- Email: [email protected]
The effect of intraluminal factors on the brush border of human intestinal absorptive cells
424
A bstrac ts of The Ne therla~,ds Socie ty of Electron Microscopy Annual Meering
THE E F F E C T OF C Y T O C H A L A S I N B A N D D ON T H E E N D O C Y T O S I S OF H O R S E R A D I S H P E R O X I D A S E A N D T R A N S P O R T OF C E L L - C O A T G L Y C O P R O T E I N S IN A B S O R P T I V E C E L L S OF C U L T U R E D HUMAN SMALL-INTESTINAL TISSUE B. A. A. S c h e v e n , J. B l o k , A. A. M u l d e r S t a p e l , W. Th. D a e m s , L. A. G i n s e l L a b o r a t o r y for E l e c t r o n M i c r o s c o p y , U n i v e r s i t y of L e i d e n , R i j n s b u r g e r w e g 10, 2 3 3 3 AA L e i d e n , The N e t h e r l a n d s
Previous autoradiographical studies s h o w e d that c e l l - c o a t g l y c o p r o t e i n s of i n t e s t i n a l a b s o r p t i v e c e l l s are t r a n s f e r r e d v i a a p i c a l v e s i c l e s and t u b u l e s from the G o l g i a p p a r a t u s to the a p i c a l cell s u r f a c e . A s m a l l a m o u n t of the r a d i o - l a b e l l e d m a t e r i a l a l s o a r r i v e d in the l y s o s o m e s , a n d a c r i n o p h a g i c f u n c t i o n of t h e s e o r g a n e l l e s in the r e g u l a t i o n of the t r a n s p o r t of c e l l - c o a t g l y c o p r o t e i n s w a s s u g g e s t e d . 1,2 However, a m e c h a n i s m by w h i c h e x o c y t o s e d c e l l - c o a t m a t e r i a l is r a p i d l y e n d o c v t o s e d and t r a n s p o r t e d to the l y s o s o m e s c o u l d n o t be excluded. R e c e n t l y , it w a s s h o w n t h a t a p i c a l e n d o c y t o s i s of h o r s e r a d i s h p e r o x i d a s e (HRP) o c c u r s in t h e s e e n t e r o c y t e s , whereas ferritin, w h i c h showed a distinct a f f i n i t y for the c e l l - c o a t g l y c o p r o t e i n s , w a s not t a k e n up b y the c e l l s . 3 T h e s e observations indicate that c e l l - c o a t m a t e r i a l is s e l e c t i v e l y e x c l u d e d f r o m the a p i c a l e n d o c y t o t i c p r o c e s s o c c u r r i n g in the a b s o r p t i v e c e l l s . 3 To c h a r a c t e r ize the e n d o c y t o t i c m e c h a n i s m f u r t h e r , w e s t u d i e d the e f f e c t of c y t o c h a l a s i n B (CB) and c y t o c h a l a s i n D (CD) on the uptake of H R P in the a b s o r p t i v e c e l l s . The s p e c i m e n s w e r e c u l t u r e d for 24 h in the p r e s e n c e of t h e s e d r u g s and the l a s t h o u r w i t h HRP. M o r p h o m e t r i c a l a n a l y s i s did n o t s h o w any e f f e c t of CB on the e n d o c y t o s i s of HRP, b u t CB c a u s e d a s i g n i f i c a n t d e c r e a s e in the n u m b e r of H R P - n e g a t i v e (exocytotic) a p i c a l vesicles and tubules. The p r e s e n c e of CD i n h i b i t e d the e n d o c y t o t i c u p t a k e of HRP c o n s i d e r a b l y w i t h o u t any e f f e c t on the n u m b e r and d i s t r i b u t i o n of the e x o c y t o t i c v e s i c l e s and t u b u l e s . To a s s e s s the e f f e c t of CD on the synt h e s i s a n d t r a n s p o r t of c e l l - c o a t g l y c o proteins, electron-microscopical autor a d i o g r a p h i c a l (20 h of l a b e l l i n g w i t h 3 H - f u c o s e , f o l l o w e d b y a 4-h c h a s e ) a n d b i o c h e m i c a l incorporation studies were performed. No e f f e c t of CD on the synt h e s i s and t r a n s p o r t of the c e l l - c o a t glycoproteins was found. The a m o u n t of the r a d i o - l a b e l l e d m a t e r i a l p r e s e n t in the b r u s h b o r d e r a n d the l y s o s o m e s w a s s i m i l a r to t h a t for the c o n t r o l c u l t u r e s . T h e s e r e s u l t s p r o v i d e e v i d e n c e of a direct t r a n s p o r t of c e l l - c o a t g l y c o p r o t e i n s to the l y s o s o m e s r a t h e r than an e x o c y t o -
tic-endocytotic mechanism. This e v i d e n c e s u p p o r t s the h y p o t h e s i s t h a t the l y s o s o m e s have a crinophagic regulatory funct i o n in the t r a n s p o r t of c e l l - c o a t g l y c o p r o t e i n s , by w h i c h e x c e s s i v e l y p r o d u c e d c e l l - c o a t m a t e r i a l is d e g r a d e d . 1.
2.
3.
L. A. Ginsel, J. J. M. Onderwater, W. Th. Daems, Virchows Arch. B Cell Pathol. 30 (1979) 245-73. J. Blok, A. A. Mulder-Stapel, L. A. Ginsel, W. Th. Daems, Cell Tiss. Res. 218 (1981) 227-51. J. Blok, A. A. Mulder-Stapel, L. A. Ginsel, W. Th. Daems, Cell Tiss. Res. 216 (1981) 1-13.
T H E E F F E C T OF I N T R A L U M I N A L F A C T O R S ON T H E B R U S H B O R D E R OF H U M A N I N T E S T I N A L ABSORPTIVE CELLS B. A. A. S c h e v e n , J. B l o k , A. A. M u l d e r S t a p e l , L. A. G i n s e l L a b o r a t o r y for E i e c t r o n M i c r o s c o p y , U n i v e r s i t y of L e i d e n , R i j n s b u r g e r w e g 10, 2 3 3 3 AA L e i d e n , T h e N e t h e r l a n d s
The cell c o a t p r e s e n t on the m i c r o v i l l i of i n t e s t i n a l a b s o r p t i v e c e l l s c o n s i s t s of g l y c o p r o t e i n s w h i c h are synthesized continuously. A f t e r completion in the G o l g i a p p a r a t u s t h e s e g l y c o p r o t e i n s are t r a n s f e r r e d to the a p i c a l p l a s m a m e m b r a n e via a p i c a l v e s i c l e s a n d t u b u l e s . I T h r e e to five h o u r s l a t e r the g l y c o p r o t e i n s are r e l e a s e d i n t o the intestinal lumen. L i t t l e is k n o w n a b o u t the f a c t o r s c o n t r o l l i n g the t u r n o v e r of the cell c o a t . B i o c h e m i c a l s t u d i e s suggest t h a t i n t r a l u m i n a l f a c t o r s s u c h as p a n c r e a t i c p r o t e a s e s and b i l e s a l t s m a y i n f l u e n c e this p r o c e s s . 2 The p r e s e n t r e p o r t c o n c e r n s the r e s u l t s of p r e l i m i n ary m o r p h o l o g i c a l and b i o c h e m i c a l e x p e r i m e n t s on the e f f e c t of e l a s t a s e , p a n c r e a t i n (a l y o p h i l i z e d p a n c r e a s e x t r a c t ) , a n d the b i l e s a l t s d e o x y c h o l a t e (DOC) and t a u r o c h o l a t e (Tch) on the a b s o r p t i v e cells. N e i t h e r DOC n o r Tch h a d any e f f e c t on the a v e r a g e l e n g t h of the m i c r o v i l l i , although ultrastructur~l alterations ( c h a n g e s in the l e n g t h ) w e r e o b s e r v e d . Biochemical data s h o w e d that t h e r e was a s l i g h t r e l e a s e of b r u s h - b o r d e r e n z y m e s such as l a c t a s e , s u c r a s e , m a l t a s e , treh a l a s e , and a l k a l i n e p h o s p h a t a s e . I n c u b a t i o n of the c e l l s in the p r e s e n c e of e l a s t a s e a n d p a n c r e a t i n led to a s i g n i f i c a n t d e c r e a s e in the h e i g h t of m i c r o v i l l i a l t h o u g h the t h i c k n e s s of the cell c o a t on the tip of t h e s e s t r u c t u r e s w a s not a f f e c t e d . Biochemical analysis s h o w e d that both substances i n d u c e d a c o n s i d e r a b l e r e l e a s e of the a b o v e -
A bstrac ts of The Ne therla~,ds Socie ty of Electron Microscopy Annual Meering
THE E F F E C T OF C Y T O C H A L A S I N B A N D D ON T H E E N D O C Y T O S I S OF H O R S E R A D I S H P E R O X I D A S E A N D T R A N S P O R T OF C E L L - C O A T G L Y C O P R O T E I N S IN A B S O R P T I V E C E L L S OF C U L T U R E D HUMAN SMALL-INTESTINAL TISSUE B. A. A. S c h e v e n , J. B l o k , A. A. M u l d e r S t a p e l , W. Th. D a e m s , L. A. G i n s e l L a b o r a t o r y for E l e c t r o n M i c r o s c o p y , U n i v e r s i t y of L e i d e n , R i j n s b u r g e r w e g 10, 2 3 3 3 AA L e i d e n , The N e t h e r l a n d s
Previous autoradiographical studies s h o w e d that c e l l - c o a t g l y c o p r o t e i n s of i n t e s t i n a l a b s o r p t i v e c e l l s are t r a n s f e r r e d v i a a p i c a l v e s i c l e s and t u b u l e s from the G o l g i a p p a r a t u s to the a p i c a l cell s u r f a c e . A s m a l l a m o u n t of the r a d i o - l a b e l l e d m a t e r i a l a l s o a r r i v e d in the l y s o s o m e s , a n d a c r i n o p h a g i c f u n c t i o n of t h e s e o r g a n e l l e s in the r e g u l a t i o n of the t r a n s p o r t of c e l l - c o a t g l y c o p r o t e i n s w a s s u g g e s t e d . 1,2 However, a m e c h a n i s m by w h i c h e x o c y t o s e d c e l l - c o a t m a t e r i a l is r a p i d l y e n d o c v t o s e d and t r a n s p o r t e d to the l y s o s o m e s c o u l d n o t be excluded. R e c e n t l y , it w a s s h o w n t h a t a p i c a l e n d o c y t o s i s of h o r s e r a d i s h p e r o x i d a s e (HRP) o c c u r s in t h e s e e n t e r o c y t e s , whereas ferritin, w h i c h showed a distinct a f f i n i t y for the c e l l - c o a t g l y c o p r o t e i n s , w a s not t a k e n up b y the c e l l s . 3 T h e s e observations indicate that c e l l - c o a t m a t e r i a l is s e l e c t i v e l y e x c l u d e d f r o m the a p i c a l e n d o c y t o t i c p r o c e s s o c c u r r i n g in the a b s o r p t i v e c e l l s . 3 To c h a r a c t e r ize the e n d o c y t o t i c m e c h a n i s m f u r t h e r , w e s t u d i e d the e f f e c t of c y t o c h a l a s i n B (CB) and c y t o c h a l a s i n D (CD) on the uptake of H R P in the a b s o r p t i v e c e l l s . The s p e c i m e n s w e r e c u l t u r e d for 24 h in the p r e s e n c e of t h e s e d r u g s and the l a s t h o u r w i t h HRP. M o r p h o m e t r i c a l a n a l y s i s did n o t s h o w any e f f e c t of CB on the e n d o c y t o s i s of HRP, b u t CB c a u s e d a s i g n i f i c a n t d e c r e a s e in the n u m b e r of H R P - n e g a t i v e (exocytotic) a p i c a l vesicles and tubules. The p r e s e n c e of CD i n h i b i t e d the e n d o c y t o t i c u p t a k e of HRP c o n s i d e r a b l y w i t h o u t any e f f e c t on the n u m b e r and d i s t r i b u t i o n of the e x o c y t o t i c v e s i c l e s and t u b u l e s . To a s s e s s the e f f e c t of CD on the synt h e s i s a n d t r a n s p o r t of c e l l - c o a t g l y c o proteins, electron-microscopical autor a d i o g r a p h i c a l (20 h of l a b e l l i n g w i t h 3 H - f u c o s e , f o l l o w e d b y a 4-h c h a s e ) a n d b i o c h e m i c a l incorporation studies were performed. No e f f e c t of CD on the synt h e s i s and t r a n s p o r t of the c e l l - c o a t glycoproteins was found. The a m o u n t of the r a d i o - l a b e l l e d m a t e r i a l p r e s e n t in the b r u s h b o r d e r a n d the l y s o s o m e s w a s s i m i l a r to t h a t for the c o n t r o l c u l t u r e s . T h e s e r e s u l t s p r o v i d e e v i d e n c e of a direct t r a n s p o r t of c e l l - c o a t g l y c o p r o t e i n s to the l y s o s o m e s r a t h e r than an e x o c y t o -
tic-endocytotic mechanism. This e v i d e n c e s u p p o r t s the h y p o t h e s i s t h a t the l y s o s o m e s have a crinophagic regulatory funct i o n in the t r a n s p o r t of c e l l - c o a t g l y c o p r o t e i n s , by w h i c h e x c e s s i v e l y p r o d u c e d c e l l - c o a t m a t e r i a l is d e g r a d e d . 1.
2.
3.
L. A. Ginsel, J. J. M. Onderwater, W. Th. Daems, Virchows Arch. B Cell Pathol. 30 (1979) 245-73. J. Blok, A. A. Mulder-Stapel, L. A. Ginsel, W. Th. Daems, Cell Tiss. Res. 218 (1981) 227-51. J. Blok, A. A. Mulder-Stapel, L. A. Ginsel, W. Th. Daems, Cell Tiss. Res. 216 (1981) 1-13.
T H E E F F E C T OF I N T R A L U M I N A L F A C T O R S ON T H E B R U S H B O R D E R OF H U M A N I N T E S T I N A L ABSORPTIVE CELLS B. A. A. S c h e v e n , J. B l o k , A. A. M u l d e r S t a p e l , L. A. G i n s e l L a b o r a t o r y for E i e c t r o n M i c r o s c o p y , U n i v e r s i t y of L e i d e n , R i j n s b u r g e r w e g 10, 2 3 3 3 AA L e i d e n , T h e N e t h e r l a n d s
The cell c o a t p r e s e n t on the m i c r o v i l l i of i n t e s t i n a l a b s o r p t i v e c e l l s c o n s i s t s of g l y c o p r o t e i n s w h i c h are synthesized continuously. A f t e r completion in the G o l g i a p p a r a t u s t h e s e g l y c o p r o t e i n s are t r a n s f e r r e d to the a p i c a l p l a s m a m e m b r a n e via a p i c a l v e s i c l e s a n d t u b u l e s . I T h r e e to five h o u r s l a t e r the g l y c o p r o t e i n s are r e l e a s e d i n t o the intestinal lumen. L i t t l e is k n o w n a b o u t the f a c t o r s c o n t r o l l i n g the t u r n o v e r of the cell c o a t . B i o c h e m i c a l s t u d i e s suggest t h a t i n t r a l u m i n a l f a c t o r s s u c h as p a n c r e a t i c p r o t e a s e s and b i l e s a l t s m a y i n f l u e n c e this p r o c e s s . 2 The p r e s e n t r e p o r t c o n c e r n s the r e s u l t s of p r e l i m i n ary m o r p h o l o g i c a l and b i o c h e m i c a l e x p e r i m e n t s on the e f f e c t of e l a s t a s e , p a n c r e a t i n (a l y o p h i l i z e d p a n c r e a s e x t r a c t ) , a n d the b i l e s a l t s d e o x y c h o l a t e (DOC) and t a u r o c h o l a t e (Tch) on the a b s o r p t i v e cells. N e i t h e r DOC n o r Tch h a d any e f f e c t on the a v e r a g e l e n g t h of the m i c r o v i l l i , although ultrastructur~l alterations ( c h a n g e s in the l e n g t h ) w e r e o b s e r v e d . Biochemical data s h o w e d that t h e r e was a s l i g h t r e l e a s e of b r u s h - b o r d e r e n z y m e s such as l a c t a s e , s u c r a s e , m a l t a s e , treh a l a s e , and a l k a l i n e p h o s p h a t a s e . I n c u b a t i o n of the c e l l s in the p r e s e n c e of e l a s t a s e a n d p a n c r e a t i n led to a s i g n i f i c a n t d e c r e a s e in the h e i g h t of m i c r o v i l l i a l t h o u g h the t h i c k n e s s of the cell c o a t on the tip of t h e s e s t r u c t u r e s w a s not a f f e c t e d . Biochemical analysis s h o w e d that both substances i n d u c e d a c o n s i d e r a b l e r e l e a s e of the a b o v e -