric culture cells demonstrated that some high potassium-responders also responded to specific TRPV2 agonist. Conclusion: These results suggest that TRPV2 is functionally expressed mainly in myenteric inhibitory motor neurons.
The vesicular inhibitory amino acid transporter (VIAAT) is responsible for vesicular storage of GABA and glycine in the GABAergic and glycinergic neurons that is the major pathway for inhibitory signal transmission in the central nervous system. In spite of the well-accepted significance of VIAAT in inhibitory neurotransmission, elucidation of the molecular mechanism of the transporter is largely unknown due to the lack of an in vitro assay procedure to assess recombinant VIAAT activity. Here, we purified VIAAT from overexpressed insect cells and reconstituted into liposomes. Upon the formation of membrane potential, the proteoliposomes actively took up GABA and glycine. E213A mutant lost uptake activity for all these substrates. The transport properties of GABA and glycine, in particular, driving force and requirement of anions were characterized and discussed with special reference to those of vesicular glutamate transporter. doi:10.1016/j.neures.2009.09.281
doi:10.1016/j.neures.2009.09.278
P1-a27 Vesicular nucleotide transporter is responsible for vesicular storage and exocytosis of ATP from astrocyte Miki Hiasa 1 , Keisuke Sawada 1 , Kayoko Fujishita 2 , Yoshinori Moriyama 1 , Syuichi Koizumi 2 1
Okayama Univ., Japan;
2
Univ. of Yamanashi, Japan
ATP is released from astrocytes and plays an important role(s) as a gliotransmitter in neural–astrocytes and astrocyte–astrocyte interactions. The issues where and how ATP is stored before secretion in astrocytes are less understood. Recently, we have identified vesicular nucleotide transporter (VNUT) that is responsible for vesicular storage of nucleotides in secretory vesicles in ATP-secreting cells (ref. Sawada et al., 2008). Here we show that VNUT is higher expressed in astrocytes in rat hippocampus and associated with secretogranine 2- or chromogranine A-containing granules. Upon stimulation with glutamate, astrocytes secrete ATP in a Ca2+ and temperature dependent fashion. Suppression of VNUT gene expression decreased the glutamate-evoked ATP secretion. These results demonstrated that ATP is stored in the dense-cored vesicles through VNUT-mediated active transport and then secreted through exocytosis.
Reference Sawada, K., et al., 2008. PNAS. doi:10.1016/j.neures.2009.09.279
P1-a28 Identification and characterization of a vesicular nucleotide transporter Keisuke Sawada, Miki Hiasa, Noriko Echigo, Narinobu Juge, Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama Okayama University Graduate School, Okayama, Japan ATP is stored in secretory vesicles, exocytozed, and acts as an intercellular transmitter upon binding to the purinoceptors. The mechanism of vesicular storage and subsequent exocytosis of ATP was far less understood. Here we identified vesicular nucleotide transporter (VNUT) that is responsible for vesicular storage of nucleotides in neurons and endocrine cells (Ref. Sawada et al., 2008). VNUT is a novel member of the SLC17 anion transporter family. The proteoliposomes containing purified VNUT actively took up ATP, ADP and GTP using membrane potential (Ψ ) as driving force. VNUT is expressed in neurons and PC12 cells. Upon stimulation with membrane depolarization, ATP is secreted in a Ca2+ -dependent fashion from these cells. Suppression of VNUT gene expression decreased ATP secretion. We concluded that VNUT plays an essential role in ATP secretion in these cells.
P1-a30 Gene expression profiling of mouse cerebellar Purkinje cells Kiyotaka Akiyama, Setsuko Nakanishi, Takayuki Naito Okinawa Institute of Science Technology, Japan Neural cells in the brain are classified into highly diverse cell types. For understanding of neural system, it is important to know precise gene expression in each of the diverse cell types. We analyzed gene expression of mouse cerebellar Purkinje cell (PC). PCs were collected by laser capture microdissection and RNAs were isolated from them. Following amplification of mRNAs, genome-wide expression profile was constructed using microarray analysis. However it is possible that these samples contain mRNAs derived from surrounding cells other than PC. In order to eliminate the effect of surrounding cells on the expression profile, a correction has been done by comparison of PC data with PC layer data. Among 377 genes which are thought to have important roles in neuronal activities (including neurotransmitter synthesizing enzyme, neuropeptide, ligand-gated channel, and G-protein coupled receptor), expression of 60 genes was detected in PC. Copy number of these genes was determined by quantitative PCR. This precise gene expression profile is important for understanding the function of PC. doi:10.1016/j.neures.2009.09.282
P1-a31 Selective depressing effects of octanol on the ionotropic receptor responses of the ganglion cells of Aplysia Shingo Kimura, Satoshi Kawasaki, Reiko Fujita, Misato Harata, Noriyuki Watanabe, Kazuhiko Sasaki Dept. Physiol., Iwate Med. Univ., Morioka, Japan To compare the effects of long-chain n-alcohols on various receptor-induced responses of neurons, we examined effect of 1-octanol on different ionotropic and metabotropic types of receptors in Aplysia ganglion cells under voltage-clamp. Application of 1-octanol significantly and reversibly decreased fast Na+ -current responses of a nicotinic- and a dopamine-receptor, fast Cl− -current responses of GABAA receptor and another type of nicotinic-receptor. In contrast, the same concentration of octanol did not affect K+ -current responses of M2-, D2-, H2-, and FMRFamidereceptors, and slow Na+ -currrent responses of D1- and 5HT-receptors. The former 4 types of receptors are known to be ionotropic and the latter 6 types are metabotropic. Therefore, all types of ionotropic receptor responses examined so far were selectively inhibited by octanol irrespective of types of receptor and ionophore, indicating that the effect is not due to competition at their receptors. doi:10.1016/j.neures.2009.09.283
P1-a32 Analysis of locomotor control using transgenic mice having controllable D1R expression Asako Sato 1 , Toshikuni Sasaoka 1,2 , Satoko Arakawa 1,3 , Tomoko Morita 1,4 , Kuniko Katsuki 1 , Motoya Katsuki 1,2 1
Reference Sawada, K., et al., 2008. PNAS 105, 5683–5686. doi:10.1016/j.neures.2009.09.280
Natl. Inst. for Basic Biol., Okazaki, Japan; 2 SOKENDAI, Japan; Med. Dent. Univ., Tokyo, Japan; 4 Univ. Tokyo, Tokyo, Japan
3
Tokyo
Dual deficiency of dopamine D1 receptor (D1R) and D2 receptor (D2R) in mice leads to impairment of locomotion and eating behavior, and the mice die prematurely. To investigate the regulation in locomotion and eating behavior, we generated transgenic mice harboring tetracycline-regulated expression of the D1R gene on the