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Calcium-dependent depolarizing potentials originating in motor nerve terminals
S153
33. Neurotransmitters IV. Presynaptic mechanisms I N H I B I T I O N OF M O N O S Y N A P T I C R E F L E X IN N E W B O R N R A T S P I N A L C O R D (i) MUSCARINIC MECHANISM. K O I C H I Y O S H I O K A , D e p a r t m e n t of P h a r m a c o l o g y r F a c u l t y of M e d i c i n e r T o k y o M e d i c a l an---d'Dental U n i v e r s i t y r Yush1"--~ma i - 5 - 4 5 t B u n k y o - k u r To-kyo 113 r Japan. A n i s o l a t e d s p i n a l c o r d - p e r i p h e r a l n e r v e p r e p a r a t i o n of n e w b o r n r a t w a s u s e d to i n v e s t i g a t e the m e c h a n i s m s of C f i b e r - i n d u c e d i n h i b i t i o n of the m o n o s y n a p t i c r e f l e x (MSR). T h e M S R e v o k e d by e l e c t r i c a l s t i m u l a t i o n of the n e r v e b r a n c h to q u a d r i c e p s f e m o r i s m u s c l e or t h o s e to h a m s t r i n g m u s c l e s w a s i n h i b i t e d for a b o u t 20 sec by c o n d i t i o n i n g s t i m u l a t i o n of h e t e r o n y m o u s n e r v e s , i n c l u d i n g the s a p h e n o u s a n d s u r a l n e r v e s F w i t h 5 p u l s e s at 50 Hz a n d a s t r e n g t h s u f f i c i e n t to activate C fibers. T h i s i n h i b i t i o n w a s p o t e n t i a t e d by e d r o p h o n i u m a n d m a r k e d l y a n t a g o n i z e d by a t r o p i n e . T h e M S R e v o k e d by the c o m m o n p e r o n e a l or t i b i a l n e r v e , by c o n t r a s t , r e c e i v e d o n l y s l i g h t i n h i b i t i o n f r o m o t h e r n e r v e s . A p p l i c a t i o n of a c e t y l c h o l i n e by p e r f u s i o n a l s o i n d u c e d an i n h i b i t i o n of m u s c a r i n i c n a t u r e of the MSR. A n a l y s i s of s y n a p t i c d e p r e s s i o n of the M S R u s i n g p a i r e d p u l s e s t i m u l a t i o n s u g g e s t e d t h a t b o t h the a c e t y l c h o l i n e - a n d n e r v e - e v o k e d i n h i b i t i o n s of M S R w e r e d u e to a p r e s y n a p t i c m e c h a n i s m . These results suggest that primary a f f e r e n t C f i b e r s e x c i t e c h o l i n e r g i c s p i n a l n e u r o n s , w h i c h then i n h i b i t the M S R through a muscarinic presynaptic mechanism.
IDENTIFICATION OF ~i AND ~2 SUBUNITS OF DIHYDROPYRIDINE-SENSITIVE CALCIUM CHANNEL OF CHICKEN HEART AND BRAIN. MASAMI TAKAHASHI* and YOSHIKO FUJIMOTO*, Department of Neurosciences t Mitsubishi Kasei Institute of Life Sciences r llMinamiooya r Machida-shi w Tokyo 194 r Japan. A BALB/c mouse was immunized with repeated injections of highly purified chicken synaptic membranes and monoclonal antibodies were produced by fusion of the spleen cells with NSI mouse myeloma cells. Hybridomas were screened for antibody production by RIA method using partially purified chicken brain dihydropyridine-sensitive Ca channels prelabeled with 3H-PN200-110. The resulting antibody, named MAC-BLI, immunoprecipitated dihydropyridine-sensitive Ca channels of chicken brain, heart, gizzard and skeletal muscles. However, to get equivalent amounts of precipitate in muscle tissues, 6-times more antibody was needed than in brain. MAC-BLI did not recognize the dihydropyridine-sensitive Ca channel from rat brain and no specific immunoprecipitation was observed even using the highest concentration of the antibody tested. These results indicate that the structure of MAC-BLI recognition sites differ among different tissues and species. Glycoprotein fractions purified from solubilized chicken cardiac sarcolemmal and synaptic plasmamembranes were labeled with 125I and im~unoprecipitated with MAC-BLI and control mouse IgG. SDS gel electrophoresis revealed that polypeptides of about 195 kDa from both tissues was specifically precipitated by MAC-BLI but not by control mouse IgG. When disulfide bonds were cleaved with DTT, the 195kDa band split into two populations of 195kba and 135kDa. These results indicate that the dihydropyridine-sensitive Ca channel of chicken heart and brain have ~I and ~2 subunits. The molecular size of the 01 subunits of these tissues is larger than that of skeletal muscle.
CALCIUM-DEPENDENT DEPOLARIZING POTENTIALS ORIGINATING IN MOTOR NERVE TERMINALS. KIICHIRO MORITA ~ 1 . ELLEN F BARRETT~ 2 and YOSHIFUMI KATAYAMA!, 1 D e p a r t m e n t of Autonomic Physiology. Niedical Research I n s t i t u t e . Tokyo Medical and Dental U n i v e r s i t y , Kandasurugadai, Chiyodaku, Tokyo lOI. 2Department of Physiology and Biophysics. U n i v e r s i t y of Miami School o_f Medicine. Miami, F l o r i d a 33101. Action p o t e n t i a l s and a f t e r p o t e n t i a l s were recorded v i a a -microelectrode i n s e r t e d into motor axons i n n e r v a t i n g l i z a r d ceratomandibularis muscle. In 1 - lO mM tetraethylammonium (TEA) the a c t i o n p o t e n t i a l was followed by a sequence of a f t e r p o t e n t i a l s w h o s e amplitude and d u r a t i o n increased with increasing proximity to the motor nerve t e r m i n a l s . These d e p o l a r i z i n g a f t e r P o t e n t i a l s were Ca-dependent: they increased with i n c r e a s i n g bath [Cal. and were abolished by s e l e c t i v e p e r f u s i o n of Cd or Mn over the terminal region. In [Ca]-free s o l u t i o n s the a f t e r p o t e n t i a l s were r e s t o r e d by s e l e c t i v e Perfusion of Ca over the terminal region, but not by Ca applied to the more proximal nerve trunk. Uhen Na i n f l u x was eliminated by l-lO ~M t e t r o d o t o x i n or by s u p e r f u s i o n of TEA f o r bath Na passage of d e p o l a r i z i n g c u r r e n t i n t o the a x o n e v o k e d Prolonged d e p o l a r i z i n g a f t e r p o t e n t i a l s t h a t were blocked by calcium blockers. These r e s u l t s suggest t h a t the i n t r a - a x o n a l l y recorded Ca-dependent afterpotentials are caused by e l e c t r o t o n i c spread of d e p o l a r i z a t i o n s produced by calcium influx into that axon's terminals.
33. Neurotransmitters IV. Presynaptic mechanisms I N H I B I T I O N OF M O N O S Y N A P T I C R E F L E X IN N E W B O R N R A T S P I N A L C O R D (i) MUSCARINIC MECHANISM. K O I C H I Y O S H I O K A , D e p a r t m e n t of P h a r m a c o l o g y r F a c u l t y of M e d i c i n e r T o k y o M e d i c a l an---d'Dental U n i v e r s i t y r Yush1"--~ma i - 5 - 4 5 t B u n k y o - k u r To-kyo 113 r Japan. A n i s o l a t e d s p i n a l c o r d - p e r i p h e r a l n e r v e p r e p a r a t i o n of n e w b o r n r a t w a s u s e d to i n v e s t i g a t e the m e c h a n i s m s of C f i b e r - i n d u c e d i n h i b i t i o n of the m o n o s y n a p t i c r e f l e x (MSR). T h e M S R e v o k e d by e l e c t r i c a l s t i m u l a t i o n of the n e r v e b r a n c h to q u a d r i c e p s f e m o r i s m u s c l e or t h o s e to h a m s t r i n g m u s c l e s w a s i n h i b i t e d for a b o u t 20 sec by c o n d i t i o n i n g s t i m u l a t i o n of h e t e r o n y m o u s n e r v e s , i n c l u d i n g the s a p h e n o u s a n d s u r a l n e r v e s F w i t h 5 p u l s e s at 50 Hz a n d a s t r e n g t h s u f f i c i e n t to activate C fibers. T h i s i n h i b i t i o n w a s p o t e n t i a t e d by e d r o p h o n i u m a n d m a r k e d l y a n t a g o n i z e d by a t r o p i n e . T h e M S R e v o k e d by the c o m m o n p e r o n e a l or t i b i a l n e r v e , by c o n t r a s t , r e c e i v e d o n l y s l i g h t i n h i b i t i o n f r o m o t h e r n e r v e s . A p p l i c a t i o n of a c e t y l c h o l i n e by p e r f u s i o n a l s o i n d u c e d an i n h i b i t i o n of m u s c a r i n i c n a t u r e of the MSR. A n a l y s i s of s y n a p t i c d e p r e s s i o n of the M S R u s i n g p a i r e d p u l s e s t i m u l a t i o n s u g g e s t e d t h a t b o t h the a c e t y l c h o l i n e - a n d n e r v e - e v o k e d i n h i b i t i o n s of M S R w e r e d u e to a p r e s y n a p t i c m e c h a n i s m . These results suggest that primary a f f e r e n t C f i b e r s e x c i t e c h o l i n e r g i c s p i n a l n e u r o n s , w h i c h then i n h i b i t the M S R through a muscarinic presynaptic mechanism.
IDENTIFICATION OF ~i AND ~2 SUBUNITS OF DIHYDROPYRIDINE-SENSITIVE CALCIUM CHANNEL OF CHICKEN HEART AND BRAIN. MASAMI TAKAHASHI* and YOSHIKO FUJIMOTO*, Department of Neurosciences t Mitsubishi Kasei Institute of Life Sciences r llMinamiooya r Machida-shi w Tokyo 194 r Japan. A BALB/c mouse was immunized with repeated injections of highly purified chicken synaptic membranes and monoclonal antibodies were produced by fusion of the spleen cells with NSI mouse myeloma cells. Hybridomas were screened for antibody production by RIA method using partially purified chicken brain dihydropyridine-sensitive Ca channels prelabeled with 3H-PN200-110. The resulting antibody, named MAC-BLI, immunoprecipitated dihydropyridine-sensitive Ca channels of chicken brain, heart, gizzard and skeletal muscles. However, to get equivalent amounts of precipitate in muscle tissues, 6-times more antibody was needed than in brain. MAC-BLI did not recognize the dihydropyridine-sensitive Ca channel from rat brain and no specific immunoprecipitation was observed even using the highest concentration of the antibody tested. These results indicate that the structure of MAC-BLI recognition sites differ among different tissues and species. Glycoprotein fractions purified from solubilized chicken cardiac sarcolemmal and synaptic plasmamembranes were labeled with 125I and im~unoprecipitated with MAC-BLI and control mouse IgG. SDS gel electrophoresis revealed that polypeptides of about 195 kDa from both tissues was specifically precipitated by MAC-BLI but not by control mouse IgG. When disulfide bonds were cleaved with DTT, the 195kDa band split into two populations of 195kba and 135kDa. These results indicate that the dihydropyridine-sensitive Ca channel of chicken heart and brain have ~I and ~2 subunits. The molecular size of the 01 subunits of these tissues is larger than that of skeletal muscle.
CALCIUM-DEPENDENT DEPOLARIZING POTENTIALS ORIGINATING IN MOTOR NERVE TERMINALS. KIICHIRO MORITA ~ 1 . ELLEN F BARRETT~ 2 and YOSHIFUMI KATAYAMA!, 1 D e p a r t m e n t of Autonomic Physiology. Niedical Research I n s t i t u t e . Tokyo Medical and Dental U n i v e r s i t y , Kandasurugadai, Chiyodaku, Tokyo lOI. 2Department of Physiology and Biophysics. U n i v e r s i t y of Miami School o_f Medicine. Miami, F l o r i d a 33101. Action p o t e n t i a l s and a f t e r p o t e n t i a l s were recorded v i a a -microelectrode i n s e r t e d into motor axons i n n e r v a t i n g l i z a r d ceratomandibularis muscle. In 1 - lO mM tetraethylammonium (TEA) the a c t i o n p o t e n t i a l was followed by a sequence of a f t e r p o t e n t i a l s w h o s e amplitude and d u r a t i o n increased with increasing proximity to the motor nerve t e r m i n a l s . These d e p o l a r i z i n g a f t e r P o t e n t i a l s were Ca-dependent: they increased with i n c r e a s i n g bath [Cal. and were abolished by s e l e c t i v e p e r f u s i o n of Cd or Mn over the terminal region. In [Ca]-free s o l u t i o n s the a f t e r p o t e n t i a l s were r e s t o r e d by s e l e c t i v e Perfusion of Ca over the terminal region, but not by Ca applied to the more proximal nerve trunk. Uhen Na i n f l u x was eliminated by l-lO ~M t e t r o d o t o x i n or by s u p e r f u s i o n of TEA f o r bath Na passage of d e p o l a r i z i n g c u r r e n t i n t o the a x o n e v o k e d Prolonged d e p o l a r i z i n g a f t e r p o t e n t i a l s t h a t were blocked by calcium blockers. These r e s u l t s suggest t h a t the i n t r a - a x o n a l l y recorded Ca-dependent afterpotentials are caused by e l e c t r o t o n i c spread of d e p o l a r i z a t i o n s produced by calcium influx into that axon's terminals.