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The regulation of choline acetyltransferase by interleukin-3 on septal cholinergic cell line
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1229
THE REGULATION OF CHOLINE ACETYLTRANSFERASE BY INTERLEUKIN-3 ON SEPTAL CHOLINERGIC CELL LINE. TATSUHIDE KUNISI-IITA. YOS. . KONISHI. KEIKICHI T-AM). De V. of Demy&x&mg Diseased
In previous studies we reported that interleukin-3 (IL-3) was a noble trophic factor for cholinergic neurons in central nervous system to promote neurite extension, neuronal survival and enhancement of choline acetyltransferase (ChAT) activity in vivo and in vitro (Kamegai et al., Neuron, 1990). Moreover, recent our works showed IL-3 could be produced in central nervous system and its receptor was specifically present in cholinergic neurons (Konishi et al., submitted). These findings suggest IL-3 directly induces the trophic events in cholinergic neurons. To clarify the mechanisms on the regulation of ChAT by IL-3 we analyzed the protein phosphorylation after IL-3 stimulation, 5’-regions of ChAT mRNAs and promoter elements of genomic ChAT cDNA (Misawa et al., J. Biol. Chem., 1992) required for IL-3 induction using a septal cholinergic cell line SN6. The results of these investigations will be discussed.
1230 TROPHIC EFFECTS OF DOPAMINE-DEPLETED STRIATAL TISSUE EXTRACT ON PC12D CELLS. HIDEKI HIDA. ATSUO FUKUDA, TAKESHI HASHITANI, KEIYA NAKAJIMA AND HIT00 NISHINO, 2nd Dept. of Phvsiol., Nagoya City Univ.. Mizuho-ku, Napova 467, Japan. In previous studies we have reported that dopamine-depleted striatal tissue extract (L-extract) promoted neurite extension in PC12D cells and that the effect of L-extract was more potent than that of intact striatal tissue extract. The mechanism to induce neurite extension was investigated by Neurite outgrowth blocking of the effect of NGF and/or FGF and by blocking signal transduction. by L-extract was slightly blocked by the pretreatment of anti-NGF and strongly blocked by addition of anti-bFGF antibody, suggesting that the action of L-extract was mostly due to bFGF activities. After inhibition of cyclic AMP-dependent protein kinase (PKA) with H-89 (a potent and selective inhibitor of PKA), the effect of L-extract was reduced but remained. However activation of protein kinase C (PKC) by phorbol ester failed to induce neurite extensions, suggesting that trophic effect of L-extract was mainly due to the signal transduction by PKA but partly others.
1231
IS NEURON-SPECIFICc-src GENE PRODUCT, pp60c-src(+)INVOLVED IN SYNAPTOGENESIS? SAOKO ATSUMI, XIU-YAN ZHAI AND TOYOKO KAWATE, Dept. -of Anat..Yamanashi Med. Univ., Tam% Yamanashi.409-38, Japan.---
Neuron- ecific c-src gene is known to be expressedmainly in neuronalcells. Its gene product pp6@-s’C(+"p b s tyrosinekinase activity and inter ts with the plasma membrane with N-terminal ?? myristicacid. Although morphologicallypp60CBsrC ' has been reportedto be localized in neuronal cell bodies and growth cones of c_srcto+qing deve neurons, its functionis still unknown. In order we raised the specificantibodyagainsthexapeptide to elucidate the function of pp60 which was a unique amino acid sequence of the neuron-specificc-src gene product, and affini yt+) purified the antibody.Using the specific antibody, we examined the localizationof pp60C'srC in cultured spinal neuronsand in co- ultures of spinal neuronsand skeletal muscle cells. Immunowas performedby the PAP technique. Neurons were dissected cytochemicaldetectionof pp6Cc-src(t'> from 7-10 day embryos and cultured in MEM containingchick embryo extract and horse serum. In lday culture, nd formed growth cones at the tip of neurites. this stage, immunoreactivity neuroblasts extended of pp60 n,e_U5'Eety was weak or moderate. As the culture proceed2 neuritesentendedand made contact with muscle cells or non-neuronalcells where severa cositieswere observed to form. At this stage, the intense immunoreactivityof pp60 observed in neuronal cell bodies and extending ne r tes, and especially in varicosities in contactareas. These findingssuggest that pp60c-srcftfplays importantroles in synaptogenesis.
1229
THE REGULATION OF CHOLINE ACETYLTRANSFERASE BY INTERLEUKIN-3 ON SEPTAL CHOLINERGIC CELL LINE. TATSUHIDE KUNISI-IITA. YOS. . KONISHI. KEIKICHI T-AM). De V. of Demy&x&mg Diseased
In previous studies we reported that interleukin-3 (IL-3) was a noble trophic factor for cholinergic neurons in central nervous system to promote neurite extension, neuronal survival and enhancement of choline acetyltransferase (ChAT) activity in vivo and in vitro (Kamegai et al., Neuron, 1990). Moreover, recent our works showed IL-3 could be produced in central nervous system and its receptor was specifically present in cholinergic neurons (Konishi et al., submitted). These findings suggest IL-3 directly induces the trophic events in cholinergic neurons. To clarify the mechanisms on the regulation of ChAT by IL-3 we analyzed the protein phosphorylation after IL-3 stimulation, 5’-regions of ChAT mRNAs and promoter elements of genomic ChAT cDNA (Misawa et al., J. Biol. Chem., 1992) required for IL-3 induction using a septal cholinergic cell line SN6. The results of these investigations will be discussed.
1230 TROPHIC EFFECTS OF DOPAMINE-DEPLETED STRIATAL TISSUE EXTRACT ON PC12D CELLS. HIDEKI HIDA. ATSUO FUKUDA, TAKESHI HASHITANI, KEIYA NAKAJIMA AND HIT00 NISHINO, 2nd Dept. of Phvsiol., Nagoya City Univ.. Mizuho-ku, Napova 467, Japan. In previous studies we have reported that dopamine-depleted striatal tissue extract (L-extract) promoted neurite extension in PC12D cells and that the effect of L-extract was more potent than that of intact striatal tissue extract. The mechanism to induce neurite extension was investigated by Neurite outgrowth blocking of the effect of NGF and/or FGF and by blocking signal transduction. by L-extract was slightly blocked by the pretreatment of anti-NGF and strongly blocked by addition of anti-bFGF antibody, suggesting that the action of L-extract was mostly due to bFGF activities. After inhibition of cyclic AMP-dependent protein kinase (PKA) with H-89 (a potent and selective inhibitor of PKA), the effect of L-extract was reduced but remained. However activation of protein kinase C (PKC) by phorbol ester failed to induce neurite extensions, suggesting that trophic effect of L-extract was mainly due to the signal transduction by PKA but partly others.
1231
IS NEURON-SPECIFICc-src GENE PRODUCT, pp60c-src(+)INVOLVED IN SYNAPTOGENESIS? SAOKO ATSUMI, XIU-YAN ZHAI AND TOYOKO KAWATE, Dept. -of Anat..Yamanashi Med. Univ., Tam% Yamanashi.409-38, Japan.---
Neuron- ecific c-src gene is known to be expressedmainly in neuronalcells. Its gene product pp6@-s’C(+"p b s tyrosinekinase activity and inter ts with the plasma membrane with N-terminal ?? myristicacid. Although morphologicallypp60CBsrC ' has been reportedto be localized in neuronal cell bodies and growth cones of c_srcto+qing deve neurons, its functionis still unknown. In order we raised the specificantibodyagainsthexapeptide to elucidate the function of pp60 which was a unique amino acid sequence of the neuron-specificc-src gene product, and affini yt+) purified the antibody.Using the specific antibody, we examined the localizationof pp60C'srC in cultured spinal neuronsand in co- ultures of spinal neuronsand skeletal muscle cells. Immunowas performedby the PAP technique. Neurons were dissected cytochemicaldetectionof pp6Cc-src(t'> from 7-10 day embryos and cultured in MEM containingchick embryo extract and horse serum. In lday culture, nd formed growth cones at the tip of neurites. this stage, immunoreactivity neuroblasts extended of pp60 n,e_U5'Eety was weak or moderate. As the culture proceed2 neuritesentendedand made contact with muscle cells or non-neuronalcells where severa cositieswere observed to form. At this stage, the intense immunoreactivityof pp60 observed in neuronal cell bodies and extending ne r tes, and especially in varicosities in contactareas. These findingssuggest that pp60c-srcftfplays importantroles in synaptogenesis.