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513 Characterization of neurotrophic factors and genes expressed in the striatum of hemiparkinsonian model rats
S81
512
EFFECTS OF ERYTHROPOIETIN
First Division, Department
ON PC12 CELLS
of Medicine, Shimane Medical University
Kunio Koshimura, Junko Tanaka, Yoshio Murakami,
Yuzuru Kato
It has been revealed that erythropoietin (EPO) receptors are located in some brain areas such as hippocampus. We investigated the effects of EPO on Ca2+ uptake, dopamine release and cell survival in PC12 cells which possess EPO receptors. EPO (10-‘3-10-10 M) increased 45Ca2+ uptake into PC12 cells (1 min, 37C), which was inhibited by nicardipine(1 microM) or anti-EPO antibody (1:lOO diluted). At the same concentrations, EPO increased dopamine release from PC12 cells (10 min, 37C), which was sensitive to nicardipine or anti-EPO antibody. After 5-day culture without serum and NGF, number of viable cells decreased to one third of that of the control cells cultured with serum and NGF. Administration of EPO to the culture medium (10-‘3-10-10 M) increased viable number of cells cultured without serum and NGF. EPO (10-13-10-‘o M) stimulated MAP kinase activity in PC12 cells. These results suggest that EPO stimulates neuronal function and viability via activation of Ca2+ channels.
513
CHARACTERIZATION OF NEUROTROPHIC FACTORS AND GENES EXPRESSED IN THE STRIATUM OF HEMIPARKINSONIAN MODEL RATS Department of Physiology, Nagoya City University Medical School, Nagoya 467, Japan’, The Center of Japan Biological Chemistry, Gifu 503-06, Japan2 YASUNOBU SHIMANO’, ICHIRO FUKUDA’, HIT00 NISHINO’
FUJIMOTO’,
KEIYA
NAKAJIMA2,
HIDEKI
HIDA’,
ATSUO
We have previously reported that the extract from dopamine-depleted striatal tissue (lesion extract) has a trophic activity on cultured pheochromocytoma (PClZD) cells and fetal dopaminergic neurons. The actions of the lesion extract promoting the neurite outgrowth on PC12D cells and the survival on fetal dopaminergic neurons were concentration-dependent. Differential display technique was used to identify novel genes encording factors associated with the trophic activity. Total mRNAs from dopamine-depleted striatum and the counter part of 6OHDA-lesioned hemi-Parkinsonian model rats were amplified by RT-PCR using 4 kinds of unique primers that we made according to the heparin binding site of several neurotrophic factors. The amount of the amplified products were displayed on sequence gel and compared between lesioned and intact side. Some fragments expressed in lesioned side more than intact side. Sequence and characteristics of these genes which were supposed to encord trophic factors on dopaminergic neurons have been discussed.
514
EFFECTS OF APGW-AMID ON [Ca2+] IN RAT PHEOCHROMOCYTOMA PC12 CELLS Dept. Neurophysiol. Inst. of Equilib. Res. ‘, Dept. Biochem., Gifu Univ. School of Medicine, Tsukasamachi 40, Gifu500, Japan2 Xiao Yan Hani, Yuzuru Ito2, Ken’ichi Matsunami’, Yoshinori Nozawa’ APGW-amide, a tetrapeptide isolated from the ganglia of Achatina fulica Ferussac, is considered as an inhibitory neurotransmitter of Achatina neurons and a neuromodulator in the Achatlna central nervous system. In the present study, the effect of APGW-amide on [Ca”+]i was investigated in Fura P-AM-loaded rat pheochromocytoma PC12 cells. APGW-amide caused a rapid [Ca2+]i elevation, which was completely inhibited by elimination of extracellular Ca2+ with EGTA and was inhibited by two L-type voltage-dependent Ca2+ channel blockers, nifedipine or varapamil. [Ca”+]i elevation was also blocked by calphostin C, a specific PKC inhibitor and pretreatment of cells with phorbol 1Zmyristate 1Sacetate (PMA) for 24 hr to down-regulate PKC. These results indicate that APGW-amide elevates [Ca2+]; in PC12 cells, possibly by Ca2+ influx via L-type Ca2+ channel activated by PKC.
512
EFFECTS OF ERYTHROPOIETIN
First Division, Department
ON PC12 CELLS
of Medicine, Shimane Medical University
Kunio Koshimura, Junko Tanaka, Yoshio Murakami,
Yuzuru Kato
It has been revealed that erythropoietin (EPO) receptors are located in some brain areas such as hippocampus. We investigated the effects of EPO on Ca2+ uptake, dopamine release and cell survival in PC12 cells which possess EPO receptors. EPO (10-‘3-10-10 M) increased 45Ca2+ uptake into PC12 cells (1 min, 37C), which was inhibited by nicardipine(1 microM) or anti-EPO antibody (1:lOO diluted). At the same concentrations, EPO increased dopamine release from PC12 cells (10 min, 37C), which was sensitive to nicardipine or anti-EPO antibody. After 5-day culture without serum and NGF, number of viable cells decreased to one third of that of the control cells cultured with serum and NGF. Administration of EPO to the culture medium (10-‘3-10-10 M) increased viable number of cells cultured without serum and NGF. EPO (10-13-10-‘o M) stimulated MAP kinase activity in PC12 cells. These results suggest that EPO stimulates neuronal function and viability via activation of Ca2+ channels.
513
CHARACTERIZATION OF NEUROTROPHIC FACTORS AND GENES EXPRESSED IN THE STRIATUM OF HEMIPARKINSONIAN MODEL RATS Department of Physiology, Nagoya City University Medical School, Nagoya 467, Japan’, The Center of Japan Biological Chemistry, Gifu 503-06, Japan2 YASUNOBU SHIMANO’, ICHIRO FUKUDA’, HIT00 NISHINO’
FUJIMOTO’,
KEIYA
NAKAJIMA2,
HIDEKI
HIDA’,
ATSUO
We have previously reported that the extract from dopamine-depleted striatal tissue (lesion extract) has a trophic activity on cultured pheochromocytoma (PClZD) cells and fetal dopaminergic neurons. The actions of the lesion extract promoting the neurite outgrowth on PC12D cells and the survival on fetal dopaminergic neurons were concentration-dependent. Differential display technique was used to identify novel genes encording factors associated with the trophic activity. Total mRNAs from dopamine-depleted striatum and the counter part of 6OHDA-lesioned hemi-Parkinsonian model rats were amplified by RT-PCR using 4 kinds of unique primers that we made according to the heparin binding site of several neurotrophic factors. The amount of the amplified products were displayed on sequence gel and compared between lesioned and intact side. Some fragments expressed in lesioned side more than intact side. Sequence and characteristics of these genes which were supposed to encord trophic factors on dopaminergic neurons have been discussed.
514
EFFECTS OF APGW-AMID ON [Ca2+] IN RAT PHEOCHROMOCYTOMA PC12 CELLS Dept. Neurophysiol. Inst. of Equilib. Res. ‘, Dept. Biochem., Gifu Univ. School of Medicine, Tsukasamachi 40, Gifu500, Japan2 Xiao Yan Hani, Yuzuru Ito2, Ken’ichi Matsunami’, Yoshinori Nozawa’ APGW-amide, a tetrapeptide isolated from the ganglia of Achatina fulica Ferussac, is considered as an inhibitory neurotransmitter of Achatina neurons and a neuromodulator in the Achatlna central nervous system. In the present study, the effect of APGW-amide on [Ca”+]i was investigated in Fura P-AM-loaded rat pheochromocytoma PC12 cells. APGW-amide caused a rapid [Ca2+]i elevation, which was completely inhibited by elimination of extracellular Ca2+ with EGTA and was inhibited by two L-type voltage-dependent Ca2+ channel blockers, nifedipine or varapamil. [Ca”+]i elevation was also blocked by calphostin C, a specific PKC inhibitor and pretreatment of cells with phorbol 1Zmyristate 1Sacetate (PMA) for 24 hr to down-regulate PKC. These results indicate that APGW-amide elevates [Ca2+]; in PC12 cells, possibly by Ca2+ influx via L-type Ca2+ channel activated by PKC.