Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Zymosan induced epithelial cells
45
Ca uptake by retinal pigment
Rocío Salceda 45
To cite this article: Rocío Salceda (1992) Zymosan induced Ca uptake by retinal pigment epithelial cells, Current Eye Research, 11:3, 195-201, DOI: 10.3109/02713689209001770 To link to this article: http://dx.doi.org/10.3109/02713689209001770
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Date: 03 April 2016, At: 19:33
Current Eye Research
Volume 1 I numbcr 3 1992. 19.5-201 -
Zymosan induced 45Ca uptake by retinal pigment epithelial cells
Rocio Salccda
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Departamento de Neurociencias, Instituto de Fisiologia Celular, UNAM, Apartado Postal 70-600, 04510 Mexico, Mexico
ABSTRACT 45Ca u p t a k e was s t u d i e d i n i s o l a t e d f r o g r e t i n a l pigment e p i t h e l i a l c e l l s i n r e s p o n s e t o t h e p h a g o c y t i c s t i m u l i , zymosan. 45Ca u p t a k e was s t r o n g l y s t i m u l a t e d immediately i n t h e p r e s e n c e of zymosan p a r t i c l e s . Calcium u p t a k e was p r o p o r t i o n a l t o t h e zymosan c o n c e n t r a t i o n s . A f t e r 60 min i n t h e p r e s e n c e of zymosan a c i d p h o s p h a t a s e and ( ? J - g l u c u r o n i d a s e a c t i v i t i e s showed a 25% and 50% i n c r e a s e , r e s p e c t i v e l y . Rod o u t e r segments induced a similar i n c r e a s e o f t h e s e enzyme a c t i v i t i e s . The zymosan-induced l y s o s o m a l enzyme a c t i v i t i e s was i n h i b i t e d by c y t o c h a l a s i n B and ruthenium r e d . The i o n o p h o r e A23187 produced a r e m a r k a b l e i n c r e a s e i n 45Ca u p t a k e b u t d i d n o t a f f e c t t h e l y s o s o m a l enzyme a c t i v i t i e s . T h e s e r e s u l t s s u g g e s t t h a t i n v i t r o TPE c e l l s a r e a b l e t o respond t o zymosan a s p h a g o c y t o s a b l e s t i m u l i and t h a t c a l c i u m m e d i a t e t h a t r e s p o n s e .
o t h e r c e l l s y s t e m s ( 1 3 , 1 4 ) i s i n f l u e n c e d by e x t e r n a l calcium, t h i s study w a s undertaken t o examine t h e p o s s i b l e r o l e of c a l c i u m f l u x e s d u r i n g zymosan i n d u c e d p h a g o c y t o s i s i n i s o l a t e d f r o g RPE c e l l s .
P r e l i m i n a r y s t u d i e s h a v e been
p r e v i o u s l y r e p o r t e d i n a b s t r a c t form ( 1 5 ) .
METHODS The r e s e a r c h w a s done i n a c c o r d a n c e w i t h t h e ARVO R e s o l u t i o n on t h e u s e of Animals i n R e s e a r c h . Frog RPE c e l l s were i s o l a t e d by a f i c o l - d e n s i t y g r a d i e n t ( 1 6 ) . The i s o l a t e d c e l l s were r e s u s p e n d e d i n a R i n g e r ' s K r e b s b i c a r b o n a t e medium c o n t a i n i n g (mM):
N a C 1 , 118; K C 1 , 4 . 7 ; KH2P04, 1.2;MgS04,
INTRODUCTION
1 . 1 7 ; CaC12, 2.5;
The r e t i n a l pigment e p i t h e l i u m (RPE) i s a u n i q u e
b i c a r b o n a t e , 3 5 ; pH 7.4. E x p e r i m e n t s were c a r r i e d
n e u r o e p i t h e l i a l t i s s u e t h a t forms a f u n c t i o n a l
o u t u n d e r dim r e d l i g h t .
u n i t witii t h e p h o t o r e c e p t o r c e l l of t h e n e u r a l r e t i n a . RPE p a r t i c i p a t e s among o t h e r f u n c t i o n s ,
g l u c o s e , 5 . 6 ; sodium
F o r measurements of 45Ca u p t a k e , RPE c e l l s (0.2-0.4
mg p r o t e i n ) w e r e i n c u b a t e d a t 37" C i n
i n t h e d e z r a d a t i o n of p h o t o r e c e p t o r o u t e r
1 m l of K r e b s medium c o n t a i n i n g 0 . 2 f l i
segment membranes (ROS) t h a t a r e shed from t h e
45CaC12 (New England N u c l e a r , B o s t o n , MA) and
t i p s of rod and cone c e l l s ( 1 ) . I n a d d i t i o n t o
d i f f e r e n t zymosan c o n c e n t r a t i o n s .
of
Zymosan A from
(Sigma Chem. Co. S t . L o u i s Mo, USA)
X O S , t h e RPE c e l l s h a v e been r e p o r t e d t o b i n d
S.cerevisiae
a n d / o r i n g e s t l a t e x b e a d s , c a r b o n p a r t i c l e s and
was p r e p a r e d a c c o r d i n g t o T a f f e t a n d R u s s e l l
r e d blood c e l l s ( 2 - 6 ) .
( 1 7 ) . A t s p e c i f i c p e r i o d s of t i m e c e l l s were
The p h a g o c y t i c p r o c e s s i s
s i m i l a r t o p h a g o c y t o s i s by o t h e r c e l l s s u c h a s
c e n t r i f u g e d i n a Beckman m i c r o f u g e f o r 20 s e c .
monocytes and macrophages ( 7 ) . The h i s t o l o g i c a l
The p e l l e t w a s r i n s e d s u p e r f i c i a l l y w i t h c o l d
c h a r a c t e r i s t i c s of p h o t o r e c e p t o r s h e d d i n g and
medium a n d t i s s u e s o l u b i l i z e d w i t h 0.1 % sodium
p h a g o c y t o s i s h a v e been s t u d i e d e x t e n s i v e l y ( 8 , 9 )
d o d e c y l s u l f a t e (SDS). The a c c u m u l a t e d
b u t t h e b i o c h e m i c a l mechanisms a r e n o t w e l l
r a d i o a c t i v i t y w a s d e t e r m i n e d a f t e r a d d i t i o n of
defined.
5 ml T r i t o s o l i n a Beckman l i q u i d s c i n t i l l a t i o n
S i n c e changes i n e x t r a c e l l u l a r c a l c i u m c a n a l t e r t h e a d h e s i v e n e s s of v a r i o u s c e l l t y p e s (10) and s i n c e p h a g o c y t o s i s i n t h e RPE (11,12)
Received on September 6, 1991; accepted
on
and
s p e c t r o p h o t o m e t e r . The v a l u e s were c o r r e c t e d f o r 45Ca b i n d i n g a t c e r o t i m e i n c u b a t i o n . F o r 45Ca e f f l u x , RPE c e l l s were l o a d e d w i t h
February 28, 1992
~
0 Oxford University Press
195
Current Eye Research 45Ca f o r 10 min a t 37" C . They w e r e c e n t r i f u g e d , and r e s u s p e n d e d i n normal medium. A f t e r s u i t a b l e i n c u b a t i o n p e r i o d a t 37" C t h e c e l l s were c e n t r i f u g e d and t h e 45Ca r e m a i n i n g i n RPE c e l l s c o u n t e d a s d e s c r i b e d above. 14C-Sucrose a c c u m u l a t i o n by RPE c e l l s was t e s t e d i n c u b a t i n g t h e RPE c e l l s a t 37" C i n 1 m l K r e b s medium c o n t a i n i n g l p C i of 14C-sucrose (New England N u c l e a r , Boston, MA.) Accumulated r a d i o a c t i v i t y was d e t e r m i n e d a s d e s c r i b e d above.
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Acid p h o s p h a t a s e and p - g l u c u r o n i d a s e a c t i v i t i e s
were t e s t e d a f t e r f r e e z i n g and thawing RPE c e l l s , by t h e h y d r o l i s i s o f p - n y t r o p h e n y l
1
I
I
1
10
30
60
p h o s p h a t e and
Time (min)
p h e n o p h t h a l e i n mono b - g l u c u r o n i c a c i d , r e s p e c t i v e l y , u s i n g a commercial k i t (Sigma Chem. Co., S t . L o u i s , Mo.). The p h a g o c y t o s i s of p h o t o r e c e p t o r o u t e r segments (ROS) was t e s t e d f o l l o w i n g t h e g e n e r a l p r o c e d u r e of Goldman e t a1 (18). R e t i n a s i n K r e b s
F i g u r e 1. Time c o u r s e of 14C-sucrose u p t a k e by RPE. Cells were i n c u b a t e d i n K r e b s medium c o n t a i n i n g 1 y C i of 14C s u c r o s e i n t h e p r e s e n c e ( ) o r a b s e n c e ( A ) of zymosan (250 p g / m l ) . Zymosan (250 r g / m l ) + r u t h e n i u m r e d ( l O O p M ) ( 0 ) . Each p o i n t r e p r e s e n t s t h e mean o f f o u r e x p e r i m e n t s f SEM.
medium were g e n t l y p i p e t e d up and down s e v e r a l times w i t h a wide-bore P a s t e u r p i p e t t e . T h e s u s p e n s i o n was t h e n f i l t e r e d t h r o u g h t e f l o n s c r e e n and ROS were c o l l e c t e d from t h e f i l t r a t e
c o n t r o l l e v e l a f t e r 1 0 min and c o n t i n u e d f o r
by low s p e e d c e n t r i f u g a t i o n (900 xg 1 0 min a t 4"
a b o u t 30 min b e f o r e l e v e l i n g o f f ( F i g . 1 ) .
C) and r e s u s p e n d e d i n K r e b s medium. RPE c e l l s
I n c u b a t i o n of RPE c e l l s i n t h e p r e s e n c e of
were i n c u b a t e d i n K r e b s medium f o r d i f f e r e n t
zymosan b r i n g s a b o u t a n a l m o s t i m m e d i a t e i n c r e a s e
p e r i o d s of t i m e i n t h e p r e s e n c e of ROS. A t t h e
i n 45Ca u p t a k e . E x p o s u r e t o zymosan i n c r e a s e d
end of t h e i n c u b a t i o n , RPE c e l l s were s e p a r a t e d
45Ca a c c u m u l a t i o n by a l m o s t t h r e e f o l d o v e r t h e
from t h e ROS by 1 5 min c e n t r i f u g a t i o n a t 1000 rpm
c o n t r o l l e v e l a f t e r two min, r e a c h i n g a p e a k a t
i n a d i s c o n t i n u o us f i c o l d e n s i t y g r a d i e n t (bottom
1 0 min. T h e r e a f t e r t h e r a t e of u p t a k e of l a b e l
l a y e r 20%, m i d d l e l a y e r 10% and u p p e r l a y e r 5 % ) .
d e c r e a s e d r e a c h i n g t h e c o n t r o l l e v e l s a t 60 min
The RPE c e l l s a t t h e bottom of t h e t u b e w e r e
( F i g . 2 ) . The u p t a k - of 45Ca i n c r e a s e d w i t h
washed w i t h c o l d medium a n d l y s o s o m a l enzyme
i n c r e a s i n g zymosan l e v e l s i n t h e e x t e r n a l medium,
a c t i v i t i e s were d e t e r m i n e d .
r e a c h i n g s a t u r a t i o n a t 500 p / m l ( F i g . 3 ) .
P r o t e i n s w e r e d e t e r m i n e d by t h e p r o c e d u r e
I n t h e p r e s e n c e of l O O p M of t h e c a l c i u m b l o c k e r , r u t h e n i u m r e d (RR) , t h e zymosan i n d u c e d 45Ca
d e s c r i b e d by Lowry e t a 1 ( 1 9 ) .
uptake w i t h i n t h e c e l l s w a s reduced t o c o n t r o l RESULTS
levels, b u t i t was n o t i n f l u e n c e d by t h e p r e s e n c e
I n o r d e r t o e s t a b l i s h zymosan a s a p h a g o c y t o s a b l e
of t h e c a l c i u m c h a n n e l b l o c k e r v e r a p a m i l
p a r t i c l e by RPE, w e f o l l o w e d t h e u p t a k e of 14C-
( T a b l e 1). While e x p o s u r e of RPE t o l O O p M R R ,
s u c r o s e and t h e a c t i v i t y of t h e l y s o s o m a l enzymes
p a r t i a l l y r e d u c e d t h e zymosan i n d u c e d 14C-sucrose
a c i d phosphatase and 6-glucuronidase.
u p t a k e ( F i g . 1).
Under normal
c o n d i t i o n s RPE c e l l s a c c u m u l a t e d l o w l e v e l s of
I n c o n t r a s t t o 45Ca u p t a k e , zymosan a d d i t i o n
14C-sucrose. Exposure of RPE t o zymosan i n c r e a s e d
t o RPE c e l l s d i d n o t c h a n g e t h e amount of 45Ca
t h e u p t a k e of 14C-sucrose by t h r e e f o l d o v e r t h e
r e l e a s e d f r o m p r e l a b e l e d c e l l s ( F i g . 4), which
196
Current Eye Research T a b l e 1. E f f e c t o f d i f f e r e n t d r u g s on t h e 45Ca u p t a k e by RPE Addition
45Ca u p t a k e (?moles /mg p r o t e i n )
--
0
0
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0.73 f 0.12 1 . 8 2 f 0.30 1 . 3 8 2 0.20
Zymosan 250 ,ug/ml A23187 ( 5 ,ug/ml) Zymosan (250 p g / m l ) + Ruthenium r e d ( 1 0 0 p M ) Zymosan (250 p g / m l ) + C y t o c h a l a s i n ( 5 +g/ml) Zymosan (250 y g / m l ) + V e r a p a m i l (10 fM)
T i m e (min) F i s u r e 2 . T i m e c o u r s e of zymosan provoked 45Ca u p t a k e by RPE, ( A ) c o n t r o l c e l l s ; ( 6 ) i n t h e p r e s e n c e of zymosan (250 ,ug/ml). 45Ca and zymosan were added a t z e r o t i m e . The d a t a a r e mean of s i x e x p e r i m e n t s f SEM.
0.94
2 0.09
1.84
2
0.26
1.65
2
0.24
RPE c e l l s were i n c u b a t e d f o r 10 min i n a Krebsb i c a r b o n a t e medium i n t h e d a r k . 45Ca w a s added a t t h e same t i m e a s t h e d r u g . Each v a l u e i s t h e mean 2 SEM of 4 t o 7 e x p e r i m e n t s .
I
I T
*
I
1
I
1
500
1000
I
1
I
10
20
30
Time (min)
Zymosan ( y g / m l ) F i g u r e 3. Dependence of 45ca2+ u p t a k e on t h e amount of zymosan. The i n c u b a t i o n t i m e i n t h e p r e s e n c e of 45Ca and zymosan w a s 10 min t h r o u g h o u t . Each p o i n t r e p r e s e n t s t h e mean of f o u r e x p e r i m e n t s f SEM.
F i g u r e 4. T i m e c o u r s e of 45Ca e f f l u x from RPE. C e l l s were l o a d e d w i t h 45Ca f o r 1 0 min a t 37OC. They w e r e c e n t r i f u g e d a n d r e s u s p e n d e d i n n o r m a l medium i n t h e p r e s e n c e ( 0 ) o r a b s e n c e ( & ) of zymosan ( 2 5 0 p g / m l ) . A t t h e i n d i c a t e d t i m e s , c e l l s were r a p i d l y c e n t r i f u g e d a n d t h e remaining r a d i a c t i v i t y counted. The d a t a are mear! of t h r e e e x p e r i m e n t s SEM.
s u g g e s t s t h a t zymosan i n d u c e d a n e t c a l c i u m
were i n c r e a s e d 25% a n d 50% r e s p e c t i v e l y , i n RPE
accumulation.
cells a f t e r one hour i n c u b a t i o n i n t h e presence
I n o r d e r t o e s t a b l i s h a r e l a t i o n s h i p between 45Ca u p t a k e and p h a g o c y t o s i s by RPE c e l l s w e
of zymosan ( F i g . 5 ) . A t s h o r t e r p e r i o d s of
examined t h e e f f e c t of zymosan on t h e l y s o s o m a l
i n c u b a t i o n , t h e s e enzymes showed a s l i g h t
enzymes a c i d p h o s p h a t a s e a n d + - g l u c u r o n i d a s e .
The
a c i d p h o s p h a t a s e and Q - g l u c u r o n i d a s e a c t i v i t i e s
i n c r e a s e . The zymosan i n d u c e d l y s o s o m a l enzyme a c t i v i t i e s w e r e reduced considerably i n t h e
197
Current Eye Research d e t a c h e d f o r s u b s e q u e n t p h a g o c y t o s i s , and d e g r a d a t i o n by a d j a c e n t p i g m e n t e p i t h e l i a l c e l l s
(1) By a n a l o g y w i t h t h e model p u t f o r t h f o r t h e macrophage, p h a g o c y t o s i s of ROS d i s c s i s o f t e n
T
considered t o be a multistep process, encompassing b i n d i n g , i n g e s t i o n a n d d e g r a d a t i o n (3.20).
I t is w e l l known t h a t t a r g e t p a r t i c l e s
i n t e r a c t w i t h macrophage c e l l membranes t h r o u g h s p e c i f i c r e c e p t o r l i g a n d bonds, and t h a t t h i s
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binding i n t u r n t r i g g e r s engulfment through t h e
+ .g
t
e x t e n s i o n of
pseudopodial processes (21,22).
P r e v i o u s s t u d i e s h a v e shown t h a t l i g h t - e v o k e d d i s c s shedding r e q u i r e s e x t r a c e l l u l a r calcium
Acid phosphatase
)-glucuronidose
(11,12). E x p o s u r e t o RPE c e l l s t o zymosan b r i n g s a b o u t a dose-dependent
F i g u r e 5. E f f e c t of d i f f e r e n t compounds and ROS on t h e a c i d p h o s p h a t a s e and (5-glucuronidase a c t i v i t i e s of RPE c e l l s . Cells were i n c u b a t e d i n Krebs medium d u r i n g 60 min a t 37" C . A f t e r i n c u b a t i o n s c e l l s were c e n t r i f u g e d and t h e enzyme a c t i v i t i e s measured a s d e s c r i b e d i n Methods. Zymosan, 250 p g / m l ; c y t o c h a l a s i n B , 5 pglrnl; A23187, 5pg/ml; r u t h e n i u m r e d , 100pM. Values a r e t h e mean f SEM ( v e r t i c a l b a r s ) of 4-7 e x p e r i m e n t s .
-
i n c r e a s e i n 45Ca- u p t a k e
( F i g . 3,4). T h i s i n c r e a s e d i n f l u x p r e c e d e s t h e o n s e t of 14C-sucrose u p t a k e and enhancement of l y s o s o m a l enzyme a c t i v i t i e s ( F i g . 1 , 5 ) . 14C-Sucrose u p t a k e r e q u i r e s 1 0 t o 30 min a f t e r i n i t i a l e x p o s u r e of t h e c e l l s t o t h e zymosan p a r t i c l e s , w h e r e a s 30-60 min a r e r e q u i r e d f o r t h e zymosan a n d ROS-induced
i n c r e a s e of t h e lysosomal
enzyme a c t i v i t i e s t e s t e d . T h e s e r e s u l t s a r e i n
p r e s e n c e of c y t o c h a l a s i n B , a d r u g which d i s r u p t s
a g r e e m e n t w i t h t h o s e d e s c r i b e d t h a t i n macrophages
a c t i n filaments (Fig. 5), without a f f e c t i n g
and L c e l l s , where e n d o c y t i c v a c u o l e s f u s e w i t h
45Ca a c c u m u l a t i o n ( T a b l e 1 ) . On t h e o t h e r h a n d ,
l y s o s o m e s w i t h i n 5-15 min a n d w h e r e by 60 min
t h e a d d i t i o n of 100 p M RR t o t a l l y i n h i b i t e d t h e
most of t h e a d s o r b e d p a r t i c l e s were i n l o c a t e d
zymosan i n d u c e d a c i d p h o s p h a t a s e a n d
l y s o s o m e s ( 2 3 , 2 4 ) . F u r t h e r m o r e , t h e k i n e t i c s of
(3-glucuronidase a c t i v i t i e s , s u g g e s t i n g t h a t
p h a g o c y t o s i s r e p o r t e d h e r e f o l l o w similar
calcium uptake mediates such response (Fig. 5 ) .
p a t t e r n s t o t h o s e r e p o r t e d by o t h e r i n v e s t i g a t o r s
S i m i l a r r e s u l t s were o b s e r v e d i n RPE c e l l s
(8,18,25,26).
exposed t o t h e n a t u r a l p h a g o c y t o s a b l e s t i m u l i ,
The i n v o l v e m e n t of membrane i n t e r n a l i z a t i o n
ROS. I n c u b a t i o n of RPE c e l l s i n t h e p r e s e n c e of
d u r i n g 45Ca u p t a k e i n d u c e d by zymosan, was
i s o l a t e d ROS l e a d s t o a n i n c r e a s e of 20% a n d 40%
examined by e x p o s i n g RPE c e l l s t o zymosan
on t h e a c i d p h o s p h a t a s e a n d ( b - g l u c u r o n i d a s e
p a r t i c l e s i n t h e p r e s e n c e of c y t o c h a l a s i n B ,
a c t i v i t i e s , respectively (Fig. 5).
which i n h i b i t s p h a g o c y t o s i s and i n t e r n a l i z a t i o n
I n t e r e s t i n g l y , e x p o s u r e of RPE c e l l s t o t h e
of t h e p l a s m a membrane b u t d o e s n o t a f f e c t
c a l c i u m i o n o p h o r e A23187, which produced a
a t t a c h m e n t of p a r t i c l e s t o t h e plasma membrane.
r e m a r k a b l e i n c r e a s e i n 45Ca u p t a k e ( T a b l e l), d i d
W h i l e c y t o c h a l a s i n B d i d n o t a f f e c t 45Ca u p t a k e ,
n o t a f f e c t t h e l y s o s o m a l enzyme a c t i v i t i e s ( F i g , 5 ) .
i t b l o c k e d t h e i n c r e a s e i n l y s o s o m a l enzyme
a c t i v i t i e s , suggesting t h a t phagocytosis and/or DISCUSSION
phagosome-lysosomal
Rod p h o t o r e c e p t o r d i s c s h e d d i n g i s t h e p r o c e s s by
i n v o l v e m e n t i s f u r t h e r s u p p o r t e d by t h e f a c t t h a t
which d i s c s from t h e o u t e r segment d i s t a l t i p a r e
t h e r e i s a n a b u n d a n c e of a c t i n f i l a m e n t s i n t h e
198
fusion was inhibited. Actin
Current Eye Research a p i c a l p r o c e s s e s o f t h e RPE c e l l s ( 2 7 , 2 8 ) , and
i n d u c e d similar i n c r e a s e on t h e l e v e l s of 45Ca,
t h a t a c t i n h a s been l o c a l i z e d underneath t h e
b u t l y s o s o m a l enzyme a c t i v i t i e s w e r e n o t
plasma membrane a t t h e p h a g o c y t i c s i t e s i n RPE
i n c r e a s e d . C o n s e q u e n t l y , a s s o c i a t i o n o f zymosan
c e l l s ( 5 , 2 8 ) . F u r t h e r m o r e , c y t o c h a l a s i n B and D
with
b l o c k b o t h l i g h t - e v o k e d and dark-primed d i s c s
calcium
shedding (29,30).
s p e c i f i c c e l l u l a r compartments.
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The most i n t e r e s t i n g f e a t u r e i s t h a t e x p o s u r e
specific surface receptor uptake
mechanism d i r e c t i n g
study
indicates
to
RPE
r e c o g n i z e s zymosan as a p h a g o c y t i c s t i m u l u s .
The
of i n c r e a s e i n l y s o s o m a l enzyme a c t i v i t i e s w i t h i n
observation
RPE c e l l s . The mechanism by which RPE r e c o g n i z e s
block
and p h a g o c y t i z e s ROS t i p s i s n o t w e l l u n d e r s t o o d .
blocks
O t h e r p h a g o c y t i c c e l l s s e l e c t i v e l y t a k e up
activities
p a r t i c l e s by s p e c i f i c l i g a n d - r e c e p t o r i n t e r a c t i o n s
uptake, suggests t h a t calcium p a r t i c i p a t e s i n the
t h o u g h t t o i n v o l v e r e c o g n i t i o n of g l y c o c o n j u g a t e s
mechanism of p h a g o c y t o s i s i n RPE c e l l s .
RR,
in
calcium
vitro,
that
that
activate
of RPE c e l l s t o ROS i n d u c e d a similar p a t t e r n
(21,22,31-33).
This
might
a n i n o r g a n i c dye know
t h e c a l c i u m u p t a k e t h r o u g h membranes the
increase
of
lysosomal
i n d u c e d by zymosan,
to (42)
enzyme
as w e l l as
45Ca
RPE p h a g o c y t o s i s of ROS h a s a l s o
been s u g g e s t e d t o i n v o l v e g l y c o c o n j u g a t e l i g a n d -
ACKNOWLEDGMENTS
r e c e p t o r i n t e r a c t i o n ( 3 , 3 4 , 3 5 ) and t h e p r e s e n c e
This
of a mannose s p e c i f i c r e c e p t o r h a s been
P228CCX891591 from CONACyT.
work
was
supported
in
part
by
grant
d e m o n s t r a t e d (36-38). CORRESPONDING AUTHOR
The zymosan and ROS i n d u c e d l y s o s o m a l enzyme a c t i v i t i e s may b e d u e b o t h t o i n t e r n a l i z a t i o n and s y n t h e s i s of l y s o s o m a l enzymes. The RPE r e l e a s e s and r e c a p t u r e s t h e l y s o s o m a l enzyme N
-
acetyl
-4
D r . Roclo S a l c e d a , Departamento d e N e u r o c ie n c ia s , I n s t i t u t o de F i s i o l o g I a C e l u l a r ,
UNAM.
P o s t a l 70-600,
MEXICO.
04510 Mzxico, D.F.
Apartado
g l u c o s a m i n i d a s e by way of a mannose 6-phosphate r e c e p t o r ( 3 9 ) . I t seems l i k e l y t h a t o t h e r enzymes
REFERENCES
may a l s o f o l l o w t h i s pathway. On t h e o t h e r h a n d ,
1.
p h a g o c y t o s i s of r e d b l o o d c e l l s and p i n o c y t o s i s of serum p r o t e i n s i n d u c e t h e s y n t h e s i s of
2.
l y s o s o m a l enzymes i n mouse macrophages a n d l e a d s to a tenfold increase i n t h e i r intracellular content (23). Those r e s u l t s i n d i c a t e t h a t t h e i n c r e a s e i n
3.
45Ca u p t a k e may p a r t i c i p a t e i n c o u p l i n g t h e i n i t i a l i n t e r a c t i o n o f t h e zymosan p a r t i c l e s w i t h
4.
s u r f a c e r e c e p t o r s t o t h e cytoplasmic phase of p h a g o c y t o s i s . T h a t i d e a a g r e e s w i t h t h e r a p i d and t r a n s i e n t e l e v a t i o n of i n t e r n a l c a l c i u m o b s e r v e d d u r i n g c o n t a c t of a macrophage w i t h a zymosan
5.
p a r t i c l e ( 4 0 ) , as w e l l a s w i t h t h e s i n g l e c a l c i u m p u l s e s o b s e r v e d d u r i n g zymosan p h a g o c y t o s i s by
6.
macrophages ( 4 1 ) . T h a t t h i s i n c r e a s e i s n o t simply a non-specific
e f f e c t on t h e c y t o p l a s m i c
c a l c i u m a s s o c i a t e d w i t h t h e o n s e t of p h a g o c y t o s i s
i s shown by t h e 45Ca u p t a k e r e s u l t i n t h e p r e s e n c e of t h e c a l c i u m i o n o p h o r e A23187. T h i s i o n o p h o r e
7.
Young, R. W. and Bok, D . (1969) P a r t i c i p a t i o n of t h e r e t i n a l pigment e p i t h e l i u m i n t h e r o d o u t e r segment r e n e w a l p r o c e s s . J . C e l l B i o l . 42, 392-402. Edwards, R.B. and S z a m i e r , R.B. (1977) D e f e c t i v e p h a g o c y t o s i s of i s o l a t e d r o d o u t e r segments by RCS rat retinal pigment e p i t h e l i u m i n c u l t u r e . S c i e n c e , 197, 10011003. Feeney, L . and Mixon, R.M. (1976) An i n v i t r o model of p. h a g o c .y t o s i s i n b o v i n e and human r e t i n a l pigment e p i t h e l i u m . Exp. Eye Res. 22, 533-548. J.G. and Ward, A. (1974). Hollyfield, P h a g o c y t i c a c t i v i t y i n t h e r e t i n a l pigment I. e p i t h e l i u m of t h e f r o g Rana p i p i e n s . Uptake of polystyrene spheres. J. U l t r a s t r u c t . Res. 46, 327-338. M.O. (1983) D e f e c t i v e C h a i t i n , M.H. a n d ? a l l , i n g e s t i o n of r o d o u t e r s e g m e n t s by c u l t u r e d dystrophic r a t pigment e p i t h e l i u m c e l l s . 812-820. I n v e s t . Ophthalmol. V i s . S c i . 6, P.L. a n d H a l l , M.O. (1986) R a t Mayerson, retinal pigment epithelium cells show s p e c i f i c i t y of p h a g o c y t o s i s i n v i t r o . J . C e l l B i o l . 103, 299-308. H.L. and S t o s s e l , T.P. (1982) The Yin, mechanism of p h a g o c y t o s i s . I n " P h a g o c y t o s i s M.L. and P a s t and F u t u r e " (Ed. Karnovsky, B o l i s , L.) Pp. 1 S 2 7 . Academic P r e s s , New York.
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Ca uptake by retinal pigment
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To cite this article: Rocío Salceda (1992) Zymosan induced Ca uptake by retinal pigment epithelial cells, Current Eye Research, 11:3, 195-201, DOI: 10.3109/02713689209001770 To link to this article: http://dx.doi.org/10.3109/02713689209001770
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Current Eye Research
Volume 1 I numbcr 3 1992. 19.5-201 -
Zymosan induced 45Ca uptake by retinal pigment epithelial cells
Rocio Salccda
Downloaded by [University of Saskatchewan Library] at 19:33 03 April 2016
Departamento de Neurociencias, Instituto de Fisiologia Celular, UNAM, Apartado Postal 70-600, 04510 Mexico, Mexico
ABSTRACT 45Ca u p t a k e was s t u d i e d i n i s o l a t e d f r o g r e t i n a l pigment e p i t h e l i a l c e l l s i n r e s p o n s e t o t h e p h a g o c y t i c s t i m u l i , zymosan. 45Ca u p t a k e was s t r o n g l y s t i m u l a t e d immediately i n t h e p r e s e n c e of zymosan p a r t i c l e s . Calcium u p t a k e was p r o p o r t i o n a l t o t h e zymosan c o n c e n t r a t i o n s . A f t e r 60 min i n t h e p r e s e n c e of zymosan a c i d p h o s p h a t a s e and ( ? J - g l u c u r o n i d a s e a c t i v i t i e s showed a 25% and 50% i n c r e a s e , r e s p e c t i v e l y . Rod o u t e r segments induced a similar i n c r e a s e o f t h e s e enzyme a c t i v i t i e s . The zymosan-induced l y s o s o m a l enzyme a c t i v i t i e s was i n h i b i t e d by c y t o c h a l a s i n B and ruthenium r e d . The i o n o p h o r e A23187 produced a r e m a r k a b l e i n c r e a s e i n 45Ca u p t a k e b u t d i d n o t a f f e c t t h e l y s o s o m a l enzyme a c t i v i t i e s . T h e s e r e s u l t s s u g g e s t t h a t i n v i t r o TPE c e l l s a r e a b l e t o respond t o zymosan a s p h a g o c y t o s a b l e s t i m u l i and t h a t c a l c i u m m e d i a t e t h a t r e s p o n s e .
o t h e r c e l l s y s t e m s ( 1 3 , 1 4 ) i s i n f l u e n c e d by e x t e r n a l calcium, t h i s study w a s undertaken t o examine t h e p o s s i b l e r o l e of c a l c i u m f l u x e s d u r i n g zymosan i n d u c e d p h a g o c y t o s i s i n i s o l a t e d f r o g RPE c e l l s .
P r e l i m i n a r y s t u d i e s h a v e been
p r e v i o u s l y r e p o r t e d i n a b s t r a c t form ( 1 5 ) .
METHODS The r e s e a r c h w a s done i n a c c o r d a n c e w i t h t h e ARVO R e s o l u t i o n on t h e u s e of Animals i n R e s e a r c h . Frog RPE c e l l s were i s o l a t e d by a f i c o l - d e n s i t y g r a d i e n t ( 1 6 ) . The i s o l a t e d c e l l s were r e s u s p e n d e d i n a R i n g e r ' s K r e b s b i c a r b o n a t e medium c o n t a i n i n g (mM):
N a C 1 , 118; K C 1 , 4 . 7 ; KH2P04, 1.2;MgS04,
INTRODUCTION
1 . 1 7 ; CaC12, 2.5;
The r e t i n a l pigment e p i t h e l i u m (RPE) i s a u n i q u e
b i c a r b o n a t e , 3 5 ; pH 7.4. E x p e r i m e n t s were c a r r i e d
n e u r o e p i t h e l i a l t i s s u e t h a t forms a f u n c t i o n a l
o u t u n d e r dim r e d l i g h t .
u n i t witii t h e p h o t o r e c e p t o r c e l l of t h e n e u r a l r e t i n a . RPE p a r t i c i p a t e s among o t h e r f u n c t i o n s ,
g l u c o s e , 5 . 6 ; sodium
F o r measurements of 45Ca u p t a k e , RPE c e l l s (0.2-0.4
mg p r o t e i n ) w e r e i n c u b a t e d a t 37" C i n
i n t h e d e z r a d a t i o n of p h o t o r e c e p t o r o u t e r
1 m l of K r e b s medium c o n t a i n i n g 0 . 2 f l i
segment membranes (ROS) t h a t a r e shed from t h e
45CaC12 (New England N u c l e a r , B o s t o n , MA) and
t i p s of rod and cone c e l l s ( 1 ) . I n a d d i t i o n t o
d i f f e r e n t zymosan c o n c e n t r a t i o n s .
of
Zymosan A from
(Sigma Chem. Co. S t . L o u i s Mo, USA)
X O S , t h e RPE c e l l s h a v e been r e p o r t e d t o b i n d
S.cerevisiae
a n d / o r i n g e s t l a t e x b e a d s , c a r b o n p a r t i c l e s and
was p r e p a r e d a c c o r d i n g t o T a f f e t a n d R u s s e l l
r e d blood c e l l s ( 2 - 6 ) .
( 1 7 ) . A t s p e c i f i c p e r i o d s of t i m e c e l l s were
The p h a g o c y t i c p r o c e s s i s
s i m i l a r t o p h a g o c y t o s i s by o t h e r c e l l s s u c h a s
c e n t r i f u g e d i n a Beckman m i c r o f u g e f o r 20 s e c .
monocytes and macrophages ( 7 ) . The h i s t o l o g i c a l
The p e l l e t w a s r i n s e d s u p e r f i c i a l l y w i t h c o l d
c h a r a c t e r i s t i c s of p h o t o r e c e p t o r s h e d d i n g and
medium a n d t i s s u e s o l u b i l i z e d w i t h 0.1 % sodium
p h a g o c y t o s i s h a v e been s t u d i e d e x t e n s i v e l y ( 8 , 9 )
d o d e c y l s u l f a t e (SDS). The a c c u m u l a t e d
b u t t h e b i o c h e m i c a l mechanisms a r e n o t w e l l
r a d i o a c t i v i t y w a s d e t e r m i n e d a f t e r a d d i t i o n of
defined.
5 ml T r i t o s o l i n a Beckman l i q u i d s c i n t i l l a t i o n
S i n c e changes i n e x t r a c e l l u l a r c a l c i u m c a n a l t e r t h e a d h e s i v e n e s s of v a r i o u s c e l l t y p e s (10) and s i n c e p h a g o c y t o s i s i n t h e RPE (11,12)
Received on September 6, 1991; accepted
on
and
s p e c t r o p h o t o m e t e r . The v a l u e s were c o r r e c t e d f o r 45Ca b i n d i n g a t c e r o t i m e i n c u b a t i o n . F o r 45Ca e f f l u x , RPE c e l l s were l o a d e d w i t h
February 28, 1992
~
0 Oxford University Press
195
Current Eye Research 45Ca f o r 10 min a t 37" C . They w e r e c e n t r i f u g e d , and r e s u s p e n d e d i n normal medium. A f t e r s u i t a b l e i n c u b a t i o n p e r i o d a t 37" C t h e c e l l s were c e n t r i f u g e d and t h e 45Ca r e m a i n i n g i n RPE c e l l s c o u n t e d a s d e s c r i b e d above. 14C-Sucrose a c c u m u l a t i o n by RPE c e l l s was t e s t e d i n c u b a t i n g t h e RPE c e l l s a t 37" C i n 1 m l K r e b s medium c o n t a i n i n g l p C i of 14C-sucrose (New England N u c l e a r , Boston, MA.) Accumulated r a d i o a c t i v i t y was d e t e r m i n e d a s d e s c r i b e d above.
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Acid p h o s p h a t a s e and p - g l u c u r o n i d a s e a c t i v i t i e s
were t e s t e d a f t e r f r e e z i n g and thawing RPE c e l l s , by t h e h y d r o l i s i s o f p - n y t r o p h e n y l
1
I
I
1
10
30
60
p h o s p h a t e and
Time (min)
p h e n o p h t h a l e i n mono b - g l u c u r o n i c a c i d , r e s p e c t i v e l y , u s i n g a commercial k i t (Sigma Chem. Co., S t . L o u i s , Mo.). The p h a g o c y t o s i s of p h o t o r e c e p t o r o u t e r segments (ROS) was t e s t e d f o l l o w i n g t h e g e n e r a l p r o c e d u r e of Goldman e t a1 (18). R e t i n a s i n K r e b s
F i g u r e 1. Time c o u r s e of 14C-sucrose u p t a k e by RPE. Cells were i n c u b a t e d i n K r e b s medium c o n t a i n i n g 1 y C i of 14C s u c r o s e i n t h e p r e s e n c e ( ) o r a b s e n c e ( A ) of zymosan (250 p g / m l ) . Zymosan (250 r g / m l ) + r u t h e n i u m r e d ( l O O p M ) ( 0 ) . Each p o i n t r e p r e s e n t s t h e mean o f f o u r e x p e r i m e n t s f SEM.
medium were g e n t l y p i p e t e d up and down s e v e r a l times w i t h a wide-bore P a s t e u r p i p e t t e . T h e s u s p e n s i o n was t h e n f i l t e r e d t h r o u g h t e f l o n s c r e e n and ROS were c o l l e c t e d from t h e f i l t r a t e
c o n t r o l l e v e l a f t e r 1 0 min and c o n t i n u e d f o r
by low s p e e d c e n t r i f u g a t i o n (900 xg 1 0 min a t 4"
a b o u t 30 min b e f o r e l e v e l i n g o f f ( F i g . 1 ) .
C) and r e s u s p e n d e d i n K r e b s medium. RPE c e l l s
I n c u b a t i o n of RPE c e l l s i n t h e p r e s e n c e of
were i n c u b a t e d i n K r e b s medium f o r d i f f e r e n t
zymosan b r i n g s a b o u t a n a l m o s t i m m e d i a t e i n c r e a s e
p e r i o d s of t i m e i n t h e p r e s e n c e of ROS. A t t h e
i n 45Ca u p t a k e . E x p o s u r e t o zymosan i n c r e a s e d
end of t h e i n c u b a t i o n , RPE c e l l s were s e p a r a t e d
45Ca a c c u m u l a t i o n by a l m o s t t h r e e f o l d o v e r t h e
from t h e ROS by 1 5 min c e n t r i f u g a t i o n a t 1000 rpm
c o n t r o l l e v e l a f t e r two min, r e a c h i n g a p e a k a t
i n a d i s c o n t i n u o us f i c o l d e n s i t y g r a d i e n t (bottom
1 0 min. T h e r e a f t e r t h e r a t e of u p t a k e of l a b e l
l a y e r 20%, m i d d l e l a y e r 10% and u p p e r l a y e r 5 % ) .
d e c r e a s e d r e a c h i n g t h e c o n t r o l l e v e l s a t 60 min
The RPE c e l l s a t t h e bottom of t h e t u b e w e r e
( F i g . 2 ) . The u p t a k - of 45Ca i n c r e a s e d w i t h
washed w i t h c o l d medium a n d l y s o s o m a l enzyme
i n c r e a s i n g zymosan l e v e l s i n t h e e x t e r n a l medium,
a c t i v i t i e s were d e t e r m i n e d .
r e a c h i n g s a t u r a t i o n a t 500 p / m l ( F i g . 3 ) .
P r o t e i n s w e r e d e t e r m i n e d by t h e p r o c e d u r e
I n t h e p r e s e n c e of l O O p M of t h e c a l c i u m b l o c k e r , r u t h e n i u m r e d (RR) , t h e zymosan i n d u c e d 45Ca
d e s c r i b e d by Lowry e t a 1 ( 1 9 ) .
uptake w i t h i n t h e c e l l s w a s reduced t o c o n t r o l RESULTS
levels, b u t i t was n o t i n f l u e n c e d by t h e p r e s e n c e
I n o r d e r t o e s t a b l i s h zymosan a s a p h a g o c y t o s a b l e
of t h e c a l c i u m c h a n n e l b l o c k e r v e r a p a m i l
p a r t i c l e by RPE, w e f o l l o w e d t h e u p t a k e of 14C-
( T a b l e 1). While e x p o s u r e of RPE t o l O O p M R R ,
s u c r o s e and t h e a c t i v i t y of t h e l y s o s o m a l enzymes
p a r t i a l l y r e d u c e d t h e zymosan i n d u c e d 14C-sucrose
a c i d phosphatase and 6-glucuronidase.
u p t a k e ( F i g . 1).
Under normal
c o n d i t i o n s RPE c e l l s a c c u m u l a t e d l o w l e v e l s of
I n c o n t r a s t t o 45Ca u p t a k e , zymosan a d d i t i o n
14C-sucrose. Exposure of RPE t o zymosan i n c r e a s e d
t o RPE c e l l s d i d n o t c h a n g e t h e amount of 45Ca
t h e u p t a k e of 14C-sucrose by t h r e e f o l d o v e r t h e
r e l e a s e d f r o m p r e l a b e l e d c e l l s ( F i g . 4), which
196
Current Eye Research T a b l e 1. E f f e c t o f d i f f e r e n t d r u g s on t h e 45Ca u p t a k e by RPE Addition
45Ca u p t a k e (?moles /mg p r o t e i n )
--
0
0
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0.73 f 0.12 1 . 8 2 f 0.30 1 . 3 8 2 0.20
Zymosan 250 ,ug/ml A23187 ( 5 ,ug/ml) Zymosan (250 p g / m l ) + Ruthenium r e d ( 1 0 0 p M ) Zymosan (250 p g / m l ) + C y t o c h a l a s i n ( 5 +g/ml) Zymosan (250 y g / m l ) + V e r a p a m i l (10 fM)
T i m e (min) F i s u r e 2 . T i m e c o u r s e of zymosan provoked 45Ca u p t a k e by RPE, ( A ) c o n t r o l c e l l s ; ( 6 ) i n t h e p r e s e n c e of zymosan (250 ,ug/ml). 45Ca and zymosan were added a t z e r o t i m e . The d a t a a r e mean of s i x e x p e r i m e n t s f SEM.
0.94
2 0.09
1.84
2
0.26
1.65
2
0.24
RPE c e l l s were i n c u b a t e d f o r 10 min i n a Krebsb i c a r b o n a t e medium i n t h e d a r k . 45Ca w a s added a t t h e same t i m e a s t h e d r u g . Each v a l u e i s t h e mean 2 SEM of 4 t o 7 e x p e r i m e n t s .
I
I T
*
I
1
I
1
500
1000
I
1
I
10
20
30
Time (min)
Zymosan ( y g / m l ) F i g u r e 3. Dependence of 45ca2+ u p t a k e on t h e amount of zymosan. The i n c u b a t i o n t i m e i n t h e p r e s e n c e of 45Ca and zymosan w a s 10 min t h r o u g h o u t . Each p o i n t r e p r e s e n t s t h e mean of f o u r e x p e r i m e n t s f SEM.
F i g u r e 4. T i m e c o u r s e of 45Ca e f f l u x from RPE. C e l l s were l o a d e d w i t h 45Ca f o r 1 0 min a t 37OC. They w e r e c e n t r i f u g e d a n d r e s u s p e n d e d i n n o r m a l medium i n t h e p r e s e n c e ( 0 ) o r a b s e n c e ( & ) of zymosan ( 2 5 0 p g / m l ) . A t t h e i n d i c a t e d t i m e s , c e l l s were r a p i d l y c e n t r i f u g e d a n d t h e remaining r a d i a c t i v i t y counted. The d a t a are mear! of t h r e e e x p e r i m e n t s SEM.
s u g g e s t s t h a t zymosan i n d u c e d a n e t c a l c i u m
were i n c r e a s e d 25% a n d 50% r e s p e c t i v e l y , i n RPE
accumulation.
cells a f t e r one hour i n c u b a t i o n i n t h e presence
I n o r d e r t o e s t a b l i s h a r e l a t i o n s h i p between 45Ca u p t a k e and p h a g o c y t o s i s by RPE c e l l s w e
of zymosan ( F i g . 5 ) . A t s h o r t e r p e r i o d s of
examined t h e e f f e c t of zymosan on t h e l y s o s o m a l
i n c u b a t i o n , t h e s e enzymes showed a s l i g h t
enzymes a c i d p h o s p h a t a s e a n d + - g l u c u r o n i d a s e .
The
a c i d p h o s p h a t a s e and Q - g l u c u r o n i d a s e a c t i v i t i e s
i n c r e a s e . The zymosan i n d u c e d l y s o s o m a l enzyme a c t i v i t i e s w e r e reduced considerably i n t h e
197
Current Eye Research d e t a c h e d f o r s u b s e q u e n t p h a g o c y t o s i s , and d e g r a d a t i o n by a d j a c e n t p i g m e n t e p i t h e l i a l c e l l s
(1) By a n a l o g y w i t h t h e model p u t f o r t h f o r t h e macrophage, p h a g o c y t o s i s of ROS d i s c s i s o f t e n
T
considered t o be a multistep process, encompassing b i n d i n g , i n g e s t i o n a n d d e g r a d a t i o n (3.20).
I t is w e l l known t h a t t a r g e t p a r t i c l e s
i n t e r a c t w i t h macrophage c e l l membranes t h r o u g h s p e c i f i c r e c e p t o r l i g a n d bonds, and t h a t t h i s
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binding i n t u r n t r i g g e r s engulfment through t h e
+ .g
t
e x t e n s i o n of
pseudopodial processes (21,22).
P r e v i o u s s t u d i e s h a v e shown t h a t l i g h t - e v o k e d d i s c s shedding r e q u i r e s e x t r a c e l l u l a r calcium
Acid phosphatase
)-glucuronidose
(11,12). E x p o s u r e t o RPE c e l l s t o zymosan b r i n g s a b o u t a dose-dependent
F i g u r e 5. E f f e c t of d i f f e r e n t compounds and ROS on t h e a c i d p h o s p h a t a s e and (5-glucuronidase a c t i v i t i e s of RPE c e l l s . Cells were i n c u b a t e d i n Krebs medium d u r i n g 60 min a t 37" C . A f t e r i n c u b a t i o n s c e l l s were c e n t r i f u g e d and t h e enzyme a c t i v i t i e s measured a s d e s c r i b e d i n Methods. Zymosan, 250 p g / m l ; c y t o c h a l a s i n B , 5 pglrnl; A23187, 5pg/ml; r u t h e n i u m r e d , 100pM. Values a r e t h e mean f SEM ( v e r t i c a l b a r s ) of 4-7 e x p e r i m e n t s .
-
i n c r e a s e i n 45Ca- u p t a k e
( F i g . 3,4). T h i s i n c r e a s e d i n f l u x p r e c e d e s t h e o n s e t of 14C-sucrose u p t a k e and enhancement of l y s o s o m a l enzyme a c t i v i t i e s ( F i g . 1 , 5 ) . 14C-Sucrose u p t a k e r e q u i r e s 1 0 t o 30 min a f t e r i n i t i a l e x p o s u r e of t h e c e l l s t o t h e zymosan p a r t i c l e s , w h e r e a s 30-60 min a r e r e q u i r e d f o r t h e zymosan a n d ROS-induced
i n c r e a s e of t h e lysosomal
enzyme a c t i v i t i e s t e s t e d . T h e s e r e s u l t s a r e i n
p r e s e n c e of c y t o c h a l a s i n B , a d r u g which d i s r u p t s
a g r e e m e n t w i t h t h o s e d e s c r i b e d t h a t i n macrophages
a c t i n filaments (Fig. 5), without a f f e c t i n g
and L c e l l s , where e n d o c y t i c v a c u o l e s f u s e w i t h
45Ca a c c u m u l a t i o n ( T a b l e 1 ) . On t h e o t h e r h a n d ,
l y s o s o m e s w i t h i n 5-15 min a n d w h e r e by 60 min
t h e a d d i t i o n of 100 p M RR t o t a l l y i n h i b i t e d t h e
most of t h e a d s o r b e d p a r t i c l e s were i n l o c a t e d
zymosan i n d u c e d a c i d p h o s p h a t a s e a n d
l y s o s o m e s ( 2 3 , 2 4 ) . F u r t h e r m o r e , t h e k i n e t i c s of
(3-glucuronidase a c t i v i t i e s , s u g g e s t i n g t h a t
p h a g o c y t o s i s r e p o r t e d h e r e f o l l o w similar
calcium uptake mediates such response (Fig. 5 ) .
p a t t e r n s t o t h o s e r e p o r t e d by o t h e r i n v e s t i g a t o r s
S i m i l a r r e s u l t s were o b s e r v e d i n RPE c e l l s
(8,18,25,26).
exposed t o t h e n a t u r a l p h a g o c y t o s a b l e s t i m u l i ,
The i n v o l v e m e n t of membrane i n t e r n a l i z a t i o n
ROS. I n c u b a t i o n of RPE c e l l s i n t h e p r e s e n c e of
d u r i n g 45Ca u p t a k e i n d u c e d by zymosan, was
i s o l a t e d ROS l e a d s t o a n i n c r e a s e of 20% a n d 40%
examined by e x p o s i n g RPE c e l l s t o zymosan
on t h e a c i d p h o s p h a t a s e a n d ( b - g l u c u r o n i d a s e
p a r t i c l e s i n t h e p r e s e n c e of c y t o c h a l a s i n B ,
a c t i v i t i e s , respectively (Fig. 5).
which i n h i b i t s p h a g o c y t o s i s and i n t e r n a l i z a t i o n
I n t e r e s t i n g l y , e x p o s u r e of RPE c e l l s t o t h e
of t h e p l a s m a membrane b u t d o e s n o t a f f e c t
c a l c i u m i o n o p h o r e A23187, which produced a
a t t a c h m e n t of p a r t i c l e s t o t h e plasma membrane.
r e m a r k a b l e i n c r e a s e i n 45Ca u p t a k e ( T a b l e l), d i d
W h i l e c y t o c h a l a s i n B d i d n o t a f f e c t 45Ca u p t a k e ,
n o t a f f e c t t h e l y s o s o m a l enzyme a c t i v i t i e s ( F i g , 5 ) .
i t b l o c k e d t h e i n c r e a s e i n l y s o s o m a l enzyme
a c t i v i t i e s , suggesting t h a t phagocytosis and/or DISCUSSION
phagosome-lysosomal
Rod p h o t o r e c e p t o r d i s c s h e d d i n g i s t h e p r o c e s s by
i n v o l v e m e n t i s f u r t h e r s u p p o r t e d by t h e f a c t t h a t
which d i s c s from t h e o u t e r segment d i s t a l t i p a r e
t h e r e i s a n a b u n d a n c e of a c t i n f i l a m e n t s i n t h e
198
fusion was inhibited. Actin
Current Eye Research a p i c a l p r o c e s s e s o f t h e RPE c e l l s ( 2 7 , 2 8 ) , and
i n d u c e d similar i n c r e a s e on t h e l e v e l s of 45Ca,
t h a t a c t i n h a s been l o c a l i z e d underneath t h e
b u t l y s o s o m a l enzyme a c t i v i t i e s w e r e n o t
plasma membrane a t t h e p h a g o c y t i c s i t e s i n RPE
i n c r e a s e d . C o n s e q u e n t l y , a s s o c i a t i o n o f zymosan
c e l l s ( 5 , 2 8 ) . F u r t h e r m o r e , c y t o c h a l a s i n B and D
with
b l o c k b o t h l i g h t - e v o k e d and dark-primed d i s c s
calcium
shedding (29,30).
s p e c i f i c c e l l u l a r compartments.
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The most i n t e r e s t i n g f e a t u r e i s t h a t e x p o s u r e
specific surface receptor uptake
mechanism d i r e c t i n g
study
indicates
to
RPE
r e c o g n i z e s zymosan as a p h a g o c y t i c s t i m u l u s .
The
of i n c r e a s e i n l y s o s o m a l enzyme a c t i v i t i e s w i t h i n
observation
RPE c e l l s . The mechanism by which RPE r e c o g n i z e s
block
and p h a g o c y t i z e s ROS t i p s i s n o t w e l l u n d e r s t o o d .
blocks
O t h e r p h a g o c y t i c c e l l s s e l e c t i v e l y t a k e up
activities
p a r t i c l e s by s p e c i f i c l i g a n d - r e c e p t o r i n t e r a c t i o n s
uptake, suggests t h a t calcium p a r t i c i p a t e s i n the
t h o u g h t t o i n v o l v e r e c o g n i t i o n of g l y c o c o n j u g a t e s
mechanism of p h a g o c y t o s i s i n RPE c e l l s .
RR,
in
calcium
vitro,
that
that
activate
of RPE c e l l s t o ROS i n d u c e d a similar p a t t e r n
(21,22,31-33).
This
might
a n i n o r g a n i c dye know
t h e c a l c i u m u p t a k e t h r o u g h membranes the
increase
of
lysosomal
i n d u c e d by zymosan,
to (42)
enzyme
as w e l l as
45Ca
RPE p h a g o c y t o s i s of ROS h a s a l s o
been s u g g e s t e d t o i n v o l v e g l y c o c o n j u g a t e l i g a n d -
ACKNOWLEDGMENTS
r e c e p t o r i n t e r a c t i o n ( 3 , 3 4 , 3 5 ) and t h e p r e s e n c e
This
of a mannose s p e c i f i c r e c e p t o r h a s been
P228CCX891591 from CONACyT.
work
was
supported
in
part
by
grant
d e m o n s t r a t e d (36-38). CORRESPONDING AUTHOR
The zymosan and ROS i n d u c e d l y s o s o m a l enzyme a c t i v i t i e s may b e d u e b o t h t o i n t e r n a l i z a t i o n and s y n t h e s i s of l y s o s o m a l enzymes. The RPE r e l e a s e s and r e c a p t u r e s t h e l y s o s o m a l enzyme N
-
acetyl
-4
D r . Roclo S a l c e d a , Departamento d e N e u r o c ie n c ia s , I n s t i t u t o de F i s i o l o g I a C e l u l a r ,
UNAM.
P o s t a l 70-600,
MEXICO.
04510 Mzxico, D.F.
Apartado
g l u c o s a m i n i d a s e by way of a mannose 6-phosphate r e c e p t o r ( 3 9 ) . I t seems l i k e l y t h a t o t h e r enzymes
REFERENCES
may a l s o f o l l o w t h i s pathway. On t h e o t h e r h a n d ,
1.
p h a g o c y t o s i s of r e d b l o o d c e l l s and p i n o c y t o s i s of serum p r o t e i n s i n d u c e t h e s y n t h e s i s of
2.
l y s o s o m a l enzymes i n mouse macrophages a n d l e a d s to a tenfold increase i n t h e i r intracellular content (23). Those r e s u l t s i n d i c a t e t h a t t h e i n c r e a s e i n
3.
45Ca u p t a k e may p a r t i c i p a t e i n c o u p l i n g t h e i n i t i a l i n t e r a c t i o n o f t h e zymosan p a r t i c l e s w i t h
4.
s u r f a c e r e c e p t o r s t o t h e cytoplasmic phase of p h a g o c y t o s i s . T h a t i d e a a g r e e s w i t h t h e r a p i d and t r a n s i e n t e l e v a t i o n of i n t e r n a l c a l c i u m o b s e r v e d d u r i n g c o n t a c t of a macrophage w i t h a zymosan
5.
p a r t i c l e ( 4 0 ) , as w e l l a s w i t h t h e s i n g l e c a l c i u m p u l s e s o b s e r v e d d u r i n g zymosan p h a g o c y t o s i s by
6.
macrophages ( 4 1 ) . T h a t t h i s i n c r e a s e i s n o t simply a non-specific
e f f e c t on t h e c y t o p l a s m i c
c a l c i u m a s s o c i a t e d w i t h t h e o n s e t of p h a g o c y t o s i s
i s shown by t h e 45Ca u p t a k e r e s u l t i n t h e p r e s e n c e of t h e c a l c i u m i o n o p h o r e A23187. T h i s i o n o p h o r e
7.
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