maitotoxin activates cation channels_biochemj_1994
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8/3/2019 Maitotoxin Activates Cation Channels_BiochemJ_1994
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B i o c h e m . J . ( 1 9 9 4 ) 3 0 1 , 4 3 7 - 4 4 1 ( P r i n t e d i n G r e a t B r i t a i n )
M a i t o t o x i n a c t i v a t e s c a t i o n c h a n n e l s d i s t i n c t f r o m t h e r e c e p t o r - a c t i v a t e dn o n - s e l e c t i v e c a t i o n c h a n n e l s o f H L - 6 0 c e l l sI a n F . M U S G R A V E , * R o l a n d S E I F E R T a n d G O n t e r S C H U L T Z
I n s t i t u t f u r P h a r m a k o l o g i e , F r e i e U n i v e r s i t A t B e r l i n , T h i e l a l l e e 6 9 - 7 3 , D - 1 4 1 9 5 B e r l i n , G e r m a n y
We i n v e s t i g a t e d w h e t h e r m a i t o t o x i n a c t i v a t e s n o n - s e l e c t i v ec a t i o n c h a n n e l s , a s w a s r e c e n t l y p r o p o s e d [ S o e r g e l , Y a s u m o t o ,D a l y a n d G u s o v s k y ( 1 9 9 2 ) M o l . P h a r m a c o l . 4 1 , 4 8 7 - 4 9 3 ] .
S t i m u l a t i o n o f d i b u t y r y l c y c l i c A M P - d i f f e r e n t i a t e d H L - 6 0 c e l l sw i t h t h e c h e m o t a c t i c p e p t i d e N - f o r m y l - L - m e t h i o n y l - L - l e u c y l - L -p h e n y l a l a n i n e ( f M L P ; 0 . 1 a M ) , t h e C a 2 + - A T P a s e i n h i b i t o rt h a p s i g a r g i n ( 0 . 1 u t M ) o r m a i t o t o x i n ( 2 5 n g / m l ) r e s u l t e d i n a n
i n c r e a s e i n c y t o p l a s m i c f r e e c a l c i u m c o n c e n t r a t i o n ( [ C a 2 + ] 1 ) .U n l i k e fMLP a n d t h a p s i g a r g i n , m a i t o t o x i n p r o d u c e d n o i n c r e a s ei n [ C a 2 + ] 1 i n t h e a b s e n c e o f e x t r a c e l l u l a r C a 2 + . T h e i n c r e a s e i n
[ C a 2 + ] ii n d u c e d b y fMLP w a s b l o c k e d b y p r e t r e a t m e n t w i t h
p e r t u s s i s t o x i n ( 1 0 0 n g / m l f o r 2 4 h ) b u t n o t t h a t i n d u c e d b ym a i t o t o x i n . S i m i l a r l y , t h e i n c r e a s e i n [ C a 2 + ] 1 p r o d u c e d b y fMLPb u t n o t t h a t p r o d u c e d b y m a i t o t o x i n w a s i n h i b i t e d b y p r e -t r e a t m e n t w i t h p h o r b o l m y r i s t a t e a c e t a t e ( 1 0 0 n g / m l ) . B o t h
I N T R O D U C T I O N
M a i t o t o x i n i s a h i g h - m o l e c u l a r - m a s s p o l y e t h e r f r o m t h e m a r i n e
o r g a n i s m G a m b i e r d i s c u s t o x i c u s . M a i t o t o x i n p r o d u c e s many
responses i n a w i d e v a r i e t y o f m a m m a l i a n c e l l s , i n c l u d i n gs t i m u l a t i o n o f h o r m o n e ( S c h e t t i n i e t a l . , 1 9 8 4 ) a n d neuro-
t r a n s m i t t e r ( T a k a h a s h i e t a l . , 1 9 8 3 ) s e c r e t i o n , c o n t r a c t i o n o f
c a r d i a c ( K o b a y a s h i e t a l . , 1 9 8 5 ) a n d s m o o t h ( O h i z u m i a n dY a s u m o t o , 1 9 8 3 ) m u s c l e , a n d s t i m u l a t i o n o f i n o s i t o l p h o s p h a t e
( B e r t a e t a l . , 1 9 8 6 ) a n d a r a c h i d o n i c a c i d r e l e a s e ( C h o i e t a l . ,
1 9 9 0 ) ( f o r f u r t h e r r e f e r e n c e s a n d r e v i e w s s e e H a m i l t o n a n d P e r e z ,1 9 8 7 ; G u s o v s k y a n d D a l y , 1 9 9 0 ) . A l l t h e s e d i v e r s e a c t i o n s ar e
c r i t i c a l l y d e p e n d e n t on s t i m u l a t i o n o f C a 2 + e n t r y c a u s e d b y
m a i t o t o x i n ( s e e H a m i l t o n a n d P e r e z , 1 9 8 7 ; G u s o v s k y a n d D a l y ,
1 9 9 0 ) . The mechanism b y w h i c h m a i t o t o x i n s t i m u l a t e s C a 2 +
e n t r y i s p o o r l y u n d e r s t o o d . G u s o v s k y a n d D a l y ( 1 9 9 0 ) h a v e
s u g g e s t e d t h a t w h i l e m a i t o t o x i n s t i m u l a t e s v o l t a g e - d e p e n d e n t
C a 2 + c h a n n e l s i n e x c i t a b l e c e l l s , t h i s i s s e c o n d a r y t o a C a 2 + _d e p e n d e n t d e p o l a r i z a t i o n a n d t h a t m a i t o t o x i n f i r s t a c t i v a t e ssome o t h e r C a 2 + e n t r y m e c h a n i s m . F u r t h e r m o r e , m a i t o t o x i n a l s o
i n d u c e s C a 2 + e n t r y i n n o n - e x c i t a b l e c e l l s ( G u s o v s k y e t a l . , 1 9 9 0 ;
Columbo e t a l . , 1 9 9 2 ; Murata e t a l . , 1 9 9 2 ; Wanatabe e t a l . ,1 9 9 3 ) . M a i t o t o x i n i s b y i t s e l f u n l i k e l y t o b e an i o n o p h o r e
( T a k a h a s h i e t a l . , 1 9 8 3 ; S l a d e c z e k e t a l . , 1 9 8 8 ; M u r a t a e t a l . ,
1 9 9 2 ) , so a p o s s i b i l i t y i s t h a t m a i t o t o x i n d i r e c t l y s t i m u l a t e s a
w i d e l y d i s t r i b u t e d C a 2 + permeant c h a n n e l . S o e r g e l e t a l . ( 1 9 9 2 )h a v e r e p o r t e d t h a t m a i t o t o x i n - i n d u c e d C a 2 + e n t r y an d i n o s i t o lp h o s p h a t e t u r n o v e r were b l o c k e d i n r a t i n s u l i n o m a ( R I N ) an d
human i n s u l i n o m a ( H I T ) c e l l s b y SKF 9 6 3 6 5 , a b l o c k e r o f
v o l t a g e - d e p e n d e n t C a 2 + c h a n n e l s a n d n o n - s e le c t i ve c a t i o n ( N S C )
fMLP- a n d m a i t o t o x i n - i n d u c e d i n c r e a s e s i n [ C a 2 l ] , w e r e b l o c k e d
b y 1 - { , - [ 3 - ( 4 - m e t h o x y p h e n y l ) p r o p o x y ] - 4 - m e t h o x y p h e n y l e t h y l } -I H - i m i d a z o l e h y d r o c h l o r i d e (SKF 9 6 3 6 5 ) i n a c o n c e n t r a t i o n -
d e p e n d e n t m a n n e r . H o w e v e r , t h e m a i t o t o x i n - i n d u c e d i n c r e a s e i n[ C a 2 + ] i w a s m o r e s e n s i t i v e t o i n h i b i t i o n b y SKF 9 6 3 6 5 t h a n t h ef M L P - i n d u c e d i n c r e a s e . f M L P - i n d u c e d i n c r e a s e s i n [ C a 2 + ] w e r e
b l o c k e d b y c a t i o n s w i t h G d 3 + b e i n g m o r e e f f e c t i v e t h a n C d 2 ,w h e r e a s f o r m a i t o t o x i n C d 2 + w a s m o r e e f f e c t i v e t h a n G d 3 + . B o t h
fMLP a n d t h a p s i g a r g i n s t i m u l a t e d q u e n c h i n g o f F u r a - 2
f l u o r e s c e n c e i n t h e p r e s e n c e o f e x t r a c e l l u l a r Mn2 , w h e r e a s
m a i t o t o x i n p r o d u c e d n o M n 2 + q u e n c h i n g . T a k e nt o g e t h e r t h e s er e s u l t s s u g g e s t t h a t m a i t o t o x i n d o e s n o t s t i m u l a t e t h e n o n -
s e l e c t i v e c a t i o n c h a n n e l a c t i v a t e d b y f M L P , b u t i n s t e a d a c t i v a t e sC a 2 + i n f l u x b y a d i f f e r e n t m e c h a n i s m .
c h a n n e l s ( M e r r i t t e t a l . , 1 9 9 0 ) , b u t n o t b y n i f e d i p i n e , a b l o c k e r o f
v o l t a g e - d e p e n d e n t C a 2 + c h a n n e l s .I n o r d e r t o d e t e r m i n e i f m a i t o t o x i n i s i n d e e d a c t i v a t i n g NSC
c h a n n e l s , we c o m p a r e d t h e e f f i c a c i e s o f SKF 9 6 3 6 5 , C d 2 + a n d
G d 3 + i n i n h i b i t i n g C a 2 + e n t r y i n d u c e d b y m a i t o t o x i n a n d
t h e c h e m o t a c t i c p e p t i d e N - f o r m y l - L - m e t h i o n y l - L - l e u c y l - L -p h e n y l a l a n i n e ( f M L P ) i n d i f f e r e n t i a t e d H L - 6 0 c e l l s . H L - 6 0 c e l l s
w e r e u s e d a s a m o d e l , a s t h e y a r e d e v o i d o f v o l t a g e - d e p e n d e n tC a 2 + c h a n n e l s , a n d h a v e a w e l l - c h a r a c t e r i z e d NSC c h a n n e l
( K r a u t w u r s t e t a l . , 1 9 9 2 , 1 9 9 3 ) , w h i c h i s a c t i v a t e d b y fMLP a n d
t h a p s i g a r g i n ( D e m a u r e x e t a l . , 1 9 9 2 ) . We r e p o r t h e r e , o n t h eb a s i s o f s e n s i t i v i t y t o SKF 9 6 3 6 5 a n d i n o r g a n i c b l o c k e r s a n d
p e r m e a b i l i t y t o Mn2 , t h a t m a i t o t o x i n d o e s n o t a c t i v a t e t h ef M L P - a c t i v a t e d NSC c h a n n e l o f H L - 6 0 c e l l s , b u t a c t i v a t e s C a 2 +
i n f l u x t h r o u g h a d i f f e r e n t m e c h a n i s m .
M A T E R I A L S A N D M E T H O D S
M a t e r i a l s
f M L P , 4 , / - p h o r b o l 1 2 - m y r i s t a t e 1 3 - a c e t a t e , 4 a - p h o r b o l 1 2 , 1 3 -d i d e c a n o a t e a n d t h a p s i g a r g i n w e r e o b t a i n e d f r o m S i g m a C h e m i e
( D e i s e n h o f e n , G e r m a n y ) . F u r a - 2 a c e t o x y m e t h y l e s t e r ( F u r a -2/AM) w a s p u r c h a s e d f r o m C a l b i o c h e m ( F r a n k f u r t / M a i n ,
G e r m a n y ) . M a i t o t o x i n w a s o b t a i n e d f r o m Wako P u r e C h e m i c a l s
I n d u s t r i e s L t d . ( W a k o , J a p a n ) . SKF 9 6 3 6 5 w a s a g i f t f r o m D r .
J . E . M e r r i t t , S m i t h K l i n e B e e c h a m ( W e l w y n , H e r t s . , U . K . ) . A l lo t h e r r e a g e n t s w e r e o f a n a l y t i c a l g r a d e o r t h e b e s t a v a i l a b l ec o m m e r c i a l g r a d e a n d o b t a i n e d f r o m s t a n d a r d c o m m e r c i a l
s u p p l i e r s . A l l b u f f e r s w e r e a d j u s t e d t o t h e s t a t e d pH w i t h 1 MNaOH. f M L P , SKF 9 6 3 6 5 , 4 , / - p h o r b o l 1 2 - m y r i s t a t e 1 3 - a c e t a t e
A b b r e v i a t i o n s u se d : [ C a 2 + 1 l , c y t o s o l i c f r e e C a 2 + c o n c e n t r a t i o n ; d b - c y c l i c A M P , d i b u t y r y l c y c l i c A M P ; D M S O , d i m e t h y l s u l p h o x i d e ; f M L P , N - f o r m y l - L -m e t h i o n y l - L - l e u c y l - L - p h e n y l a l a n i n e ; F u r a - 2 / A M , F u r a - 2 a c e t o x y m e t h y l e s t e r ; NSC c h a n n e l s , n o n - s e l e c t i v e c a t i o n c h a n n e l s ; SKF 9 6 3 6 5 , 1 - { f - [ 3 - ( 4 -m e t h o x y p h e n y l ) p r o p o x y ] - 4 - m e t h o x y p h e n y l e t h y l } - 1 H - i m i d a z o l e h y d r o c h l o r i d e .
* T o whom c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d .
4 3 7
8/3/2019 Maitotoxin Activates Cation Channels_BiochemJ_1994
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4 3 8 1 . F . M u s g r a v e , R . S e i f e r t a n d G . S c h u l t z
( b )a )
0 C a 2 +
t TG ( 0 . 1 uM)
1 0 0 _
3 0
I fMLP ( 0 . 1 u M )
( c )
_ 0 C a 2 +
I MTX ( 2 5 n g / m I )
1 0 Ll-l1 m i n
F i g u r e 1 E f f e c t s o f f M L P , t h a p s i g a r g i n a n d m a i t o t o x i n o n [ C a 2 + ] 1 i n d b - c y c l i c A M P - d i f f e r e n t i a t e d H L - 6 0 c e l l s
T h e f i g u r e s h o w s o r i g i n a l t r a c i n g s . C e l l s w e r e l o a d e d w i t h F u r a - 2 a s d e s c r i b e d i n t h e M a t e r i a l s a n d m e t h o d s s e c t i o n a n d i n c u b a t e d i n m e a s u r e m e n t b u f f e r c o n t a i n i n g e i t h e r 1 m M C a 2 + ( + C a 2 + )o r 1 mM E G T A ( 0 C a 2 + ) a t 3 7 O C f o r 3 m i n b e f o r e t h e a d d i t i o n o f t h e s t i m u l u s . T h e e f f e c t s o f f M L P ( a ) , t h a p s i g a r g i n ( T G ) ( b ) a n d m a i t o t o x i n ( M T X ) ( c ) o n [ C a 2 + ] i a r e s h o w n . F l u o r e s c e n c e w a s
m o n i t o r e d o n t h e R a t i o I I f l u o r i m e t e r w i t h a n e x c i t a t i o n w a v e l e n g t h o f 3 4 0 nm a n d a n e m i s s i o n w a v e l e n g t h o f 5 1 0 n m . S i m i l a r r e s u l t s w e r e s ee n i n a t l e a s t t h r e e f u r t h e r i n d e p e n d e n t e x p e r i m e n t sp e r f o r m e d i n d u p l i c a t e o r t r i p l i c a t e .
a n d t h a p s i g a r g i n were d i s s o l v e d i n d i m e t h y l s u l p h o x i d e (DMSO)t o g i v e s t o c k s o l u t i o n s o f 1 0 m M . Fura-2/AM was d i s s o l v e d i nDMSO t o g i v e a 2 mM s t o c k . H e a v y m e t a l s were d i s s o l v e d i nd e i o n i z e d w a t e r t o g i v e 1 0 mM s t o c k s o l u t i o n s . M a i t o t o x i n was
d i s s o l v e d i n d e i o n i z e d w a t e r t o g i v e a s t o c k s o l u t i o n o f 5 0 , u g / m l .S t o c k s o l u t i o n s were s t o r e d as a l i q u o t s a t -2 0 ' C . F u r t h e r
d i l u t i o n s were made i n d e i o n i z e d w a t e r .
C e l l c u l t u r e
HL-60 c e l l s were grown i n s u s p e n s i o n c u l t u r e i n RPMI-1640
medium s u p p l e m e n t e d w i t h 10% ( v / v ) h o r s e serum , 1 % ( v / v )n o n - e s s e n t i a l a m i n o a c i d s , 2 mM L - g l u t a m i n e , 5 0 u n i t s o f
p e n i c i l l i n / m l a n d 5 0 , u g o f s t r e p t o m y c i n / m l i n a h u m i d i f i e d
a t m o s p h e r e w i t h 7 % CO2 a t 3 7 ' C . To i n d u c e d i f f e r e n t i a t i o n ,H L - 6 0 c e l l s were s e e d e d a t a d e n s i t y o f 1 x 1 0 6 l / m l a n d were
c u l t u r e d f o r 4 8 h i n t h e presence o f 0 . 2 mM d i b u t y r y l c y c l i c AMP( d b - c y c l i c A M P ) ( C h a p l i n s k i a n d N i e d e l , 1 9 8 2 ; S e i f e r t a n d
S c h a c h t e l e , 1 9 8 8 ) . C e l l s were h a r v e s t e d b y c e n t r i f u g a t i o n f o rO m i n a t 2 5 0 g . C e l l p r e p a r a t i o n s c o n t a i n e d more t h a n 9 0 0 %
v i a b l e c e l l s as j u d g e d b y T r y p a n B l u e e x c l u s i o n .
M e a s u r e m e n t o f c y t o s o l i c f r e e C a 2 + c o n c e n t r a t i o n ( [ C a 2 + ] , )
[ C a 2 + ] 1 was d e t e r m i n e d w i t h t h e d y e Fura-2/AM as p r e v i o u s l y
d e s c r i b e d ( S c h w a n e r e t a l . , 1 9 9 2 ; S e i f e r t e t a l . , 1 9 9 2 ) w i t h m i n o r
m o d i f i c a t i o n s . B r i e f l y , HL-60 c e l l s were s u s p e n d e d a t 1 x 1 0 7c e l l s / m l i n l o a d i n g b u f f e r c o n s i s t i n g o f 1 3 8 mM N a C l , 6 mM
K C l , 1 mM C a C l 2 , 1 mM MgSO4, 1 mM Na2HPO4, 5 mM
NaHCO3, 5 . 5 mM g l u c o s e a n d 2 0 mM H e p e s , pH 7 . 4 ,s u p p l e m e n t e d w i t h 1 % ( w / v ) BSA. Fura-2/AM was a d d e d t o
g i v e a f i n a l c o n c e n t r a t i o n o f 4 ,M, a n d c e l l s were i n c u b a t e d f o r
3 0 m i n a t 3 7 ' C . A f t e r t h i s i n c u b a t i o n p e r i o d , t h e c e l l s were
d i l u t e d t o 0 . 5 x 1 0 6 c e l l s / m l a n d were c e n t r i f u g e d f o r 1 0 m i n a t
2 5 0 g t o remove e x t r a c e l l u l a r d y e . The c e l l s were t h e n r e s u s p e n d e d
a t 2 x 1 0 6 c e l l s / m l i n measurement b u f f e r c o n s i s t i n g o f 1 3 8 mM
N a C l , 6 mM K C l , 1 mM M g C l 2 , 5 . 5 mM g l u c o s e a n d 2 0 mM
H e p e s , pH 7 . 4 , s u p p l e m e n t e d w i t h 1 0 0 ,M C a C l 2 , a n d k e p t a t
room t e m p e r a t u r e u n t i l measurement o f [ C a 2 + 1 ] . T h i s p h o s p h a t e -
a n d b i c a r b o n a t e - f r e e b u f f e r wa s r e q u i r e d f o r e x p e r i m e n t s w i t h
h e a v y m e t a l s , a n d was u s e d t h r o u g h o u t f o r purposes o f c on-
s i s t e n c y ( M e r r i t t e t a l . , 1 9 8 9 ) . A s a m p l e ( 0 . 5 m l ) o f c e l l s u s p e n s i o n
was a d d e d t o a c r y l f l u o r e s c e n c e c u v e t t e s ( S a r s t e d t , N i i m b r e c h t ,G e r m a n y ) , f u rt h e r d i l ut e d w i t h 1 . 5 m l o f t h e measurement b u f f e ra n d C a C l 2 a d d e d t o g i v e a f i n a l c o n c e n t r a t i o n o f 1 m M .F l u o r e s c e n c e was d e t e r m i n e d a t 3 7 ° C u n d e r c o n s t a n t s t i r r i n g a t
1 0 0 0 r e v . / m i n , u s i n g a LS 5 0 B d u a l - w a v e l e n g t h s p e c t r o -
f l u o r i m e t e r ( P e r k i n - E l m e r , B e a c o n s f i e l d , B u c k s . , U . K . ) . T h e
e x c i t a t i o n w a v e l e n g t h s were 3 4 0 nm a n d 3 8 0 nm a n d t h e e m i s s i o n
w a v e l e n g t h was 5 1 0 nm. I n some e x p e r i m e n t s , a R a t i o I I
s p e c t r o f l u o r i m e t e r ( A m i n c o , S i l v e r S p r i n g , MD, U . S . A . ) was
u s e d . The e x c i t a t i o n w a v e l e n g t h was 3 4 0 n m , a n d t h e e m i s s i o n
w a v e l e n g t h was 5 1 0 nm. C e l l s were i n c u b a t e d f o r 3 m i n a t 3 7 0 C
b e f o r e a d d i t i o n o f s t i m u l i . F l u o r e s c e n c e s i g n a l s were c a l i b r a t e da f t e r l y s i s o f t h e c e l l s w i t h 0 . 1 % ( w / v ) T r i t o n X - 1 0 0 ( m a x i m a l
f l u o r e s c e n c e ) a n d s u b s e q u e n t a d d i t i o n o f 2 0 mMEGTA ( m i n i m a l
f l u o r e s c e n c e ) . [ C a 2 + ] 1 was c a l c u l a t e d a c c o r d i n g t o eqn. 5 ( d u a lw a v e l e n g t h ) or eqn. 6 ( s i n g l e w a v e l e n g t h ) i n G r y n k i e w i c z e t a l .
( 1 9 8 5 ) . C a 2 " e n t r y was e s t i m a t e d b y s u b t r a c t i n g i n c r e a s e s i n
[ C a 2 + ] i o b s e r v e d i n t h e a b s e n c e o f e x t r a c e l l u l a r C a 2 + f r o m
responses i n t h e presence o f e x t r a c e l l u l a r C a 2 + . The v a l u e so
o b t a i n e d appears t o b e m a i n l y d u e t o e n t r y o f e x t r a c e l l u l a r C a 2 +
w i t h n e g l i g i b l e c o n t r i b u t i o n s f r o m o t h e r sources ( s e e K r a u t w u r s t
e t a l . , 1 9 9 2 a n d t h e D i s c u s s i o n ) .
M e a s u r e m e n t o f M n 2 + q u e n c h i n g
Mn2+ q u e n c h i n g e x p e r i m e n t s were p e r f o r m e d i n n o m i n a l l y C a 2 + -
f r e e measurement b u f f e r on t h e R a t i o I I s p e c t r o f l u o r i m e t e r w i t h
an e x c i t a t i o n w a v e l e n g t h o f 3 6 0 nm and an e m i s s i o n w a v e l e n g t h
o f 5 1 0 nm ( M e r r i t t e t a l . , 1 9 8 9 ; K r a u t w u r s t e t a l . , 1 9 9 2 ) . I n some
1 0 0 0 0
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4 4 0 1 . F . M u s g r a v e , R . S e i f e r t a n d G . S c h u l t z
1 0 0
( a ) O MT X* f M L P S h o u l d e r
O f M L P P e a k0 F
6 0 _
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1 0 0 F
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fMLP
F i g u r e 4 E f f e c t s o f S K F 9 6 3 6 5 o r h e a v y m e t a l s o n t h e f M L P - a n dm a l t o t o x i n - i n d u c e d I n c r e a s e s I n C a 2 + e n t r y i n d b - c y c l l c A M P - d i f f e r e n f t a t e dH L - 6 0 c e l l s
T h e f i g u r e s h o w s t h e m e a n + S . D . f r o m t h r e e s e p a r a t e e x p e r i m e n t s p e r f o r m e d i n d u p l i c a t e .
C e l l s w e r e l o a d e d w i t h F u r a - 2 a s d e s c r i b e d i n t h e M a t e r i a l s a n d m e t h o d s s e c t i o n a n d i n c u b a t e di n m e a s u r e m e n t b u f f e r c o n t a i n i n g 1 m M C a 2 + w i t h e i t h e r s o l v e n t ( c o n t r o l ) o r b l o c k e r a t 3 7 O Cf o r 3 m i n b e f o r e t h e a d d i t i o n o f t h e s t i m u l u s [ f M L P , 0 . 1 , u M ( U , O ) ; m a i t o t o x i n ( M T X ) ,2 5 n g / m l ( 0 ) ] . F l u o r e s c e n c e w a s m o n i t o r e d o n t h e L S 5 0 B s p e c t r o f l u o r i m e t e r . ( a ) f M L P -i n d u c e d C a 2 + e n t r y v a l u e s w e r e c a l c u l a t e d e i t h e r 2 0 s ( p e a k ) o r 8 0 s ( s h o u l d e r ) a f t e r a d d i t i o n
o f s t i m u l u s . B y 8 0 s a f t e r t h e a d d i t i o n o f f M L P , t h e r e l e a s e o f i n t r a c e l l u l a r C a 2 + h a d r e t u r n e dt o b a s a l . M a i t o t o x i n - i n d u c e d C a 2 + e n t r y v a l u e s w e r e c a l c u l a t e d 8 0 s a f t e r a d d i t i o n o f m a i t o t o x i n ,
b y w h i c h t i m e p l a t e a u v a l u e s h a d b e e n r e a c h e d . ( b ) D i f f e r e n c e s b e t w e e n C a 2 + e n t r y i n t h e
p r e s e n c e o f G d 3 + o r C d 2 + ( 1 0 , u M ) . M e a n C a 2 + e n t r y m e a s u r e d a t 8 0 s w a s 2 1 4 + 1 1 2 n M
( n = ) f o r f M L P ( 0 . 1
# M )a n d 9 9 8 + 4 9 9 n M ( n =) f o r m a i t o t o x i n ( 2 5 n g / m l ) .
7 2 0 + 2 7 5 nM ( n = ) , m e a s u r e d 8 0 s a f t e r s t i m u l u s a d d i t i o n ] .4 a - P h o r b o l 1 2 , 1 3 - d i d e c a n o a t e was w i t h o u t e f f e c t on t h e fMLP-
i n d u c e d i n c r e a s e i n [ C a 2 + ] 1 ( d a t a n o t s h o w n ) . As p h o r b o l e s t e r
p r e t r e a t m e n t c an i n t e r f e r e w i t h r e c e p t o r c o u p l i n g t o
p h o s p h o l i p a s e C ( S m i t h e t a l . , 1 9 8 7 ) , t h i s r e s u l t s u g g e s t s t h a t
m a i t o t o x i n a c t s a t a s i t e b e y o n d p h o s p h o l i p a s e C .
B o t h fMLP- ( 0 . 1 , u M ) a n d m a i t o t o x i n - ( 2 5 n g / m l ) i n d u c e d
i n c r e a s e s i n [ C a 2 + ] , were b l o c k e d b y SKF 9 6 3 6 5 ( F i g u r e 2 ) . SKF9 6 3 6 5 was l e s s e f f e c t i v e a t b l o c k i n g t h e p e a k c o m p o n e n t o f t h e
f M L P - i n d u c e d i n c r e a s e i n [ C a 2 + ] 1 t h a n t h e ' s h o u l d e r ' , i . e . t h e
p e r s i s t e n t response a t t h e t i m e when i n t r a c e ll ul a r r e l e a s e had
- a 3 0 0 0 0. _
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MTX
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( 2 5 n g / m l )B a s a l B a s a l
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1 m i n
F i g u r e 5 E f f e c t s o f f M L P , t h a p s i g a r g i n a n d m a l t o t o x i n o n M n 2 + q u e n c h i n go f F u r a - 2 f l u o r e s c e n c e i n d b - c y c l i c A M P - d i f f e r e n t i a t e d H L - 6 0 c e l l s
F l u o r e s c e n c e w a s m e a s u r e d w i t h t h e R a t i o 1 1 s p e c t r o f l u o r i m e t e r a t a n e x c i t a t i o n w a v e l e n g t h o f
3 6 0 nm a n d a n e m i s s i o n w a v e l e n g t h o f 5 1 0 n m . T h e f i g u r e s h o w s o r i g i n a l t r a c i n g s . C e l l s w e r e
l o a d e d w i t h F u r a - 2 a s d e s c r i b e d i n t h e M a t e r i a l s a n d m e t h o d s s e c t i o n a n d i n c u b a t e d i n
n o m i n a l l y C a 2 + - f r e e m e a s u r e m e n t b u f f e r a t 3 7 ° C f o r 3 m i n b e f o r e a d d i t i o n o f t h e s t i m u l u s[ f M L P ( a ) ; t h a p s i g a r g i n ( T G ) ( b ) ; m a i t o t o x i n ( M T X ) ( c ) ] o r s o l v e n t ( B a s a l ) . A f t e r t h e a d d i t i o n
o f s t i m u l u s ( 3 m i n ) , M n 2 + ( 5 0 , u M ) w a s a d d e d a n d t h e f a l l i n f l u o r e s c e n c e w a s f o l l o w e d f o r
a f u r t h e r 2 - 3 m i n . S i m i l a r r e s u l t s w e r e s e e n i n a t l e a s t t h r e e f u r t h e r i n d e p e n d e n t e x p e r i m e n t s .
r e t u r n e d t o b a s a l v a l u e s ( F i g u r e 3 ) . SKF 9 6 3 6 5 was more
e f f e c t i v e a t b l o c k i n g m a i t o t o x i n - i n d u c e d [ C a 2 + ] 1 i n c r e a s e s t h a n
e i t h e r component o f t h e f M L P - i n d u c e d i n c r e a s e i n [ C a 2 + ] i( F i g u r e 4 ) .
G d 3 + ( 1 0 M) s t r o n g l y i n h i b i t e d f M L P - i n d u c e d i n c r e a s e s i n[ C a 2 + ] 1 , w h i l e C d 2 + ( 1 0 ,M) was v i r t u a l l y i n e f f e c t i v e ( s e e F i g u r e s
3 a n d 4 ) . As w i t h t h e e f f e c t s o f SKF 9 6 3 6 5 , G d 3 + was more
e f f e c t i v e on t h e s h o u l d e r t h a n on t h e p e a k response. I n c o n t r a s t ,
G d 3 + ( 1 0 # M ) h a d no e f f e c t on t h e m a i t o t o x i n - i n d u c e d i n c r e a s e s
i n [ C a 2 + ] 1 , w h i l e C d 2 + ( 1 0 M) p r o d u c e d a m o d e r a t e i n h i b i t i o n .B o t h fMLP ( 0 . 1 , u M ) a n d t h a p s i g a r g i n ( 0 . 1 , u M ) p r o d u c e d
s u b s t a n t i a l q u e n c h i n g o f F u r a - 2 f l u o r e s c e n c e a t 3 6 0 nm i n t h e
presence o f e x t r a c e l l u l a r Mn2+ ( F i g u r e 5 ) , p r e s u m a b l y d u e t o
e n t r y o f Mn2+ t h r o u g h NSC c h a n n e l s ( M e r r i t t e t a l . , 1 9 8 9 ) . I n
c o n t r a s t , m a i t o t o x i n p r o d u c e d no q u e n c h i n g o f f l u o r e s c e n c e ,u s i n g e i t h e r t h e reverse ( s e e F i g u r e 5 ) or t h e f o r w a r d m e t h o d ( d a t a
n o t s h o w n ) . U s i n g t h e reverse m e t h o d o l o g y w i t h C a 2 + ( i . e .
a d d i n g t h e s t i m u l u s t o c e l l s s u s p e n d e d i n n o m i n a l l y C a 2 + - f r e eb u f f e r , t h e n a d d i n g C a 2 + 2 m i n l a t e r , e x c i t a t i o n w a v e l e n g t h
3 4 0 n m ) [ C a 2 + ] 1 i n c r e a s e d r a p i d l y a f t e r m a i t o t o x i n a p p l i c a t i o n t o
t h e c o n c e n t r a t i o n s a c h i e v e d u s i n g t h e f o r w a r d m e t h o d ( d a t a n o t
s h o w n ) . The c o n c e n t r a t i o n - r e s p o n s e curves t o f M L P f o r i n -c r e a s i n g [ C a 2 + ] , a n d Mn2+ q u e n c h i n g were a l m o s t super-
i m p o s a b l e , an d t h e s e n s i t i v i t i e s o f s t i m u l u s - i n d u c e d C a 2 + e n t r y
a n d Mn2+ q u e n c h i n g t o SKF 9 6 3 6 5 were s i m i l a r ( d a t a n o t
s h o w n ) , s u g g e s t i n g t h a t i n t h e s e c e l l s s t i m u l u s - i n d u c e d
f l u o r e s c e n c e q u e n c h i n g b y Mn2+ was an i n d e x o f s t i m u l u s -
i n d u c e d C a 2 + e n t r y .
D I S C U S S I O N
M a i t o t o x i n h a s b e e n r e p o r t e d t o s t i m u l a t e C a 2 + e n t r y i n a l l c e l l s
r e p o r t e d so f a r e x c e p t 3 T 3 f i b r o b l a s t s ( s e e r e v i e w b y G u s o v s k ya n d D a l y , 1 9 9 0 ) , i n c l u d i n g b o t h e x c i t a b l e a n d n o n - e x c i t a b l e c e l l s .
I n a g r e e m e n t w i t h a p r e v i o u s r e p o r t ( G u s o v s k y e t a l . , 1 9 9 0 ) , we
r e p o r t h e r e t h a t m a i t o t o x i n i n c r e a s e s [ C a 2 + ] 1 i n d b - c y c l i c AMP-d i f f e r e n t i a t e d HL-60 c e l l s . The q u e s t i o n i s , h o w i s m a i t o t o x i n
p r o d u c i n g t h i s i n c r e a s e i n [ C a 2 ' ] i ? A s m a i t o t o x i n n e i t h e r a c t s t o
p e r m e a b i l i z e l i p o s o m e s ( T a k a h a s h i e t a l . , 1 9 8 3 ; Murata e t a l . ,
1 9 9 2 ) or m i t o c h o n d r i a ( T a k a h a s h i e t a l . , 1 9 8 3 ) n o r f o r m s p o r e s
i n b l a c k - l i p i d membranes ( S l a d e c z e k e t a l . , 1 9 8 8 ) , m a i t o t o x i n i s
u n l i k e l y t o b e a c t i n g as an i o n o p h o r e . F u r t h e r m o r e , i n t h e p r e s e n t
-0
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M a i t o t o x i n a n d n o n - s e l e c t i v e c a t i o n c h a n n e l s 4 4 1
s t u d y m a i t o t o x i n - i n d u c e d [ C a 2 + ] i i n c r e a s e s c a n n o t b e s e c o n d a r y
t o i n o s i t o l t r i s p h o s p h a t e - i n d u c e d e m p t y i n g o f i n t r a c e l l u l a r C a 2 +
s t o r e s , a s i n t h e a b s e n c e o f e x t r a c e l l u l a r C a 2 + n o i n c r e a s e s i n
[ C a 2 + ] , a r e s e e n , e v e n t h o u g h i n o s i t o l t r i s p h o s p h a t e - r e l e a s a b l es t o r e s a r e p r e s e n t , a s s h o w n b y t h e r e s p o n s e s t o fMLP a n d
t h a p s i g a r g i n ( s e e F i g u r e 1 ) . T h i s i s i n a g r e e m e n t w i t h p r e v i o u sr e p o r t s ( G u s o v s k y e t a l . , 1 9 9 0 ; s e e a l s o G u s o v s k y a n d D a l y ,
1 9 9 0 ) .S o e r g e l e t a l . ( 1 9 9 2 ) h a v e s u g g e s t e d t h a t m a i t o t o x i n d i r e c t l y
a c t i v a t e s a n NSC c h a n n e l ( s ) . I n t h e p r e s e n t s t u d y t h e m a i t o t o x i n -
i n d u c e d C a 2 + e n t r y w a s i n h i b i t e d b y SKF 9 6 3 6 5 ( s e e F i g u r e s 2a n d 4 ) , w h i c h b l o c k s b o t h v o l t a g e - d e p e n d e n t C a 2 + c h a n n e l s a n d
NSC c h a n n e l s ( M e r r i t t e t a l . , 1 9 9 0 ) . A s H L - 6 0 c e l l s h a v e a n SKF9 6 3 6 5 - i n h i b i t a b l e NSC c h a n n e l a n d a r e d e v o i d o f v o l t a g e -d e p e n d e n t C a 2 + c h a n n e l s ( K r a u t w u r s t e t a l . , 1 9 9 2 ) , m a i t o t o x i n
c o u l d p l a u s i b l y b e a c t i v a t a t i n g NSC c h a n n e l s i n t h e s e c e l l s . T h i s
f i n d i n g i s i n a g r e e m e n t w i t h t h e r e p o r t o f S o e r g e l e t a l . ( 1 9 9 2 )t h a t SKF 9 6 3 6 5 , b u t n o t n i f e d i p i n e , i n h i b i t s C a 2 + e n t r y i n HI T
a n d R IN c e l l s .
H o w e v e r , t h e m a i t o t o x i n - i n d u c e d i n c r e a s e i n [ C a 2 1 i ] w a s m o r e
s e n s i t i v e t o SKF 9 6 3 6 5 t h a n t h e f M L P - i n d u c e d i n c r e a s e i n
[ C a 2 + ] , , r a i s i n g t h e p o s s i b i l i t y t h a t t h e m e c h a n i s m s a c t i v a t e d b ym a i t o t o x i n a n d fMLP a r e n o t i d e n t i c a l . F u r t h e r e v i d e n c e t h a tm a i t o t o x i n a n d fMLP a c t i v a t e d i f f e r e n t m e c h a n i s m s f o r i n -c r e a s i n g [ C a 2 + ] 1 c o m e s f r o m i n h i b i t i o n b y h e a v y - m e t a l i o n s . I n
e l e c t r o p h y s i o l o g i c a l s t u d i e s t h e f M L P - a c t i v a t e d NSC c h a n n e l i s
d i f f e r e n t i a l l y i n h i b i t e d b y h e a v y - m e t a l i o n s , w i t h G d 3 + b e i n gm o r e p o t e n t t h a n C d 2 + ( K r a u t w u r s t e t a l . , 1 9 9 3 ) . T h i s w a s
c o n f i r m e d i n t h e p r e s e n t s t u d y , w h e r e t h e f M L P - i n d u c e d i n c r e a s e si n C a 2 + e n t r y a r e b l o c ke d b y 1 0 ,M G d 3 + b u t n o t b y 1 0 , u M C d 2 +
( s e e F i g u r e s 3 a n d 4 ) . I n c o n t r a s t , t h e m a i t o t o x i n - i n d u c e d
i n c r e a s e s i n C a 2 + e n t r y w e r e u n a f f e c t e d b y 1 0 #M G d 3 + , w h e r e a s
1 0 ,M C d 2 + p r o d u c e d a m o d e s t b u t s i g n i f i c a n t b l o c k .T h a p s i g a r g i n - i n d u c e d i n c r e a s e s i n C a 2 + e n t r y h a v e a s e n s i t i v i t yt o h e a v y - m e t a l i o n s s i m i l a r t o t h a t o f fMLP ( D e m a u r e x e t a l . ,1 9 9 2 ) .
I n t h e p r e s e n t s t u d y C a 2 + e n t r y w a s e s t i m a t e d b y s u b t r a c t i n gi n c r e a s e s i n [ C a 2 + ] , m e a s u r e d i n t h e a b se n c e o f e x t r a c e l l u l a r C a 2 +
f r o m t h a t m e a s u r e d i n i t s p r e s e n c e , a n d i t may b e a r g u e d t h a t t h ed i f f e r e n c e s i n s e n s i t i v i t y t o b l o c k e r s a r e a n a r t e f a c t o f t h i sp r o c e d u r e . H o w e v e r , t h e s e n s i t i v i t y o f f M L P - i n d u c e d C a 2 + e n t r yt o SKF 9 6 3 6 5 , G d 3 + a n d C d 2 + a t 8 0 s a f t e r a d d i t i o n o f s t i m u l u s ,b y w h i c h t i m e r e l e a s e o f C a 2 + f r o m t h e i n t r a c e l l u l a r s t o r e h a d
r e t u r n e d t o b a s e l i n e , a g r e e s r e a s o n a b l y w e l l w i t h e l e c t r o -p h y s i o l o g i c a l s t u d i e s ( K r a u t w u r s t e t a l . , 1 9 9 3 ) .We s t u d i e d a n i n d e x o f C a 2 + e n t r y u n c o m p l i c a t e d b y C a 2 +
r e l e a s e f r o m i n t r a c e l l u l a r s t o r e s , i . e . s t i m u l u s - i n d u c e d q u e n c h i n go f F u r a - 2 f l u o r e s c e n c e ( M e r r i t t e t a l . , 1 9 8 9 ; D e m a u r e x e t a l . ,1 9 9 2 ; K r a u t w u r s t e t a l . , 1 9 9 2 ) . H o w e v e r , a l t h o u g h fMLP a n d
t h a p s i g a r g i n s t i m u l a t e d s u b s t a n t i a l q u e n c h i n g o f F u r a - 2
f l u o r e s c e n c e i n t h e p r e s e n c e o f e x t r a c e l l u l a r Mn2 , m a i t o t o x i n
p r o d u c e d n o q u e n c h i n g a t a l l u n d e r a v a r i e t y o f p r o t o c o l s ( s e eF i g u r e 5 ) . T h i s s u g g e s t s t h a t t h e m e c h a n i s m b y w h i c h m a i t o t o x i n
i n c r e a s e s [ C a 2 + ] 1 i s p o o r l y p e r m e a b l e t o M n 2 + , e s p e c i a l l y s i n c e t h et h a p s i g a r g i n - i n d u c e d M n 2 ' q u e n c h i n g c o u l d b e c l e a r l y s e e n ,a l t h o u g h t h e l a t t e r C a 2 + e n t r y d e v e l o p s e v e n more s l o w l y t h a n
t h a t o f m a i t o t o x i n a n d i s a l s o s m a l l e r i n e x t e n t ( s e e F i g u r e s 1 a n d
5 ) . M u r a t a e t a l . ( 1 9 9 2 ) r e p o r t e d t h a t m a i t o t o x i n i n d u c e s an
i n f l u x o f 5 4 M n 2 + i n t o HI T c e l l s . H o w e v e r , t h e i n f l u x o f 5 4 M n 2 +
w a s m o r e t h a n 2 0 t i m e s s m a l l e r t h a n 4 5 C a 2 + - i n f l u x a n d t h i s r e p o r ti s t h u s c o n s i s t e n t w i t h o u r r e s u l t s .
D o e s m a i t o t o x i n p r o d u c e i n c r e a s e s i n [ C a 2 + ] 1 i n o u r H L - 6 0
c e l l s b y b l o c k i n g C a 2 l e f f l u x , r a t h e r t h a n i n c r e a s i n g C a 2 + e n t r y ?T h i s i s u n l i k e l y s i n c e , a s d e s c r i b e d a b o v e , t h e i n c r e a s e i n [ C a 2 + 1 ]i s b l o c k e d b y SKF 9 6 3 6 5 , w h i c h d o e s n o t a f f e c t C a 2 + e f f l u x i n t h ec o n c e n t r a t i o n s we h a v e u se d ( M e r r i t t e t a l . , 1 9 9 0 ) . S i m i l a r l y , t h ei n h i b i t i o n o f t h e e f f e c t o f m a i t o t o x i n b y l o w c o n c e n t r a t i o n s o f
C d 2 + w o u l d a l s o a r g u e a g a i n s t a n e f f e c t on t h e e f f l u x m e c h a n i s m .
F i n a l l y , m a i t o t o x i n a p p e a r s t o o p e n v o l t a g e - i n d e p e n d e n tc h a n n e l s i n H L - 6 0 c e l l s ( D . K r a u t w u r s t , u n p u b l i s h e d w o r k ) .T a k e n t o g e t h e r t h e s e r e s u l t s s u g g e s t t h a t m a i t o t o x i n i s i n d e e d
i n c r e a s i n g C a 2 + e n t r y i n d b - c y c l i c A M P - d i f f e r e n t i a t e d H L - 6 0
c e l l s .
To s u m m a r i z e , m a i t o t o x i n a n d fMLP a c t i v a t e two s e p a r a t eC a 2 + e n t r y p a t h w a y s , b o t h o f w h i c h a r e s e n s i t i v e t o SKF 9 6 3 6 5
b u t h a v e d i f f e r e n t s e n s i t i v i t i e s t o h e a v y - m e t a l i o n s a n d d i f f e r e n tp e r m e a b i l i t i e s t o M n 2 + . T h i s s u g g e s t s t h a t t h e m a i t o t o x i n -a c t i v a t e d C a 2 + e n t r y p a t h w a y i n H L - 6 0 c e l l s i s n o t t h e r e c e p t o r -d e p e n d e n t NSC c h a n n e l t h a t i s s t i m u l a t e d b y fMLP a n d
t h a p s i g a r g i n . W h e t h e r m a i t o t o x i n s t i m u l a t e s C a 2 + e n t r y v i aa c t i v a t i o n o f a n o v e l v o l t a g e - i n d e p e n d e n t C a 2 + - p e r m e a n t c h a n n e l
o r some o t h e r m e c h a n i s m , r e m a i n s t o b e d e t e r m i n e d .
W e w i s h t o t h a n k E v e l y n G l a B f o r e x c e l l e n t t e c h n i c a la s s i s t a n c e . T h i s
w o r kw a s
s u p p o r t e d b y f u n d s f r o m t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t a n d F o n d s d e rC h e m i s c h e n I n d u s t r i e . I . F . M . i s a n A u s t r a l i a n N H & M R C R e s e a r c h F e l l o w .
R E F E R E N C E S
B e r t a , P . , S l a d e c z e k , F . , D e r a n c o u r t , J . , D u r a n d , M . , T r a v o , P . a n d H a i e c h , J . ( 1 9 8 6 ) F E B SL e t t . 1 9 7 , 3 4 9 - 3 5 2
C h a p l i n s k i , T . J . a n d N i e d e l , J . E . ( 1 9 8 2 ) J . C l i n . I n v e s t . 7 0 , 9 5 3 - 9 6 4C h o i , 0 . H . , P a d g e t t , W . L . , N i s h i z a w a , Y . , G u s o v s k y , F . , Y a s u m o t o , T . a n d D a l y , J . W .
( 1 9 9 0 ) M o l . P h a r m a c o l . 3 7 , 2 2 2 - 2 3 0C o l u m b o , M . , T a g l i a l a t e l a , M . , W a r n e r , J . A . , M a c G l a s h a n , D . W . J . , Y a s u m o t o , T . ,
A n n u n z i a t o , L . a n d M a r o n e , G . ( 1 9 9 2 ) J . P h a r m a c o l . E x p . T h e r . 2 6 3 , 9 7 9 - 9 8 6D e m a u r e x , N . , L e w , D . P . a n d K r a u s e , K . H . ( 1 9 9 2 ) J . B i o l . C h e m . 2 6 7 , 2 3 1 8 - 2 3 2 4G r y n k i e w i c z , G . , P o e n i e , M . a n d T s i e n , R . Y . ( 1 9 8 5 ) J . B i o l . C h e m . 2 6 0 , 3 4 4 0 - 3 4 5 0G u s o v s k y , F . a n d D a l y , J . W . ( 1 9 9 0 ) B i o c h e m . P h a r m a c o l . 3 9 , 1 6 3 3 - 1 6 3 9
G u s o v s k y , F . , B i t r a n , J . A . , Y a s u m o t o , T . a n d D a l y , J . W . ( 1 9 9 0 ) J . P h a r m a c o l . E x p . T h e r .
2 5 2 , 4 6 6 - 4 7 3H a m i l t o n , S . L . a n d P e r e z , M . ( 1 9 8 7 ) B i o c h e m . P h a r m a c o l . 3 6 , 3 3 2 5 - 3 3 2 9K o b a y a s h i , M . , M i y a k o d a , G . , N a k a m u r a , T . a n d O h i z u m i , Y . ( 1 9 8 5 ) E u r . J . P h a r m a c o l .
1 1 1 , 1 2 1 - 1 2 3K r a u t w u r s t , D . , S e i f e r t , R . , H e s c h e l e r , J . a n d S c h u l t z , G . ( 1 9 9 2 ) B i o c h e m . J . 2 8 8 ,
1 0 2 5 - 1 0 3 5K r a u t w u r s t , D . , H e s c h e l e r , J . , A r n d t s , D . , L o s e l , W . , H a m m e r , R . a n d S c h u l t z , G . ( 1 9 9 3 )
M o l . P h a r m a c o l . 4 3 , 6 5 5 - 6 5 9M e r r i t t , J . E . , J a c o b , R . a n d H a l l a m , T . J . ( 1 9 8 9 ) J . B i o l . C h e m . 2 6 4 , 1 5 2 2 - 1 5 2 7M e r r i t t , J . E . , A r m s t r o n g , W . P . , B e n h a m , C . D . , H a l l a m , T . J . , J a c o b , R . , J a x a C h a m i e c , A . ,
L e i g h , B . K . , M c C a r t h y , S . A . , M o o r e s , K . E . a n d R i n k , T . J . ( 1 9 9 0 ) B i o c h e m . J . 2 7 1 ,5 1 5 - 5 2 2
M u r a t a , M . , G u s o v s k y , F . , Y a s u m o t o , T . a n d D a l y , J . W . ( 1 9 9 2 ) E u r . J . P h a r m a c o l . 2 2 7 ,4 3 - 4 9
O h i z u m i , Y . a n d Y a s u m o t o , T . ( 1 9 8 3 ) J . P h y s i o l . ( L o n d o n ) 3 3 7 , 7 1 1 - 7 2 1
S c h e t t i n i , G . , K o i k e , K . , L o g i n , I . S . , J u d d , A . M . , C r o n i n , M . J . , Y a s u m o t o , T . a n d M a c L e o d ,R . M . ( 1 9 8 4 ) A m . J . P h y s i o l . 2 4 7 , E 5 2 0 - E 5 2 5
S c h w a n e r , I . , S e i f e r t , R . a n d S c h u l t z , G . ( 1 9 9 2 ) B i o c h e m . J . 2 8 1 , 3 0 1 - 3 0 7S e i f e r t , R . a n d S c h a c h t e l e , C . ( 1 9 8 8 ) B i o c h e m . B i o p h y s . R e s . C o m m u n . 1 5 2 , 5 8 5 - 5 9 2S e i f e r t , R . , S e r k e , S . , H u h n , D . , B e s s l e r , W . G . , H a u s c h i l d t , S . , M e t z g e r , J . , W e i s m u l l e r ,
K . H . a n d J u n g , G . ( 1 9 9 2 ) E u r . J . B i o c h e m . 2 0 3 , 1 4 3 - 1 5 1S l a d e c z e k , F . , S c h m i d t , B . H . , A l o n s o , R . , V i a n , L . , T e p , A . , Y a s u m o t o , T . , C o r y , R . N . a n d
B o c k a e r t , J . ( 1 9 8 8 ) E u r . J . B i o c h e m . 1 7 4 , 6 6 3 - 6 7 0
S m i t h , C . D . , U h i n g , R . J . a n d S y n d e r m a n , R . ( 1 9 8 7 ) J . B i o l . C h e m . 2 6 2 , 6 1 2 1 4 1 2 7S o e r g e l , D . G . , Y a s u m o t o , T . , D a l y , J . W . a n d G u s o v s k y , F . ( 1 9 9 2 ) M o l . P h a r m a c o l . 4 1 ,
4 8 7 - 4 9 3T a k a h a s h i , M . , T a t s u m i , M . , O h i z u m i , Y . a n d Y a s u m o t o , T . ( 1 9 8 3 ) J . B i o l . C h e m . 2 5 8 ,
1 0 9 4 4 - 1 0 9 4 9W a t a n a b e , A . , I s h i d a , Y . , H o n d a , H . , K o b a y a s h i , M . a n d O h i z u m i , Y . ( 1 9 9 3 ) B r . J .
P h a r m a c o l . 1 0 9 , 2 9 - 3 6
R e c e i v e d 2 5 O c t o b e r 1 9 9 3 / 1 8 F e b r u a r y 1 9 9 4 ; a c c e p t e d 2 3 F e b r u a r y 1 9 9 4