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Inositol 1,4,5-trisphosphate (IP3) receptor and calcium release
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2. Symposia II. Inositol phosphate and calcium signaling in nerve cells B R A D Y K I N I N - E V O K E D A C E T Y L C H O L I N E R E L E A S E VIA INOSITOL T R I S P H O S P H A T E DEPENDENT CALCIUM RISE..HARUHIRQ HIGASHIDA! AND AKIHIKQ.Q(~URA~,IDeoartment of Bioohvsics. Neuroinformation Research Insitute. Kanazawa University School of Medicine. Kanazawa 920 and -2DeDarlTnent of Neuroscience. Mit~ubishi Kasei Institute of Life Science. Machida 194. Japan. The mechanism of the bradykinin (BK)-induced increase of acetylcholine was studied in NG10815 mouse neuroblastoma x rat glioma hybrid celts and their synapses formed onto mouse muscle cells under culture condition. The BK- induced facilitation was observed even if the extracellular medium was changed to a lower Ca 2+ concentration or Cd 2+ (0.5-2 mM) added. Ba 2+ (2-4 mM) bloked the membrane hypeq~olarization of hybrid cells in response to BK, but still the characteristic facilitation of MEPPs was observed in myotybes. Ionophoretic injection of inositol 1.4.5-trisphosphate (InsP3) into the cytoplasm of an NG108-15 cell briefly evoked MEPPs during the InsP3-induced hyperpolarizing phase. However. the InsP3-dependent facilitation w a s also o b s e r v e d w h e t h e r or not InsP3 hyperpolarized the cell. Real-time quantitative monitoring of the intraceIIular free Ca 2+ concentration ([Ca2+]i) with fura-2 on a single NG108-15 cell showed that BK induced an elevation of the [Ca2+]i under any conditions mentioned above. These data suggest that the BK-induced initial facilitation results from the inositol 1,4,5-trisphosphate-dependent elevation of residual Ca 2+.
INOSITOL 1,4,5-TRISPHOSPHATE ( I P 3 ) RECEPTOR AND CALCIUM RELEASE KATSUHIKO MIKOSHIBA, T E I I C H I FURUICHt, ATSUSHI MIYAWAKI, NOBUAKI MAEDA, SHtNGO YOSHIKAW~, MICHIO NIINOB~, AND $HINJI NAKADE. Institute for Protein Research, Osaka U n i v e r ~ i t v , 3 - 2 Yamadaoka S u i t a , Osaka 5 6 5 , and National (n~titute for B a s i c B i o l o g y , Mvodai i i ~ h o , O k a z a k i 4 4 4 , J a p a n . Inositol 1,4,5-trisphosphate (IP3) is a second messenger, p r o d u c e d by t h e phosphoinositide t u r n o v e r s y s t e m in r e s p o n s e t o h o r m o n e s , n e u r o t r a n s m i t t e r s , or growth factors in a w i d e v a r i e t y of c e l l t y p e s . E v i d e n c e has been a c c u m u l a t e d for the presence of high-affinity, saturabl9 IP3 b i n d i n g sites and t h e I P 3 m e d i a t e d Ca Z÷ r e l e a s e from n o n m i t o c h o d r i a l Ca Z+ s t o r e s . R e c e n t l y , P400 p r o t e i n , originally f o u n d as a c e r e b e l l a r glycoproteln of relative m o l e c u l a r w e i g h t Mr 250K, has been shown t o be i d e n t i c a l t o t h e IP3 r e c e p t o r p r o t e i n ( I P 3 - R ) . The IP3-R is a b u n d a n t in c e r e b e l l a r Purkinje c e l l s and is m o s t l y a b s e n t from c e r e bella of Purkinje cell degenerate mutant mice. I m m u n o h i s t o c h e m i c a l a n a l y s i s u s i n g i m m u n o g o l d has shown t h a t IP3-R is h i g h l y c o n d e n s e d on t h e s m o o t h s u r f a c e d endoplasmic reticulum ( E R ) , and v e r y low a m o u n t on t h e r o u g h s u r f a c e d ER, b u t n o t f o u n d i n t h e c y t o p l a s m i c membrane, Golgi apparatus, and m i t o c h o n d r i a . Cross linking experiment using purified r e c e p t o r IP3-R f o r m s a h o m o t e t r a m e r i c s t r u c t u r e . We o b t a i n e d t h e c e r e b e l l a r t y p e IP3-R cDNA and g e n e r a t e d an L c e l l transfectant ( L 1 5 ) t h a t p r o d u c e s cDNAderived IP3-R. This protein displays high affinity, specificity, and c a p 3 c i ~ y for I P 3 , as d o e s t h e c e r e b e l l a r IP3-R. IP3 can a l s o i n d u c e g r e a t e r 4bCaZ+ r e l e a s e from t h e membrane v e s i c l e s of L|5 cells than from those of control L cells. These r e s u l t s provide direct e v i d e n c e t h a t t h e c D N A - d e r i v e d IP3-R p r o t e i n Is a c t u a l l y i n v o l v e d in p h y s i o l o g i c a l Ca Z+ m o b i l i z a t i o n , through binding to IP3 m o l e c u l e s in t h e same m a n n e r as t h e c e r e b e l l a r I P 3 - R . From t h e s e r ~ suits, we c o n c l u d e d that I P 3 - R is an IP3 b i n d i n g protein and f o r m s a Ca Z+ c h a n n e l m a k i n g h o m o t e t r a m e r , w h i c h is l o c a t e d s p e c i f i c a l l y on t h e s m o o t h - s u r f a c e d ER. R e f : N a t u r e 34? 3 2 - 3 8 ( 1 9 8 9 ) , EMBO J. ~ 6 1 - 6 7 ( 1 9 9 0 ) , Neuron ~ 1]] 8 ( 1 9 9 0 ) , C e l l S i r . F u n c t . 15 1 6 3 - 1 7 3 ( 1 9 9 0 ) .
2. Symposia II. Inositol phosphate and calcium signaling in nerve cells B R A D Y K I N I N - E V O K E D A C E T Y L C H O L I N E R E L E A S E VIA INOSITOL T R I S P H O S P H A T E DEPENDENT CALCIUM RISE..HARUHIRQ HIGASHIDA! AND AKIHIKQ.Q(~URA~,IDeoartment of Bioohvsics. Neuroinformation Research Insitute. Kanazawa University School of Medicine. Kanazawa 920 and -2DeDarlTnent of Neuroscience. Mit~ubishi Kasei Institute of Life Science. Machida 194. Japan. The mechanism of the bradykinin (BK)-induced increase of acetylcholine was studied in NG10815 mouse neuroblastoma x rat glioma hybrid celts and their synapses formed onto mouse muscle cells under culture condition. The BK- induced facilitation was observed even if the extracellular medium was changed to a lower Ca 2+ concentration or Cd 2+ (0.5-2 mM) added. Ba 2+ (2-4 mM) bloked the membrane hypeq~olarization of hybrid cells in response to BK, but still the characteristic facilitation of MEPPs was observed in myotybes. Ionophoretic injection of inositol 1.4.5-trisphosphate (InsP3) into the cytoplasm of an NG108-15 cell briefly evoked MEPPs during the InsP3-induced hyperpolarizing phase. However. the InsP3-dependent facilitation w a s also o b s e r v e d w h e t h e r or not InsP3 hyperpolarized the cell. Real-time quantitative monitoring of the intraceIIular free Ca 2+ concentration ([Ca2+]i) with fura-2 on a single NG108-15 cell showed that BK induced an elevation of the [Ca2+]i under any conditions mentioned above. These data suggest that the BK-induced initial facilitation results from the inositol 1,4,5-trisphosphate-dependent elevation of residual Ca 2+.
INOSITOL 1,4,5-TRISPHOSPHATE ( I P 3 ) RECEPTOR AND CALCIUM RELEASE KATSUHIKO MIKOSHIBA, T E I I C H I FURUICHt, ATSUSHI MIYAWAKI, NOBUAKI MAEDA, SHtNGO YOSHIKAW~, MICHIO NIINOB~, AND $HINJI NAKADE. Institute for Protein Research, Osaka U n i v e r ~ i t v , 3 - 2 Yamadaoka S u i t a , Osaka 5 6 5 , and National (n~titute for B a s i c B i o l o g y , Mvodai i i ~ h o , O k a z a k i 4 4 4 , J a p a n . Inositol 1,4,5-trisphosphate (IP3) is a second messenger, p r o d u c e d by t h e phosphoinositide t u r n o v e r s y s t e m in r e s p o n s e t o h o r m o n e s , n e u r o t r a n s m i t t e r s , or growth factors in a w i d e v a r i e t y of c e l l t y p e s . E v i d e n c e has been a c c u m u l a t e d for the presence of high-affinity, saturabl9 IP3 b i n d i n g sites and t h e I P 3 m e d i a t e d Ca Z÷ r e l e a s e from n o n m i t o c h o d r i a l Ca Z+ s t o r e s . R e c e n t l y , P400 p r o t e i n , originally f o u n d as a c e r e b e l l a r glycoproteln of relative m o l e c u l a r w e i g h t Mr 250K, has been shown t o be i d e n t i c a l t o t h e IP3 r e c e p t o r p r o t e i n ( I P 3 - R ) . The IP3-R is a b u n d a n t in c e r e b e l l a r Purkinje c e l l s and is m o s t l y a b s e n t from c e r e bella of Purkinje cell degenerate mutant mice. I m m u n o h i s t o c h e m i c a l a n a l y s i s u s i n g i m m u n o g o l d has shown t h a t IP3-R is h i g h l y c o n d e n s e d on t h e s m o o t h s u r f a c e d endoplasmic reticulum ( E R ) , and v e r y low a m o u n t on t h e r o u g h s u r f a c e d ER, b u t n o t f o u n d i n t h e c y t o p l a s m i c membrane, Golgi apparatus, and m i t o c h o n d r i a . Cross linking experiment using purified r e c e p t o r IP3-R f o r m s a h o m o t e t r a m e r i c s t r u c t u r e . We o b t a i n e d t h e c e r e b e l l a r t y p e IP3-R cDNA and g e n e r a t e d an L c e l l transfectant ( L 1 5 ) t h a t p r o d u c e s cDNAderived IP3-R. This protein displays high affinity, specificity, and c a p 3 c i ~ y for I P 3 , as d o e s t h e c e r e b e l l a r IP3-R. IP3 can a l s o i n d u c e g r e a t e r 4bCaZ+ r e l e a s e from t h e membrane v e s i c l e s of L|5 cells than from those of control L cells. These r e s u l t s provide direct e v i d e n c e t h a t t h e c D N A - d e r i v e d IP3-R p r o t e i n Is a c t u a l l y i n v o l v e d in p h y s i o l o g i c a l Ca Z+ m o b i l i z a t i o n , through binding to IP3 m o l e c u l e s in t h e same m a n n e r as t h e c e r e b e l l a r I P 3 - R . From t h e s e r ~ suits, we c o n c l u d e d that I P 3 - R is an IP3 b i n d i n g protein and f o r m s a Ca Z+ c h a n n e l m a k i n g h o m o t e t r a m e r , w h i c h is l o c a t e d s p e c i f i c a l l y on t h e s m o o t h - s u r f a c e d ER. R e f : N a t u r e 34? 3 2 - 3 8 ( 1 9 8 9 ) , EMBO J. ~ 6 1 - 6 7 ( 1 9 9 0 ) , Neuron ~ 1]] 8 ( 1 9 9 0 ) , C e l l S i r . F u n c t . 15 1 6 3 - 1 7 3 ( 1 9 9 0 ) .