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syntaxin 1A reduced PC12h cell
S88
616
Localization
and characterization
of a novel StePO-related protein kinase
Dept. Anat. Fat. Med. Mie Univ., 2-174 Edobashi, Tsu, 514, Japan Tomonari Tsutsumi, Hiroshi Ushiro, Tetsuro Kayahara, Katsuma Nakano We have cloned a novel protein kinase cDNA from rat brain. The deduced amino acid sequence of the catalytic domain indicates that it belongs to the Ste20-related protein kinase family, the members of which have been reported to act upstream of MAP kinase pathways. The distribution of the kinase in rat organs was examined with a rabbit polyclonal antibody against the C-terminal non-catalytic region. Immunoblot analysis demonstrated ubiquitous expression of the enzyme, except in liver and skeletal muscle where no immunoreactive band was detected. Immunohistochemical analysis revealed cell type specific distribution of the enzyme. In the brain, the most prominent immunoreactivity was found in epithelial cells of choroid plexus. A moderate immunoreactivity was seen in large neurons such as motor neurons in brainstem, cerebellar Purkinje cells and pyramidal cells of cerebral cortex.
617
Activation
of dopamine release from HPC-l/syntaxin
1A reduced PC12h cell
Dept. Physiology1 , Neuropsychiatry, Kyorin Univ. School of Medicine, Shinkawa, Mitaka, Tokyo 181, Japan2, Discovery Res. Labs I, Takeda Chem Ind., Wadai, Tsukuba 300-42, Japan3, Dept. Anatomy, Saitama Medical School, Moroyama, Iruma, Saitama 350-04, Japan4 Kimio Akagawa’ , Takuya Watanabe3, Tomonori Fujiwara’ , Shinji Komazaki4, Osamu Tajima’, Kazuhiko Yamaguchi’ To elucidate the function of HPC-l/syntaxin 1A in exocytosis, we have constructed HPC-l/syntaxin lAreduced PC12h cells (PClZh/(syn) that were stably transfected with a plasmid for antisense HPC-l/syntaxin 1A expression. In these PC12h/(syn cells, the expression of HPC-l/syntaxin 1A protein was decreased at least 55% of PClSh, but the organelles of these cells especially vesicles appeared normal by electromycroscopical study. PClPh/(syn and PC12h cells were treated with high potassium solution and released dopamine was measured by electrochemical detector. We have observed that dopamine release was increased in PClPh/(syn cells compared with PC12h. Furthermore, with the stimulation of carbachol as well as high potassium solution, the amount of dopamine released was higher in PClPh/Asyn cells than in PC12h. In different clones of PC12h/(syn cells, there was a correlation between the extent of HPC-l/syntaxin 1A reduction and the increase of dopamine release. These results indicate that HPC-l/syntaxin 1A have inhibitory function in exocytosis.
618
ROLE OF ORP150 INISCHEMIC/HYPOXIC
Department of Anatomy Osaka 565, Japan
and Neuroscience, Osaka University
NEURONAL
CELL DEATH
Medical School, 2-2 Yamadaoka,
Suita,
MICHIO TAMATANI, SATOSHI OGAWA, KENTARO OZAWA, MASAYA TOHYAMA Although neurotrophic factoros and cytokines were known to protect CNS neurons aginst hypoxic/ischemic insults, the mechnanisms in their neuroprotective roles are unknown. We have investigated a role of ORP150, initially characterized based on its expression in astrocytes subjected to oxygen deprivation, in hypoxic neuronal cell death. Immunohistchemical and in situ hybridization studies showed the early and marked increase of ORP150 in neurons and astrocytes in ischemic regions of a rat MCA occlusion model, whereas exposure of cultured cortical neurons to hypoxia induced no increase in ORP150 protein level. However, incubation of the cultured neurons with TNFa, which was upregulated in MCA-occluded cerebral hemisphere and showed neuroprotectability in cultured neurons against hypoxic insult, induced lo-fold increase of the antigen, while incubation of either IGF-1, basic FGF or EGF was without effect. These data indicate that ORP150 may participate in neuroprotective role of TNFa in ischemic/hypoxic brain.
616
Localization
and characterization
of a novel StePO-related protein kinase
Dept. Anat. Fat. Med. Mie Univ., 2-174 Edobashi, Tsu, 514, Japan Tomonari Tsutsumi, Hiroshi Ushiro, Tetsuro Kayahara, Katsuma Nakano We have cloned a novel protein kinase cDNA from rat brain. The deduced amino acid sequence of the catalytic domain indicates that it belongs to the Ste20-related protein kinase family, the members of which have been reported to act upstream of MAP kinase pathways. The distribution of the kinase in rat organs was examined with a rabbit polyclonal antibody against the C-terminal non-catalytic region. Immunoblot analysis demonstrated ubiquitous expression of the enzyme, except in liver and skeletal muscle where no immunoreactive band was detected. Immunohistochemical analysis revealed cell type specific distribution of the enzyme. In the brain, the most prominent immunoreactivity was found in epithelial cells of choroid plexus. A moderate immunoreactivity was seen in large neurons such as motor neurons in brainstem, cerebellar Purkinje cells and pyramidal cells of cerebral cortex.
617
Activation
of dopamine release from HPC-l/syntaxin
1A reduced PC12h cell
Dept. Physiology1 , Neuropsychiatry, Kyorin Univ. School of Medicine, Shinkawa, Mitaka, Tokyo 181, Japan2, Discovery Res. Labs I, Takeda Chem Ind., Wadai, Tsukuba 300-42, Japan3, Dept. Anatomy, Saitama Medical School, Moroyama, Iruma, Saitama 350-04, Japan4 Kimio Akagawa’ , Takuya Watanabe3, Tomonori Fujiwara’ , Shinji Komazaki4, Osamu Tajima’, Kazuhiko Yamaguchi’ To elucidate the function of HPC-l/syntaxin 1A in exocytosis, we have constructed HPC-l/syntaxin lAreduced PC12h cells (PClZh/(syn) that were stably transfected with a plasmid for antisense HPC-l/syntaxin 1A expression. In these PC12h/(syn cells, the expression of HPC-l/syntaxin 1A protein was decreased at least 55% of PClSh, but the organelles of these cells especially vesicles appeared normal by electromycroscopical study. PClPh/(syn and PC12h cells were treated with high potassium solution and released dopamine was measured by electrochemical detector. We have observed that dopamine release was increased in PClPh/(syn cells compared with PC12h. Furthermore, with the stimulation of carbachol as well as high potassium solution, the amount of dopamine released was higher in PClPh/Asyn cells than in PC12h. In different clones of PC12h/(syn cells, there was a correlation between the extent of HPC-l/syntaxin 1A reduction and the increase of dopamine release. These results indicate that HPC-l/syntaxin 1A have inhibitory function in exocytosis.
618
ROLE OF ORP150 INISCHEMIC/HYPOXIC
Department of Anatomy Osaka 565, Japan
and Neuroscience, Osaka University
NEURONAL
CELL DEATH
Medical School, 2-2 Yamadaoka,
Suita,
MICHIO TAMATANI, SATOSHI OGAWA, KENTARO OZAWA, MASAYA TOHYAMA Although neurotrophic factoros and cytokines were known to protect CNS neurons aginst hypoxic/ischemic insults, the mechnanisms in their neuroprotective roles are unknown. We have investigated a role of ORP150, initially characterized based on its expression in astrocytes subjected to oxygen deprivation, in hypoxic neuronal cell death. Immunohistchemical and in situ hybridization studies showed the early and marked increase of ORP150 in neurons and astrocytes in ischemic regions of a rat MCA occlusion model, whereas exposure of cultured cortical neurons to hypoxia induced no increase in ORP150 protein level. However, incubation of the cultured neurons with TNFa, which was upregulated in MCA-occluded cerebral hemisphere and showed neuroprotectability in cultured neurons against hypoxic insult, induced lo-fold increase of the antigen, while incubation of either IGF-1, basic FGF or EGF was without effect. These data indicate that ORP150 may participate in neuroprotective role of TNFa in ischemic/hypoxic brain.