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1230 Immunohistochemistry of dopamine-transporter in neurons using a monoclonal antibody
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IMMUNOHISTOCHEMISTRY O F DOPAMINE-TRANSPORTER IN NEURONS USING A MONOCLONAL ANTIBODY. TOMOKO KADOTA 1, KAYOKO MOROI 1, YUMIKO SAITO 2, SEI-ICHI K A W A S H I M A z AND KEN KADOTA 3, tSch. Med., Chiba University, Chiba 260, 2"I'okyo Metropolitan Inst. Med. Sci., Bunkyo-ku, Tokyo 113, and 3Chiba Center of Social Welfare for Mental Retardation, Sodegaura 299-02, Japan.
Neurotransmitter transporters have been pharmacologically identified to localize in the plasmamembrane of nerve endings and shown working in re-uptake of neurotransmitters released into the intercellular space. Dopamine transporter (DAT) has been recently cloned. The authors have prepared an antibody against DAT using a synthesized peptide, and immunohistochemically examined PC12 cells along the developmental stages of the neurite outgrowth. PC12 cells were cultured in the M E M medium containing the nerve growth factor (NGF) or a protease inhibitor for 1-7 days. The neurite outgrowth was significantly activated in the medium containing N G F or a protease inhibitor. It has been shown that DAT was e x p r e s s e d . i n the P C I 2 cells and localized in the plasmamembrane of the cell body at the first stage of culture and then in the growth cone during the stage of neurite outgrowth.
] 23]
Subpopulation of smaller diameter primary sensory neurons defined by expression of a 29-kD protein recognized by monoclonal antibody PC3.1. KEIKO TAKIGUCHI-HAYASHI AND YASUYOSHI ARIMATSU, Lab. of Neuromorphol., Mitsubishi K a s e i I n s t . o f Life S c i . , 1 l - M i n a m i o o v a , M a c h i d a - s h i , T o k y o 194, J a p a n Monoclonal ~ntibody PC3.1 recognizes a novel 29-kD protein of adult rat brain. In the developing rat nervous systems, PC3.1 antigen was detected in subpopulation of primary sensory neurons from prenatal stages. PC3.1-immunopositive n e u r o n s b e l o n g to s m a l l e r diameter neurons and partially coincide with some of neuropeptide-containing neurons like c a l c i t o n i n g e n e - r e l a t e d p e p t i d e , s u b s t a n c e P, s o m a t o s t a t i n , a n d c h o l e k y s t o k i n i n , r e s p e c t i v e l y . PC3.1 antigen was detected in both nucleus and cytoplasm of the cell body, and in axonal f i b e r s . S t r o n g i m m u n o r e a c t i v i t y w a s a l s o d e t e c t e d i n t h e l a m i n a IT o f t h e s p i n a l c o r d . These results indicate that a subpopulation of smaller diameter primary sensory neurons can be classified by the expression of the 29-kD protein recognized by PC3.1. Function of this protein and physiological significance of these neurons are discussed.
1232
GAP-43-DERIVEDPEPT1DES MODULATINGGo ACTIVATIONCAN ALTER THE SENSITIVIFY OF THE GROWTHCONE TO A COLLAPSING FACTOR. MICHIHIRO IGARASHIt. YOSHIAKI SUDO2. AND MARK C. FISIIMAN. Developmental Biology Lab., Masschusetts Gem Hosp.-East. Charlesto~n. MA 02129. USA; Present addresses, ~nept. Mol. Cell. Neurob i o l . , Gunma Univ. Sch. Med., Maebashi, Gunma 371; and Sumitomo Pharmac. Co., Takarazuka, Hyogo 665, Japan.
Currently. inhibitory signals for nerve growth are recognized as important regulators as well as signals for promoting neurite outgrowth. Such molecules cause growth cone collapse in in v i t r o experiments. We ha~e suggested that G protein-coupled receptors are involved in growth cone collapse induced by t~o d i f f e r e n t membrane-bound inhibitory signals. GAP-43. a protein enriched in growth cone membrane, d i r e c t l y a c t i v a t e s Go protein. We have recently demonstrated that an amino terminus decapeptide of GAP-43 (I-10 peptide) is e s s e n t i a l to i t s activation of Go. Thus. we have investigated the i n t e r a c t i o n between the 1-10 peptide and other i n i h i b i t o r y molecules. The 1-10 peptide p o t e n t i a t e s the ef[oct of a collapsing factor, brain membrane extract (BME) from chick embryos. BME is known to collapse growth cones from dorsal root ganglia (DRG) and r e t i n a l neurons. This s e n s i t i z a t i o n by 1-10 peptide is completely blocked by p e r t u s s i s toxin at a low dose (20 ng/mt) less than which blocks the a c t i v i t y of B~IE. Among several peptides ~ith different amino acids substituted f~r two cysteines of 1-i0. two peptides, those substituted with tyrosine (Y), or , i t h methionine (M). inhibit Go activation. These peptides also inhibit DRG growth cone collapse by BME. These r e s u l t s suggest that native and modified 1-10 peptides, which have a c t i v i t i e s to a c t i v a t e or inhibit Go activation. also potentiate o r - a t t e n u a t e the i n h i b i t i o n of nerve growth.
IMMUNOHISTOCHEMISTRY O F DOPAMINE-TRANSPORTER IN NEURONS USING A MONOCLONAL ANTIBODY. TOMOKO KADOTA 1, KAYOKO MOROI 1, YUMIKO SAITO 2, SEI-ICHI K A W A S H I M A z AND KEN KADOTA 3, tSch. Med., Chiba University, Chiba 260, 2"I'okyo Metropolitan Inst. Med. Sci., Bunkyo-ku, Tokyo 113, and 3Chiba Center of Social Welfare for Mental Retardation, Sodegaura 299-02, Japan.
Neurotransmitter transporters have been pharmacologically identified to localize in the plasmamembrane of nerve endings and shown working in re-uptake of neurotransmitters released into the intercellular space. Dopamine transporter (DAT) has been recently cloned. The authors have prepared an antibody against DAT using a synthesized peptide, and immunohistochemically examined PC12 cells along the developmental stages of the neurite outgrowth. PC12 cells were cultured in the M E M medium containing the nerve growth factor (NGF) or a protease inhibitor for 1-7 days. The neurite outgrowth was significantly activated in the medium containing N G F or a protease inhibitor. It has been shown that DAT was e x p r e s s e d . i n the P C I 2 cells and localized in the plasmamembrane of the cell body at the first stage of culture and then in the growth cone during the stage of neurite outgrowth.
] 23]
Subpopulation of smaller diameter primary sensory neurons defined by expression of a 29-kD protein recognized by monoclonal antibody PC3.1. KEIKO TAKIGUCHI-HAYASHI AND YASUYOSHI ARIMATSU, Lab. of Neuromorphol., Mitsubishi K a s e i I n s t . o f Life S c i . , 1 l - M i n a m i o o v a , M a c h i d a - s h i , T o k y o 194, J a p a n Monoclonal ~ntibody PC3.1 recognizes a novel 29-kD protein of adult rat brain. In the developing rat nervous systems, PC3.1 antigen was detected in subpopulation of primary sensory neurons from prenatal stages. PC3.1-immunopositive n e u r o n s b e l o n g to s m a l l e r diameter neurons and partially coincide with some of neuropeptide-containing neurons like c a l c i t o n i n g e n e - r e l a t e d p e p t i d e , s u b s t a n c e P, s o m a t o s t a t i n , a n d c h o l e k y s t o k i n i n , r e s p e c t i v e l y . PC3.1 antigen was detected in both nucleus and cytoplasm of the cell body, and in axonal f i b e r s . S t r o n g i m m u n o r e a c t i v i t y w a s a l s o d e t e c t e d i n t h e l a m i n a IT o f t h e s p i n a l c o r d . These results indicate that a subpopulation of smaller diameter primary sensory neurons can be classified by the expression of the 29-kD protein recognized by PC3.1. Function of this protein and physiological significance of these neurons are discussed.
1232
GAP-43-DERIVEDPEPT1DES MODULATINGGo ACTIVATIONCAN ALTER THE SENSITIVIFY OF THE GROWTHCONE TO A COLLAPSING FACTOR. MICHIHIRO IGARASHIt. YOSHIAKI SUDO2. AND MARK C. FISIIMAN. Developmental Biology Lab., Masschusetts Gem Hosp.-East. Charlesto~n. MA 02129. USA; Present addresses, ~nept. Mol. Cell. Neurob i o l . , Gunma Univ. Sch. Med., Maebashi, Gunma 371; and Sumitomo Pharmac. Co., Takarazuka, Hyogo 665, Japan.
Currently. inhibitory signals for nerve growth are recognized as important regulators as well as signals for promoting neurite outgrowth. Such molecules cause growth cone collapse in in v i t r o experiments. We ha~e suggested that G protein-coupled receptors are involved in growth cone collapse induced by t~o d i f f e r e n t membrane-bound inhibitory signals. GAP-43. a protein enriched in growth cone membrane, d i r e c t l y a c t i v a t e s Go protein. We have recently demonstrated that an amino terminus decapeptide of GAP-43 (I-10 peptide) is e s s e n t i a l to i t s activation of Go. Thus. we have investigated the i n t e r a c t i o n between the 1-10 peptide and other i n i h i b i t o r y molecules. The 1-10 peptide p o t e n t i a t e s the ef[oct of a collapsing factor, brain membrane extract (BME) from chick embryos. BME is known to collapse growth cones from dorsal root ganglia (DRG) and r e t i n a l neurons. This s e n s i t i z a t i o n by 1-10 peptide is completely blocked by p e r t u s s i s toxin at a low dose (20 ng/mt) less than which blocks the a c t i v i t y of B~IE. Among several peptides ~ith different amino acids substituted f~r two cysteines of 1-i0. two peptides, those substituted with tyrosine (Y), or , i t h methionine (M). inhibit Go activation. These peptides also inhibit DRG growth cone collapse by BME. These r e s u l t s suggest that native and modified 1-10 peptides, which have a c t i v i t i e s to a c t i v a t e or inhibit Go activation. also potentiate o r - a t t e n u a t e the i n h i b i t i o n of nerve growth.