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Effect of N-methylisoquinolinium ion (NMIQ+) on catecholamine metabolism
$144 SECRETION OF CATECHOLAMINE FROM CULTURED ADRENAL CHROMAFFIN CELLS BY MASTOPARAN. KONOSUKE KUMAKURA1 , MICA 0 . - I M A I Z U M I 1 . AND HISATAKA KASAI 2 " , ~ L l f e S c i e n c e Institute, S o p h i a U n i v e r s i t y , 7-1, K i o i - c h o , C h i y o d a - k u , T o k y o 102, a n d = D e p a r t m e n t o f C h e m i s t r y , T o k y o M e t r o p o l i t a n U n i v e r s i t y , Japan. Mastoparan (MAST), a venom peptide from wasp, evokes the secretion of cateeholamlnes (CA) f r o m a d r e n a l c h r o m a f f i n c e l l s as w e l l as the r e l e a s e of h i s tamlne f r o m m a s t o c e l l s by u n k n o w n m e c h a n i s m s . To c l a r i f y the m e c h a n i s m s w h e r e b y M A S T e v o k e s CA s e c r e t i o n f r o m t h e s e c e l l s , we s t u d i e d character]stlcs of M A S T evoked secretory process in c u l t u r e d b o v i n e a d r e n a l c h r o m a f f i n c e l l s by direct monitoring using cell-bed perfusion c o u p l e d to an a m p e r o m e t r i c detector. A pulsatlle injection of M A S T i n t o the f l o w s t r e a m r e s u l t e d in a t r a n s i e n t and dosed e p e n d e n t s e c r e t o r y r e s p o n s e . P e r s i s t e n t e x p o s u r e of t h e s e c e l l s to M A S T r e s u l t e d in c o n t i n u o u s s e c r e t i o n of CA. S t i m u l a t i o n w i t h M A S T a f t e r w i t h d r a w a l of the ext r a c e l l u l a r Ca 2. e v o k e d CA s e c r e t i o n 5-10 tlmes that in the p r e s e n c e of Ca =+, a n d this enhanced secretion returned to the c o n t r o l level by r e a d d i t i o n of Ca =÷. In the a b s e n c e of the e x t r a c e l l u l a r Ca 2., the s e c r e t o r y r e s p o n s e was o b t a i n e d either by repetitive stimulation w l t h M A S T or by M A S T a f t e r a c h a l l e n g e w i t h 20 m M caffeine, w i t h a r e l a t i v e l y c o n s t a n t m a g n i t u d e . T h e s e r e s u l t s s u g g e s t that m e c h a n i s m s for MAST-evoked secretion in t h e s e c e l l s m a y i n v o l v e a step or s t e p s a b s o l u t e l y i n d e p e n d e n t of Ca 2~.
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~ T OF N~MEI1tYLISO~UIN0~INIUM ION (NNIQ) ~N CATECHOLAMINEMETABOLISM, TSUTOMUTAKAHASHI 1. MAKOTO NAOI% YOKO HIHATA* J , MASAYASU~ _ ~ . *~. AND T0~IIHARU NAGATSU~, ~ D e p a ~ t m m t o f Food and N u t r i t i o n , Konan ~ C o l l e g e , Konan, D e p a ~ t m ~ t o f B t ~ s t z ~ , Nagoya U n i v e r s i t ~ _School o f Medicine, Nagoya, aDepartment of Hygiene and Public Health, Nippon Medical School, Tokyo, Japan. In human brains, 1 , 2 , 3 , ~ - t e t r ~ i s o q u i n o l i n e was identified, and an increase of its concentration was confirmed in a parkinsortian brain. Recently, a tetrahydroisoquinoline derivative, N-methyl-l,2,3,q-tetrahydrolsoqu/noline, was found to be oxidized by both type A and B m0~o~m~ne oxidase (MAO) in human brain synaptosmms [Nsoi et a~., J. Neurochem. 52, 653 (1989)]. Effects of its oxidative product, N-methyl-isoquinolinium ion (N~IQ+), on enzymes involved in catecholam/ne metabolism were studied using clonal rat pheochromocytome PCI~h cell line. In vft~o, NNIQ + dose-dependently inhibited tyrosine hydroxylase (TH) activity in the cells, end IC50 was obta/ned around 75 ~M. NMIQ + inhibited aromatic L-amlnoacid decarboxylase (AADC) activity in competition to a co-factor, pyridoxal-5-phosphate, and type A MAO activity in competition to substrate; K i values were ~0 ~M and 11 ~ , respectively. To see the effects of + NMIQ on enzymes ~n the cells, the cells were cultured for 6 days in the presence of 100 nM to 1 mM NMIQ +. Protein amount, or cell number, and AADC and MA0 activity were reduced only with 1 ~4 NMIQ +, but TH activity was r e d u c e d With MMIQ + even at i0 ~M, which shows that NNIQ + can reduce dopamine synthesis at concentrations lower than requiPed for cell death. These data sugEest that NMIQ + is a naturally-occurrin~ neurotoxin, which perturbs catecholamine metabolism in dopmminerEic neurons, as N-methyl-~-phenyl-pyrid/n/um ion.
I ~ I B I T I O ~ OF HUMAN~
~ _ _ ~ _ r ~, ~ o ~ D e ~ t
N~oya 4~,
MONO~XT_~ 01IDASE } ~ ~ r ~ Y C I ~ O
~AOI~. ~ J I
w~Y~*
~. T ~ _
~ .
~ *
TAKAHIK0 K O J M *1,
2. AND ~ u a S U
~A~TSU~.
of B i o c h e m i s t r y . NaEoya University S c h o o l o f M e d i c i n e , 65 T s u z ~ , - - ~ - c h o , S h o v a - k u ,
and ~ N a t t o n ~
Cancer Center Research Institute,
Tokyo 104 t J a p a n .
Discovery of l-nethyl-~-- ~ - l , 2 , 3 , 6 - t e t r a h ~ n ~ I d i n e , a neuz~toxin that elicits p a ~ s m by specific deKeneration of d o p ~ z ~ c neurons in the nlKro-striatal system,
initiated intensive studies to find a naturally-occurrir~ n e u r o t o x i n t h a t stay c a u s e p a r k i n s o ~ m n i n humans. R e c e n t l y we r e p o r t e d i n h i b i t i o n o f dop-m~ne m e t a b o l i s m by c a r c i n o & ~ n i c h e t e r o c y c l i c m t t n e s . Our p r e s e n t s t u d y i s on t h e e f f e c t s o f a s e r i e s o f h e t e r o c y c l i c a m i n e s on a c t i v i t y o f t y p e A and B nonoR=~ne o x i d a s e (HAO-A and HAO-B} i n human b r a i n s y n a p t o a n u e s . A l l t h e h e t e r o c y c l i c m a i n e s i n h i b i t e d NAO-A c o s ~ t i t i v e l y in terms of subst~te. 3-At~no-l-smthyl~-pyrido[4,3-b]indole ( T r p - P - 2 ) was t h e m o s t p o t e n t i n h i b i t o r ; i t s K~ v a l u e toward NAO-A was 0 . 8 4 ~ , w h i c h was a b o u t 1 / 5 0 o f i t s Km v a l u e toward a n maine s u b s t r a t e , k ~ n u ~ m ~ n e , ~ 6 . ~ ~ . 3-Amino-l,4-dlmethyl-SH-pyrldo[~,3-b]indole (Trp-P-I) was also a potent inhibitor of MA0-A. 3or 2-Amino-pyrldo-indoles or -Imldazoles inhibited both NAO-A and NAO-B competitively to substrato, but smines with 2-amino-3-methylimidazo ring and amines structurally-~elated to carboline inh~bited MAO-B mm-cu~petltively. The K i values of hetez~cycllc - m ~ e e to MAO-B were in Eeneral much larger than those to NA0-A. Inb/bltlon of MAO-A by Trp-P-2 p~oved to be reversible, and Trp-P-2 was not metabolized by MA0. Potency and type of inhibition by these amines were discussed in Pelatiml to their chemical structures.
÷
~ T OF N~MEI1tYLISO~UIN0~INIUM ION (NNIQ) ~N CATECHOLAMINEMETABOLISM, TSUTOMUTAKAHASHI 1. MAKOTO NAOI% YOKO HIHATA* J , MASAYASU~ _ ~ . *~. AND T0~IIHARU NAGATSU~, ~ D e p a ~ t m m t o f Food and N u t r i t i o n , Konan ~ C o l l e g e , Konan, D e p a ~ t m ~ t o f B t ~ s t z ~ , Nagoya U n i v e r s i t ~ _School o f Medicine, Nagoya, aDepartment of Hygiene and Public Health, Nippon Medical School, Tokyo, Japan. In human brains, 1 , 2 , 3 , ~ - t e t r ~ i s o q u i n o l i n e was identified, and an increase of its concentration was confirmed in a parkinsortian brain. Recently, a tetrahydroisoquinoline derivative, N-methyl-l,2,3,q-tetrahydrolsoqu/noline, was found to be oxidized by both type A and B m0~o~m~ne oxidase (MAO) in human brain synaptosmms [Nsoi et a~., J. Neurochem. 52, 653 (1989)]. Effects of its oxidative product, N-methyl-isoquinolinium ion (N~IQ+), on enzymes involved in catecholam/ne metabolism were studied using clonal rat pheochromocytome PCI~h cell line. In vft~o, NNIQ + dose-dependently inhibited tyrosine hydroxylase (TH) activity in the cells, end IC50 was obta/ned around 75 ~M. NMIQ + inhibited aromatic L-amlnoacid decarboxylase (AADC) activity in competition to a co-factor, pyridoxal-5-phosphate, and type A MAO activity in competition to substrate; K i values were ~0 ~M and 11 ~ , respectively. To see the effects of + NMIQ on enzymes ~n the cells, the cells were cultured for 6 days in the presence of 100 nM to 1 mM NMIQ +. Protein amount, or cell number, and AADC and MA0 activity were reduced only with 1 ~4 NMIQ +, but TH activity was r e d u c e d With MMIQ + even at i0 ~M, which shows that NNIQ + can reduce dopamine synthesis at concentrations lower than requiPed for cell death. These data sugEest that NMIQ + is a naturally-occurrin~ neurotoxin, which perturbs catecholamine metabolism in dopmminerEic neurons, as N-methyl-~-phenyl-pyrid/n/um ion.
I ~ I B I T I O ~ OF HUMAN~
~ _ _ ~ _ r ~, ~ o ~ D e ~ t
N~oya 4~,
MONO~XT_~ 01IDASE } ~ ~ r ~ Y C I ~ O
~AOI~. ~ J I
w~Y~*
~. T ~ _
~ .
~ *
TAKAHIK0 K O J M *1,
2. AND ~ u a S U
~A~TSU~.
of B i o c h e m i s t r y . NaEoya University S c h o o l o f M e d i c i n e , 65 T s u z ~ , - - ~ - c h o , S h o v a - k u ,
and ~ N a t t o n ~
Cancer Center Research Institute,
Tokyo 104 t J a p a n .
Discovery of l-nethyl-~-- ~ - l , 2 , 3 , 6 - t e t r a h ~ n ~ I d i n e , a neuz~toxin that elicits p a ~ s m by specific deKeneration of d o p ~ z ~ c neurons in the nlKro-striatal system,
initiated intensive studies to find a naturally-occurrir~ n e u r o t o x i n t h a t stay c a u s e p a r k i n s o ~ m n i n humans. R e c e n t l y we r e p o r t e d i n h i b i t i o n o f dop-m~ne m e t a b o l i s m by c a r c i n o & ~ n i c h e t e r o c y c l i c m t t n e s . Our p r e s e n t s t u d y i s on t h e e f f e c t s o f a s e r i e s o f h e t e r o c y c l i c a m i n e s on a c t i v i t y o f t y p e A and B nonoR=~ne o x i d a s e (HAO-A and HAO-B} i n human b r a i n s y n a p t o a n u e s . A l l t h e h e t e r o c y c l i c m a i n e s i n h i b i t e d NAO-A c o s ~ t i t i v e l y in terms of subst~te. 3-At~no-l-smthyl~-pyrido[4,3-b]indole ( T r p - P - 2 ) was t h e m o s t p o t e n t i n h i b i t o r ; i t s K~ v a l u e toward NAO-A was 0 . 8 4 ~ , w h i c h was a b o u t 1 / 5 0 o f i t s Km v a l u e toward a n maine s u b s t r a t e , k ~ n u ~ m ~ n e , ~ 6 . ~ ~ . 3-Amino-l,4-dlmethyl-SH-pyrldo[~,3-b]indole (Trp-P-I) was also a potent inhibitor of MA0-A. 3or 2-Amino-pyrldo-indoles or -Imldazoles inhibited both NAO-A and NAO-B competitively to substrato, but smines with 2-amino-3-methylimidazo ring and amines structurally-~elated to carboline inh~bited MAO-B mm-cu~petltively. The K i values of hetez~cycllc - m ~ e e to MAO-B were in Eeneral much larger than those to NA0-A. Inb/bltlon of MAO-A by Trp-P-2 p~oved to be reversible, and Trp-P-2 was not metabolized by MA0. Potency and type of inhibition by these amines were discussed in Pelatiml to their chemical structures.