European Journal of Cell Biq|f>gy m

Volume 13 • Number 1 > June 1&7#

C O N T E N T S V O L U M E 1 3 III

A M E L U N X E N , F . , E. T H I O T I A N G N I O , E . SPIESS

Untersuchungen zur Z e l l w a n d b i l d u n g . I. Elektronenmikroskopische Beobachtungen zur Feinstruktur der Rank en von Cucurbi ta m a x i m a L .

I n v e s t i g a t i o n s o n c e l l w a l l f o r m a t i o n . I . E l e c t r o n m i c r o s c o p i c a l o b s e r v a t i o n s oft t h e f i n e s t r u c t u r e of t h e t e n d r i l s of C u c u r b i t a m a x i m a L .

2 3 3

A M E L U N X E N , F., siehe SPIESS , E . 2 5 1

A M E L U N X E N , F., E. SPIESS , E. T H I O T I A N G N I O

Untersuchungen zur Z e l l w a n d b i l d u n g . III. Autoradiographie von Dünnschnitten und biochemische Analyse isolierter Dictyosomen der Ranken von Cucurbi ta m a x i m a L . 2 6 0

I n v e s t i g a t i o n s o n c e l l w a l l f o r m a t i o n . III. I n v e s t i g a t i o n s o n t h i n s e c t i o n s b y a u t o r a d i o g r a p h y a n d b i o c h e m i c a l a n a l y s i s of i s o l a t e d d i c t y o s o m e s of t e n d r i l s of C u c u r b i t a m a x i m a L .

A N D R I E S , J . C.

Variat ions ultrastructurales au sein des cellules epitheliales mesenteriques d'Aeshna cyanea (Insecte, Odonate) en fonction de la prise de nourriture 4 5 1

M i d g u t c e l l s u l t r a s t r u c t u r a l m o d i f i c a t i o n s d u r ' m g d i g e s t i o n in A e s b n a c y a n e a ( I n s e c t e , O d o n a t a )

B A R A N O W S K I , Z .

Three-dimensional analysis of movement in Physarum polycephalum plasmodia 1 1 8

D r e i d i m e n s i o n a l e A n a l y s e d e r B e w e g u n g b e i P h y s a r u m p o l y c e p h a l u m - P l a s m o d i e n

B R U N K , U . T . , siehe S C H E L L E N S , J . P. M . 9 3

C H E S C O E , D . , siehe D U C K E T T , J . G . 3 2 2

C O O K E , P., A . M . P O I S N E R

Microf i laments in bovine adrenal chromaffin cells 4 4 2 M i k r o f i l a m e n t e i n d e n chromaffinen Z e l l e n d e r R i n d e r - N e b e n n i e r e

D A H L , G . , siehe S C H U D T , C H . 2 1 1

D E I C H G R Ä B E R , G . , siehe S C H N E P F , E . 3 4 1

D E U M L I N G , B . , J . S I N C L A I R , J . N . T I M M I S , J . I N G L E

Demonstrat ion of satellite D N A components in several plant species w i t h the Ag +-Cs2S04 gradient technique 2 2 4 V o r k o m m e n v o n S a t e l l i t e n - D N S i n e i n i g e n P f l a n z e n a r t e n b e i A n w e n d u n g d e r Ag+-Cs2SÖ4 G r a d i e n t e n t e c h n i k

D E X H E I M E R , J .

Etüde de la secretion de mucilage par les cellules des glandes digestives de Drosera (D. rotundifol ia L . ; et D . capensis L.) . Appl ica t ion de quelques techniques cytochimiques 3 0 7 S t u d y of t h e s e c r e t i o n of m u c i l a g e i n d i g e s t i n g g l a n d c e l l s of D r o s e r a

D ' H A E S E , J . , siehe H I N S S E N , H . 1 3 2

D I E T E R I C H , C. E. , siehe D I E T E R I C H , H . J . 5 7

I V C O N T E N T S V O L U M E 13

D I E T E R I C H , H . J . , K . A . R O S E N B A U E R , C . E . D I E T E R I C H

Das Blutgefäßsystem des Pecten oculi beim Haussperl ing (Passer domesticus). Architektonik und Ultrastruktur nach licht-, transmissions- und rasterelektronen-mikroskopischen Untersuchungen 57 T h e b l o o d v e s s e l S y s t e m of t h e p e c t e n o c u l i i n t h e h o u s e s p a r r o w ( P a s s e r d o m e s t i c u s ) . Its a r c h i t e c t u r e a n d f i n e s t r u c t u r e s t u d i e d b y l i g h t , t r a n s m i s s i o n , a n d s c a n n i n g e l e c t r o n m i c r o s c o p y

D U C K E T T , J . G . , D . C H E S C O E

A combined ultrastructural and X - r a y microanalytical study of spermatogenesis in the bryophyte, Phaeoceros laevis (L.) Prosk. 322 K o m b i n i e r t e u l t r a s t r u k t u r e l l e u n d röntgenmikroanalytische U n t e r s u c h u n g d e r S p e r m a t o g e n e s e i m L e b e r m o o s P h a e o c e r o s l a e v i s ( L . ) P r o s k .

E L D E R , J . H . , J . M O R R E , T H . W . K E E N A N (Short Communicat ion)

Distr ibut ion of glycoproteins among subcellular fractions f rom rat liver 279

G l y c o p r o t e i n - M u s t e r v o n Z e l l f r a k t i o n e n a u s R a t t e n l e b e r

E L L I S , R . A . , C . C . G O E R T E M I L L E R JR.

Scanning electron microscopy of intercellular Channels and the local izat ion of ouabain sensitive p-nitrophenyl Phosphatase activity in the salt-secreting lacrymal glands of the marine turtle Chelonia mydas 1 R a s t e r e l e k t r o n e n m i k r o s k o p i s c h e D a r s t e l l u n g d e r I n t e r z e l l u l a r s p a l t e n u n d L o k a l i s a t i o n v o n O u a b a i n - s e n s i t i v e r p-Nitrophenyl-phosphatase-Aktivität i n d e n l a c r i m a l e n S a l z ­drüsen d e r Meeresschildkröte C h e l o n i a m y d a s

F R A N K E , W . W . , T H . W . K E E N A N , J . S T A D L E R , R . G E N Z , E . - D . J A R A S C H , J . K A R T E N B E C K

Nuclear membranes f rom mammal ian liver. VII . Characteristics of highly puri f ied nuclear membranes in comparison w i t h other membranes 28 Z u s a m m e n s e t z u n g h o c h g e r e i n i g t e r K e r n m e m b r a n f r a k t i o n e n a u s d e r R a t t e n l e b e r i m V e r g l e i c h m i t a n d e r e n M e m b r a n e n

F R A N K E , W . W . , U . S C H E E R , M . F . T R E N D E L E N B U R G , H . S P R I N G , H . Z E N T G R A F

Absence of nucleosomes in transcriptionally active chromatin 401 C h r o m a t i n während d e r T r a n s k r i p t i o n : d a s F e h l e n v o n N u c l e o s o m e n

F R I E D L A N D E R , M . , J . G E R S H O N , A . R E I N H A R T Z

Atypica l cycle of the nucleolus in spermatocytes of the insect Locusta migratoria 171 B e s o n d e r h e i t e n d e s N u c l e o l u s - Z y k l u s i n S p e r m a t o c y t e n d e r W a n d e r h e u s c h r e c k e L o c u s t a m i g r a t o r i a

G E N Z , R . , siehe F R A N K E , W . W . 28

G E R S H O N , J . , siehe F R I E D L A N D E R , M . 171

G H O S H , S. (Short Communicat ion)

Nucleo lar development in induced binucleate cells of A l l i u m cepa L . 163 N u c l e o l u s - E n t w i c k l u n g i n künstlich i n d u z i e r t e n z w e i k e r n i g e n Z w i e b e l z e l l e n

G O E R T E N M I L L E R JR. , C . C , siehe E L L I S , R . A . 1

G R A T Z L , M . , siehe S C H U D T , C H . 211

C O N T E N T S V O L U M E 1 3 V

G R A T Z L , M . , D . S C H W A B

T h e effect of microtubular inhibitors on secretion f r o m liver into b lood plasma and bile 1 9 9 D i e W i r k u n g mikrotubulärer Hemmstoffe auf d i e S e k r e t i o n d e r L e b e r i n d a s B l u t p l a s m a u n d d i e G a l l e

G U N N I N G , B . E . S., siehe R O B A R D S , A . W . 8 5

H A U S M A N N , K . , K . - H . M O C I K A T

Direct evidence for a surface coat on the plasma membrane of the ciliate Li tonotus duplostriatus 4 6 9 D i r e k t e r N a c h w e i s e i n e s »surface coat« auf d e r P l a s m a m e m b r a n d e s C i l i a t e n L i t o n o t u s d u p l o s t r i a t u s

H E B A N T , C , R . P. C . J O H N S O N

Ultrastructural features of freeze-etched water-conducting cells i n Polytr ichum (Polytrichales, Musc i ) 3 5 4 U l t r a s t r u k t u r d e r w a s s e r l e i t e n d e n Z e l l e n v o n P o l y t r i c h u m n a c h Gefrierätzung

H E R L A N , G . , F . W U N D E R L I C H (Short Communicat ion)

Isolation of a nuclear protein matrix f rom Tetrahymena macronuclei 2 9 1

I s o l a t i o n e i n e r K e r n p r o t e i n m a t r i x a u s T e t r a h y m e n a m a c r o n u c l e i

H I N S S E N , H . , J . D ' H A E S E

Synthetic f ibrils f rom Physarum actomyosin - seif assembly, Organization and contraction 1 3 2

S y n t h e t i s c h e F i b r i l l e n a u s i s o l i e r t e m S c h l e i m p i l z a c t o m y o s i n - E n t s t e h u n g , S t r u k t u r

u n d K o n t r a k t i o n

I N G L E , J . , siehe D E U M L I N G , B . 2 2 4

J A R A S C H , E . - D . , siehe F R A N K E , W . W . 2 8

J O H N S O N , R . P. C , siehe H E B A N T , C . 3 5 4

K A R T E N B E C K , J . , siehe F R A N K E , W . W . 2 8

K E E N A N , T H . W . , siehe E L D E R , J . H . 2 7 9

K E E N A N , T H . W . , siehe F R A N K E , W . W . 2 8

K L O P P S T E C H , K . , H . G . S C H W E I G E R

In vitro translation of poly ( A ) R N A from Acetabularia 3 9 4

I n v i t r o T r a n s l a t i o n v o n P o l y ( A ) R N A a u s A c e t a b u l a r i a

L I N D G R E N , A . , siehe S C H E L L E N S , J . P. M . 9 3

L J U B E S I C , N . , siehe S C H N E P F , E . 3 4 1

M E Y E R - R O C H O W , V . B . (Short Communicat ion) A tri-directional microvi l lus orientation in the monocel lular , distal rhabdom of a nocturnal beetle 4 7 6

I n d r e i v e r s c h i e d e n e n R i c h t u n g e n a n g e o r d n e t e M i k r o v i l l i i m monozellulären, d i s t a l e n R h a b d o m e i n e s n a c h t a k t i v e n Käfers

M O C I K A T , K . - H . , siehe H A U S M A N N , K . 4 6 9

V I C O N T E N T S V O L U M E 13

M O L L E N H A U E R , H . H . , D . J . M O R R E

Trans i t ion elements between endoplasmic reticulum and G o l g i apparatus in plant cells 297 U b e r g a n g s s t r u k t u r e n z w i s c h e n e n d o p l a s m a t i s c h e m R e t i k u l u m u n d G o l g i - A p p a r a t

i n p f l a n z l i c h e n Z e l l e n

M O O R , H . , siehe N I E D E R M E Y E R , W . 364

M O R R E , D . J . , siehe E L D E R , J . H . 279

M O R R E , D . J . , siehe M O L L E N H A U E R , H . H . 297

N I E D E R M E Y E R , W . , G . R . P A R I S H , H . M O O R

The elasticity of the yeast cell tonoplast related to its ultrastructure and chemical composi t ion. I. Induced swell ing and shr inking; a freeze-etch membrane study 364 D i e Elastizität d e s H e f e t o n o p l a s t e n i n b e z u g auf s e i n e U l t r a s t r u k t u r u n d c h e m i s c h e Z u s a m m e n s e t z u n g . I . I n d u z i e r t e s D e h n e n u n d S c h r u m p f e n ; M e m b r a n u n t e r s u c h u n g e n m i t d e r Gefrierätztechnik

N I E D E R M E Y E R , W .

The elasticity of the yeast cell tonoplast related to its ultrastructure and chemical composi t ion. II. Chemical and cytochemical investigations 380 D i e Elastizität d e s H e f e t o n o p l a s t e n i n b e z u g auf s e i n e U l t r a s t r u k t u r u n d c h e m i s c h e Z u s a m m e n s e t z u n g . I L C h e m i s c h e u n d c y t o c h e m i s c h e U n t e r s u c h u n g e n

N E H L S , R . , G . S C H A F F N E R (Short Communicat ion)

Specific negative staining of proteins in situ w i t h i ron tannin 285 P r o t e i n s p e z i f i s c h e r N e g a t i v k o n t r a s t i n s i t u m i t E i s e n - T a n n i n

P A R I S H , G . R . , siehe N I E D E R M E Y E R , W . 364

P A Y N E , H . L . , siehe R O B A R D S , A . W . 85

P E T T E , D . , siehe S C H U D T , C H . 74

P I G O N , A .

C e l l surface and medium condit ioning in Acanthamoeba culture 107

Zelloberfläche u n d M e d i u m - c o n d i t i o n i n g i n A c a n t h a m o e b a - K u l t u r e n

P I P A N , N .

Death and phagocytosis of epithelial cells in developing mouse kidney 435

T o d u n d P h a g o z y t o s e d e r E p i t h e l z e l l e n während d e r E n t w i c k l u n g d e r Mäuseniere

P O I S N E R , A . M . , siehe C O O K E , P . 442

P R Z E L E C K A , A . , A . S O B O T A

C a l c i u m dependent deposits at the plasma membrane dur ing development of the oocyte of Gal ler ia mellonella 182 Verändertes Ca-Bindungsvermögen d e s P l a s m a l e m m a s während d e r O o c y t e n -E n t w i c k l u n g v o n G a l l e r i a m e l l o n e l l a

R E I N H A R T Z , A . , siehe F R I E D L A N D E R , M . 1 7 1

C O N T E N T S V O L U M E 13 VII

R O B A R D S , A . W . , H . L . P A Y N E , B . E . S. G U N N I N G

Isolation of the endodermis using wall -degrading enzymes 85 I s o l i e r u n g d e r E n d o d e r m i s m i t Hilfe Z e l l w a n d - a b b a u e n d e r E n z y m e

R O S E N B A U E R , K . A . , siehe D I E T E R I C H , H . J . 57

S C H A F F N E R , G . , siehe N E H L S , R . 285

S C H E E R , U . , siehe F R A N K E , W . W . 401

S C H E L L E N S , J . P. M . , U . T . B R U N K , A . L I N D G R E N

Influence of serum on ruff l ing activity, pinocytosis and proliferation of i n v i t ro cultivated human glia cells 93 D e r Einfluß d e s S e r u m s auf Ruffling-Aktivität, P i n o c y t o s e u n d Z e l l v e r m e h r u n g v o n k u l t i v i e r t e n m e n s c h l i c h e n G l i a - Z e l l e n

S C H N E P F , E . , G . D E I C H G R Ä B E R , N . L J U B E S I C

T h e effects of colchicine, ethionine, and deuterium oxide on microtubules in y o u n g Sphagnum leaflets. A quantitative study 341 D i e W i r k u n g v o n C o l c h i c i n , Äthionin u n d D e u t e r i u m o x i d auf d i e M i k r o t u b u l i i n j u n g e n Sphagnum-Blättchen

S C H U D T , C H . , G . D A H L , M . G R A T Z L

Calc ium- induced fusion of plasma membranes isolated f rom myoblasts g r o w n in culture 211

C a l c i u m - i n d u z i e r t e F u s i o n v o n P l a s m a m e m b r a n e n , i s o l i e r t a u s M y o b l a s t e n i n K u l t u r

S C H U D T , C H . , D . P E T T E

Influence of monosaccharides, medium factors and enzymatic modif icat ion on fusion of myoblasts in vitro 74 Einflüsse v o n M o n o s a c c h a r i d e n , M e d i u m f a k t o r e n u n d e n z y m a t i s c h e r M o d i f i k a t i o n

auf d i e F u s i o n v o n M y o b l a s t e n i n v i t r o

S C H W A B , D . , siehe G R A T Z L , M . 199

S C H W E I G E R , H . G . , siehe K L O P P S T E C H , K . 394

S I N C L A I R , J . , siehe D E U M L I N G , B . 224

S O B O T A , A . , siehe P R Z E L E C K A , A . 182

SPIESS, E . , siehe A M E L U N X E N , F . 233

SPIESS , E . , siehe A M E L U N X E N , F . 260

SPIESS, E . , E . T H I O T I A N G N I O , F . A M E L U N X E N

Untersuchungen zur Z e l l w a n d b i l d u n g . II. Nachweis der Zellwandsynthese und der Synthese von Cellulose, Hemicellulose und Pektin in den Ranken v o n Cucurb i ta maxima L . 251 I n v e s t i g a t i o n s o n c e l l w a l l f o r m a t i o n . II. P r o v e f o r c e l l w a l l s y n t h e s i s a n d s y n t h e s i s of c e l l u l o s e , h e m i c e l l u l o s e a n d p e c t i n i n t e n d r i l s of C u c u r b i t a m a x i m a L .

S P R I N G , H . , siehe F R A N K E , W . W . 401

S T A D L E R , J . , siehe F R A N K E , W . W . 28

S T I E M E R L I N G , R. , siehe S T O C K E M , W . 158

VIII C O N T E N T S V O L U M E 13

S T O C K E M , W . , R . S T I E M E R L I N G

Intracellular segregation of endocytotically ingested substances 1 5 8 Intrazelluläre S e g r e g a t i o n e n d o c y t o t i s c h a u f g e n o m m e n e r S u b s t a n z e n

S T O C K E R T , J . C .

Metachromatic staining of nucleolar differentiations by toluidine blue-molybdate in Chironomus salivary glands 191 M e t a c h r o m a t i s c h e Färbung v o n N u c l e o l u s - D i f f e r e n z i e r u n g e n i n C h i r o n o m u s -Speicheldrüsen d u r c h T o l u i d i n b l a u - M o l y b d a t

T H I O T I A N G N I O , E . , siehe A M E L U N X E N , F . 2 3 3

T H I O T I A N G N I O , E . , siehe A M E L U N X E N , F . 2 6 0

T H I O T I A N G N I O , E. , siehe SPIESS , E . 2 5 1

T I M M I S , J . N . , siehe D E U M L I N G , B. 2 2 4

T R E N D E L E N B U R G , M . F . , siehe F R A N K E , W . W . 4 0 1

W O L B U R G , H .

Differences in distributions of 3 H-proline and 3 H-uridine-labelled Compounds in the fish visual System during ouabain-induced Wal ler ian degeneration 1 3

U n t e r s c h i e d l i c h e V e r t e i l u n g v o n 3 H - P r o l i n - u n d 3 H - U r i d i n - m a r k i e r t e n V e r b i n d u n g e n i m S e h - S y s t e m d e r K a r a u s c h e während d e r d u r c h O u a b a i n i n d u z i e r t e n W a l l e r s c h e n D e g e n e r a t i o n

W U N D E R L I C H , F . , siehe H E R L A N , G . 2 9 1

Z E N T G R A F , H . , siehe F R A N K E , W . W . 4 0 1

BOOK REVIEWS

B O S C H K E , F. (Hrsg.): Photochemistry 1 6 9

D O S E , K . , H . R A U C H F U S S : Chemische Evolut ion und der Ursprung lebender Systeme 1 7 0

D R E W S , U . : Cholinesterase in Embryonic Development 4 8 2

F L O R K I N , M . , E. H . S T O T Z (Hrsg.): Comprehensive Biochemistry 168

H O L T , D . B. , M . D . M U I R , P. R . G R A N T , I. M . B O S W A R V A (Hrsg.) : Quantitat ive Scanning

Electron Microscopy 1 6 8

M O D I S , L . : Topo-opt i ca l Investigations of Mucopolysaccharides ( A c i d Glycosaminoglycans) 4 8 2

O R C I , L . , A . P E R R E L E T : Freeze-Etch Histo logy 1 7 0

C Y T O B I O L O G I E 13, 199-210 (1976) § Wissenschaftliche Verlagsges. m b H • Stuttgart

The effect of microtubular inhibitors on secretion from liver into blood plasma and bile

Die Wirkung mikrotubulärer Hemmstoffe auf die Sekretion der Leber in das Blutplasma und die Galle

M A N F R E D G R A T Z L l ) a n d DIETER SCHWAB

Fachbereich Theoretische M e d i z i n der Universität des Saarlandes,

Homburg/Saar , Germany

Received A p r i l 10, 1976

A b s t r a c t

L i v e r - m i c r o t u b u l a r i n h i b i t o r s - s e c r e t i o n - p l a s m a - b i l e

Injection of either colchicine or vinblastine into rats in v i v o reduced the levels of coagula-t ion factors V and VII and triglycerides i n b lood plasma. In contrast, lumicolchicine, a structural isomer of colchicine which does not disrupt microtubules, had no effect on the levels of coagulation factors or triglycerides i n b lood plasma. The reduction in the levels of proteins and triglycerides in b lood is accompanied by an accumulation of vesicles wi th in the hepato-cyte, as seen under a light microscope. These vesicles have been identified as Golgi-derived vesicles by electron microscopy. Golgi-derived secretory vesicles isolated f rom rat liver were found to contain more protein, coagulation factor V and triglycerides after injection of colchicine or vinblastine. These findings suggest an involvement of microtubules in secretion from liver into b lood plasma. O n the other hand, excretion of bi l irubin-glucuronides and f luid into bile was not affected by these chemicals. This indicates that glucuronides are not transferred to the extracellular space by vesicular transport.

Introduction

T h e inter ference of m i c r o t u b u l a r i n h i b i t o r s w i t h the secre t ion of p r o t e i n s a n d l i p o -p r o t e i n s f r o m the hepatocyte i n t o the b l o o d p l a s m a s t u d i e d i n d i f ferent l a b o r a t o r i e s [11, 12, 19, 22 , 23] suggest an i n v o l v e m e n t of the m i c r o t u b u l a r System i n this process . C o l c h i c i n e a n d v i n b l a s t i n e reduces the s e r u m t r i g l y c e r i d e l eve l i n rats a n d c o n c o m i t a n t l y

l) Ass . Prof. D R . M . G R A T Z L , Department of Physiological Chemistry, University of the Saarland, D-6650 Homburg/Saar , Germany.

200 M A N F R E D G R A T Z L and D I E T E R S C H W A B

inhibits the release of proteins associated with serum very low density lipoproteins (VLDL) and serum high-density lipoproteins (HDL) [22]. As shown recently [19] micro­tubular inhibitors decrease the secretion of albumin and other unidentified proteins in vivo and in rat liver slices. Also, hepatocytes, incubated in Suspension with colchicine, released fibrinogen at reduced rates [4], Since these drugs do not impede the early steps in the secretory process, namely, the synthesis and the movement of secretory products from the rough endoplasmic reticulum to the Golgi complex, it was concluded that they inhibit discharge of proteins and lipids accumulated in Golgi-derived secretory vesicles [11, 12, 19, 22, 23]. These findings were further strengthened by morphological studies, since the accumulation of secretory vesicles filled with V L D L particles can be easily identified by electronmicroscopy [11, 12, 19, 22].

The antimitotic drugs colchicine and vinblastine are characterized by a specific binding to a protein, named tubulin, which in its polymeric State builds up microtubules. Therefore most of the effects evoked by these agents have been ascribed to micro­tubules.

The final Steps of secretion, which seem to be impaired by microtubular inhibitors, involve the fusion of Golgi-derived vesicles with the plasma membrane. Therefore the interaction of the drugs colchicine and vinblastine with membranes is of particular interest. Nonspecific binding of colchicine has been reported for subcellular membranes of different tissues including liver (cf. [21]). Colchicine also seems to affect the distribu-tion of intramembraneous particles in T e t r a h y m e n a p y r i f o r m i s [25] as well as redistribu-tion of lectin binding sites on the membrane of leukocytes [17].

To distinguish whether the effects of antimitotic drugs on the secretion of the liver are related to microtubules or are due to interaction with membranes, we used lumi-colchicine which binds to membranes nearly as well as does colchicine [21] but lacks specific binding to tubulin [24]. The action of this structural isomer of colchicine on the secretion of triglycerides and proteins from rat liver was investigated and compared with that of colchicine and vinblastine. To study whether the secretion of proteins other than albumin [19], fibrinogen [4], or those associated with V L D L or H D L [22] are reduced after treatment of rats with microtubular inhibitors, we determined the levels of coagulation factors in the blood plasma. The accumulation of triglycerides and proteins inside the hepatocyte was followed by isolation of Golgi-derived secretory vesicles from rats injected with antimitotic drugs. To obtain Information about the pathway leading from the hepatocyte to the bile, the excretion of fluid and bilirubin glucuronide was measured after injection of microtubular inhibitors.

Materials and methods

Female Sprague Dawley rats (180 to 220 g) were used throughout. The rats were fasted for 12 hours and anaesthetized with hexobarbital (100 mg/kg) by intraperitoneal injection. The general bile duct and the right jugular vein were cannulated with P V C tubing. T o prevent hyperthermic alteration of the bile flow [10] the animals were warmed by means of a heating lamp. 0.9 ml blood was withdrawn every hour with a syringe containing 0.1 ml 0.1 M sodium citrate. In order to keep the blood volume constant, 0.9 ml 0.9 °/o sodium chloride was injected after each withdrawal. In separate experiments, after the first sampling the replacement Solution contained colchicine (0.5 mg/100 g body weight, E . M e r c k , Darmstadt, Germany) or vinblastine (1 mg/100 g body weight, a gift from E l i L i l l y G m b H , Giessen, Germany) . The remaining replacement Solutions contained only sodium chloride. After removal of cells by centrifugation plasma was assayed for Pro thrombin time, factor V , factor VII and triglycerides:

M i c r o t u b u l a r inhibitors and secretion f rom liver 201

Pro thrombin time was determined by adding to 0.1 m l of prewarmed plasma 0.2 m l of 1 : 1 mixture of 25 m M C a C U and thromboplast in (Hof fmann-La Roche, Basel, Switzerland). Factor V activity was tested by m i x i n g in prewarmed test tubes 0.1 m l of a Solution of plasma deficient in factor V (Behring Werke A G , M a r b u r g , Germany) w i t h 0.1 m l plasma (diluted 1 : 20 in 0.05 M diethyl barbiturate-acetate buffer, p H 7.6) incubating for exactly 30 sec. at 37° C and then adding 0.2 ml prewarmed calcium thromboplast in Solution (Behring Werke A G , M a r b u r g , Germany) . Factor VH-act iv i ty was determined in a s imilar way using factor VII -deficient plasma (obtained f r o m M e r z + Dade A G , Bern, Switzerland) and a 1 : 10 d i lut ion of the plasma to be tested in a Solution containing 2.8 X 10" 2 M sodium barbital , 1.25 M X 10" 1 M sodium chloride, buffered at p H 7.35. T o Start the reaction, 0.2 m l of 1 : 1 mixture of 25 m M C a C b and thromboplast in (Merz + Dade A G , Basel, Switzerland) were added. T h e time of clot format ion after the addit ion of C a 2 + was measured in a coagulometer according to S C H N I T G E R and G R O S S (Heinrich A m e l u n g , 492 Lemgo-Brake, Germany) and compared to a Standard curve prepared by testing dilutions of normal pooled plasma (mixture of five plasmas) and plott ing their clotting times on log-log paper. 100 units of coagulation factors were taken to be the amount present in 1 m l of pooled plasma.

Triglycerides were assayed by the enzymatic determination of glycerol obtained after alkaline hydrolysis [5].

Bile was collected in cooled and darkened Polyethylene vessels containing 0.2 m l 10 m M E D T A . The vessels were changed every hour. Azopigments derived f r o m conjugated bile pigments were analyzed by coupl ing w i t h the diazonium salt of ethyl anthranilate [9].

Golgi- fract ions were isolated f r o m rat liver homogenates of control rats and of drug-ireated rats four hours after intraperitoneal injection of 0.5 mg/100 g body weight colchicine or 1 mg/100 g body weight vinblastine. As a slight modif icat ion of the isolation procedure [6] we buffered all Solutions with 0.01 M cacodylate at p H 7.4. Lumicolchic ine was prepared by Irradiation of colchicine w i t h ultraviolet light [24]. Protein was determined as described [13] using crystalline bovine serum albumin as a Standard. A l l chemicals not specified were of the purest grade commercial ly available.

For electron microscopic investigations, l iver slices of both the control rats and those injected four hours previously w i t h microtubular inhibitors were f ixed at r o o m temperature by treatment w i t h 2 % glutaraldehyde in 0.25 M sucrose, 0.01 M cacodylate at p H 7.4 for 60 min. After postf ixation in 1 % osmium tetroxide in the same buffer the slices were dehydrated w i t h ethanol and embedded in E p o n 812. T h i n sections were cut w i t h a Reichert ultramicrotome, stained w i t h uranyl acetate and lead citrate in the conventional manner and

Figure 1 see page 202, Figure 2 see page 203.

Fig. 1. Semithin-sectioned rat hepatocytes observed under the light microscope. - H Hepato­cyte. - N Nucleus . - E Erythrocytes. - a. Hepatocytes of control rats. O n l y a few l i p i d droplets (arrows) are visible in the dense homogenous cytoplasm. - 720 X . - b. Hepatocytes after treatment w i t h colchicine. N o t e the considerable increase of the number of the l ip id droplets (arrows) in the cytoplasm of each cell . - 720 X . - c. Hepatocytes after treatment w i t h v i n ­blastine. In these cells too the amount of l i p i d droplets (arrows) is higher than in control c e l l s . - 720 X .

Fig. 2. T w o adjacent hepatocytes of vinblastine-treated rats. - a. Secretory vesicles ( a r r o w s ) of varying size assembled in Clusters can be demonstrated throughout the cytoplasm. -N Nucleus. - M M i t o c h o n d r i a . - E R Endoplasmic ret iculum. - g Glycogen. - B Bile capil lary. -10 500 X . - b. In higher magnif icat ion inside the secretory vesicles (V) beside the V L D L particles electron dense material ( a r r o w s ) presumably secretory protein can be seen. - M M i t o -chondrion. - g Glycogen. - 50 000 X .

202 M A N F R E D G R A T Z L and D I E T E R S C H W A B

M i c r o t u b u l a r inhibitors and secretion f r o m liver

Legend see page 201.

Fig . 3. Hepatocytes of colchicine-treated rats. Clusters of secretory vesicles ( a r r o w s ) are scattered throughout the cytoplasm. - N Nucleus. - E R Endoplasmic reticulum. - g Glycogen. - 10 500 X .

M i c r o t u b u l a r inhibitors and secretion f rom liver 205

c x a m i n e d w i t h a Siemens E lmiskop 101. For light microscopy epon embedded material was cut into sections of 1 um and stained wi th methylene blue/azur according to R I C H A R D S O N . Photographs were taken wi th a Zeiss Photomicroscope II on Agfapan 25 f i l m .

Results

In comparison to control animals the injection of colchicine or vinblastine into rats led to an increase of lipid droplets within the hepatocyte as seen under a light microscope (Fig. 1 a to c). Electron microscopic examination of the same liver section revealed that most of these droplets contain VLDL-particles (Fig. 2, 3). In the untreated hepatocyte, secretory vesicles filled with V L D L particles are found in the Golgi-regions or scattered throughout the cytoplasm. Secretory vesicles discharge their content into the space of Disse and can easily be detected there in controls.

Microtubules were found in the peripheral cytoplasm of the hepatocytes as well as close to the nucleus. However, the number of identifiable microtubules was so small that changes in their amounts with or without drugs could not be determined.

Vinblastine treatment leads to an apparent increase of secretory vesicles containing V L D L particles inside the hepatocyte (Fig. 2) and a concomitant decrease of V L D L particles in the space of Disse. These secretory vesicles vary in size and seem to contain more electron dense material than do those of the controls. Presumably this represents enclosed secretory proteins. This material was also seen in the livers of colchicine-treated rats, but was most prominent after treatment with vinblastine. These observa-tions indicate an impairment in the transfer of secretory products from the intracellular to the extracellular fluid.

It has been demonstrated that vinblastine leads to a conversion of microtubules to paracrystals [1, 14]. In the present investigation paracrystals were observed occasionally in Kupffer cells but never in hepatocytes.

The ultrastructure of hepatocytes from rats treated with colchicine was similar to that observed in rats after injection of vinblastine (cf. Figs. 2, 3). The amount of secretory vesicles containing V L D L particles increased and very few VLDL-particles were seen in the space of Disse which often seems to be collapsed.

The VLDL-particles shown to accumulate within the hepatocyte after injection of microtubular inhibitors are rieh in triglycerides [8]. As demonstrated in Figure 4 the increase of the amount of the secretory vesicles in the hepatocyte is aecompanied by a decrease of plasma triglyceride levels in the intact rat. This finding aecords with data presented by others [11, 12, 22, 23]. To investigate whether lumicolchicine, a drug known not to interfere with microtubules, exhibits the same effect, this structural isomer of colchicine was prepared and injected into rats. After injection of this drug (0.5 mg/ 100 g body weight) the levels of triglycerides did not differ from those of the control rats during the experiment (Fig. 4). The secretion of proteins was also unaffected by lumicolchicine as followed by assay of the coagulation factors (Prothrombin time) in plasma (Fig. 5). Colchicine and vinblastine, in contrast, led to a drastic decrease in the levels of coagulation factors.

Assaying Prothrombin time, the sum of several coagulation factors is determined. Those exhibiting the shortest half-life should decay rapidly if secretion is blocked. Therefore we assayed factor V and factor VII, which are known to fall to half in the plasma within hours [20] after administration of colchicine and vinblastine to rats (Figs. 6, 7). Compared to controls both drugs diminish the amount of these two coagulation factors present in blood plasma.

2 >

50

PI o O

M i c r o t u b u l a r inhibitors and secretion f rom liver 207

To examine, whether the decreased levels of triglycerides and coagulation factors in plasma in fact are due to an impaired secretion, we isolated Golgi-derived secretory vesicles before and after treatment with microtubular inhibitors. Golgi fraction 1 represents primarily trans-Golgi elements from the secretory Golgi face [2, 6, 7] and these vesicles should accumulate inside the hepatocyte if secretion is blocked. Colchicine and vinblastine indeed cause an increase in the amount of secretory vesicles unable to discharge their content into the extracellular space (Tab. I). To analyze the intraluminal portion, which represents the material to be exported, the secretory vesicles were opened using a pH-jump method as follows: After titration of the Suspension to p H 9 . 5 the vesicles were incubated for 20 min. at 37° C and than titrated back to p H 7.4. This represents a slight modification of a procedure already successfully applied to this fraction [6]. To remove Golgi membranes we centrifuged for 60 min. at 105 000 g and analyzed the supernatant for protein, triglycerides and coagulation factors. All secretory products were found to be at least doubled in isolated Golgi-derived secretory vesicles after treatment with colchicine or vinblastine (Tab. I). Unfortunately factor VII could not be determined in the extracted Golgi fraction because of the considerable inactiva-tion of this protein during the incubation in alkaline medium.

Tab. I. Effect of colchicine or vinblastine (4 hours, intraperitoneally) on the recovery of G o l g i I fraction and intra luminal Compounds. Average of three experiments.

G o l g i 1 Intraluminal

Triglycerides J A M o l / g liver

Protein mg/g liver

Protein mg/g liver

Factor V U / g liver

Triglycerides u. M o l / g l iver

Control 0.037 0.049 0.030 0.43 0.031

Vinblastine 0.131 0.090 0.069 0.79 0.097

Colchicine 0.104 0.088 0.061 0.89 0.082

Fig. 8. Effect of colchicine or vinblastine on bile f low and bil irubin-glucuronide excretion. After a control period of one hour colchi­cine (0.5 mg/100 g body weight), vinblastine (1 m g / 100 g body weight) or the solvent of these drugs, 0.9 % N a C l (control) were injected intravenously. Each point is the mean value of 4 experiments (SEM).

3.0

£ 2.0

1.0

o Control

^ T Colchicine

Vinblastine

Vinblastine

Time (hours)

Effect of Antimicrotubular Drugs on Fluid - and Bilirubinglucuronide Excretion in Bile.

208 M A N F R E D G R A T Z L and D I E T E R S C H W A B

The lack of effect of colchicine on biliary excretion of phospholipids and cholesterol indicates that this transfer is not dependent on vesicular transport [22]. To find out whether this holds also for glucuronides we determined the excretion of fluid and bilirubin glucuronide into the bile. As shown in Figure 8 bile flow and transfer of bilirubin glucuronide is unchanged by microtubular inhibitors. Therefore, glucuronides, like biliary lipids, are not dependent on vesicular transport.

Discussion

The experiments here reported show that injection of microtubular inhibitors such as colchicine or vinblastine into rats in vivo decreases the secretion of coagulation factors from the liver. This decrease in secretion is accompanied by accumulation of secretory products within the hepatocyte. One of the coagulation factors tested could be traced back to its intracellular storage site. Together with undischarged triglycerides and proteins, coagulation factor V was found to have increased in the fraction of isolated secretory vesicles. Similar results were reported recently by others [19] who were able to show an increased accumulation of albumin inside secretory vesicles isolated from rat liver after injection of colchicine. From these studies and the Observa­tion of an increased protein content of isolated secretory vesicles as described [19] and as confirmed in this paper, it may be concluded that all plasma proteins except v-globulins [15], accumulate in secretory vesicles inside the hepatocyte after administra-tion of microtubular inhibitors. This conclusion is in accord with the Observation of decreased secretion of fibrinogen by isolated hepatocytes [4] and of decreased levels of proteins associated with H D L and V L D L [22] in the plasma of rats treated with microtubular inhibitors. Also, it has been reported [11] that direct application of micro­tubular inhibitors to perfused mouse liver resulted in lowered release of albumin and other proteins into the perfusate. This study shows that the accumulation of secretory products can be detected by observations using a light microscope. However, under the higher magnification of an electron microscope, the nature of the vesicles observed is easily revealed by their load of VLDL-particles (cf. Figs. 1 b, c, 2, 3). The electron dense material observed inside the secretory vesicles in this study may represent accumulated proteins (Fig. 2 b).

In most of the recent studies, effects upon secretion observed after administration of colchicine or vinblastine were attributed to its effect on microtubules [11, 12, 22]. However, experiments concerning the binding of these drugs to subcellular membranes [21] cast some doubt on this concept especially since the release of proteins and lipids from the hepatocyte involves specific functions of the membrane of Golgi-derived vesicles as well as the plasma membrane. To examine the possible action of these drugs on subcellular membranes we used the technique of freeze-cleaving electron microscopy (G. D A H L and M . G R A T Z L , unpublished observations). Changes in the array of intra-membranous particles as a function of temperature observed in membranes by others [25], or by interaction with colchicine or vinblastine [25] could not be observed in the contiguous surface of the plasma membrane. The microvillar surface, which would have been most interesting to observe, was unfortunately only exposed in small areas by this technique. Therefore the array of intramembranous particles in that part of the plasma membrane specialized for secretion could not be adequately determined. Thus, freeze-cleaving of hepatic tissue provided no evidence for the inhibitory effect of antimicro-tubular drugs on secretion by interaction with membranes.

Lumicolchicine is a structural isomer of colchicine, which behaves like colchicine in its nonspecific binding to intracellular membranes but unlike colchicine lacks specific

M i c r o t u b u l a r inhibitors and secretion from liver 209

binding to tubulin [21]. If the secretory process is affected by interaction of colchicine with membranes, comparable effects should occur with lumicolchicine. Since this was not the case our results favour the involvement of microtubules in the secretory processes studied.

The relationship between secretion and the microtubular System still remains to be clarified. Because of the relatively small number of morphologically identifiable micro­tubules in an untreated hepatocyte we could not relate the amount of microtubules after treatment with colchicine or vinblastine to the decrease in secretion as others have suggested [11, 12, 22]. In addition, the paracrystalline structures which appear if microtubules are treated with vinblastine [1, 14] were never observed in the liver parenchyma cells, although they were occasionally seen in Kupffer cells. Definitive proof for the existence of microtubules in the liver has been obtained from recent studies showning that tubulin, which binds antimicrotubular drugs is present in liver homo-genates and can be purified therefrom [18].

The most striking Observation of the present study is that drugs such as colchicine and vinblastine known to interfere with vesicular transport in the hepatocyte [11, 12, 19, 22, 23], do not inhibit fluid or bilirubin-glucuronide release into the bile. From the latency of the enzyme UDP-glucuronyl-transferase it has often been speculated that this enzyme is arranged in the endoplasmic membrane to allow the release of products into the intracisternal Space. Due to the vectorial transfer during their formation, glucuronides could be carried to the secretory surface in membrane-bound vesicles (cf. [3]). Our results, however, clearly show that the transfer of glucuronides to the extracellular fluid is not dependent on vesicular transport. Studies concerning the permeability of glucuronides synthesized by isolated microsomal membranes [16] also suggest that these products are released into the cytoplasm. Another example of non-vesicular transport from the hepatocyte to the bile is the secretion of lecithin and cholesterol [22].

A c k n o w l e d g e m e n t s . W e thank D R . G . D A H L for the Performance of freeze-cleaving electron microscopy and its interpretation. The authors are indebted to D R . P. T R A Y L O R for expert help during the preparation of this manuscript.

Parts of this paper have been presented at the lOth Meet ing of the Federation of European Biochemical Societies, Paris, July 20-25, 1975 and the "International Symposium on micro­tubules and microtubular inhib i tors" , Beerse (Belgium) September 2-5, 1975.

This w o r k was supported by the Deutsche Forschungsgemeinschaft, mainly by the Sonder­forschungsbereich 38 " M e m b r a n f o r s c h u n g " .

References

[1] B E N S C H , K . G . , and S. E. M A L A W I S T A : M i c r o t u b u l a r crystals in mammalian cells. J . Cel l Biol . 40, 95-107 (1969).

[2] B E R G E R O N , J . J . M . , J . H . E H R E N R E I C H , P. S I E K E W I T Z , and G . E. P A L A D E : G o l g i fractions

prepared f rom rat liver homogenates. II. Biochemical characterization. J . Ce l l B i o l . 59, 73-88 (1973).

[3] B E R R Y , C , and T . H A L L I N A N : " C o u p l e d transglucuronidat ion" : A new tool for studying the latency of U D P - g l u c u r o n y l transferase. FEBS Letters 42, 73-76 (1974).

[4] C R A N E , L . J . , and D . L . M I L L E R : Synthesis and secretion of fibrinogen and albumin by isolated rat hepatocytes. B iochim. Biophys. Res. C o m m . 60, 1269-1276 (1974).

[5] E G G S T E I N , M . , and E. K U H L M A N N : Triglyceride and Glyceride. In: H . U . B E R G M E Y E R (ed.):

Methoden der enzymatischen Analyse, p. 1871-1878, 3. Auflage. Verlag Chemie, W e i n h e i m / Bergstraße 1974.

210 M A N F R E D G R A T Z L and D I E T E R S C H W A B

[6] E H R E N R E I C H , J . H . , J . J . M . B E R G E R O N , P . S I E K E V I T Z , and G . E . P A L A D E : G o l g i fractions prepared f r o m rat liver homogenates. I. Isolation procedure and morphologica l characteriza-t ion. J . C e l l B i o l . 59, 45-72 (1973).

[7] F A R Q U H A R , M . G . , J . J . M . B E R G E R O N , and G . E. P A L A D E : Cytochemistry of G o l g i fractions

prepared f r o m rat liver. J . C e l l B i o l . 60, 8-25 (1974).

[8] G L A U M A N N , H . , A . B E R G S T R A N D , and J . L . E . E R I C S S O N : Studies on the synthesis and intra­cellular transport of l ipoprotein particles in rat liver. J . C e l l B i o l . 64, 356-377 (1975).

[9] H E I R W E G H , K . P . M . , G . P . V A N H E S S , P . L E R O Y , F . P . V A N R O Y , and F . H . J A N S E N : Hetero-

geneity of bile pigment conjugates as revealed by chromatography of their ethyl anthranilate azopigments. Biochem. J . 120, 877-890 (1970).

[10] K A R U P , W . , and J . A . L A R S E N : T h e effect of slight hyperthermia on liver funct ion as measured by the el imination rate of ethanol, the hepatic uptake and excretion of indocyanine green and bile format ion. Ac ta Physiol . Scand. 84, 396-407 (1972).

[11] L E M A R C H A N D , Y . , C . P A T Z E L T , F . A S S I M A C O P O U L O S - J E A N N E T , E . G . L O T E N , and B . J E A N -

R E N A U D : Evidence for a role of the microtubular System in the secretion of newly synthesized a l b u m i n and other proteins by the liver. J . C l i n . Invest. 53, 1515-1517 (1974).

[12] L E M A R C H A N D , Y . , A . S I N G H , F . A S S I M A C O P O U L O S - J E A N N E T , L . O R C I , C . R O U I L L E R , and

B . J E A N R E N A U D : A role for the microtubular System i n the release of very l o w density l ipo­protein by perfused mouse livers. J . B i o l . C h e m . 248, 6862-6870 (1973).

[13] L O W R Y , O . H . , N . J . R O S E B R O U G H , A . L . F A R R , and R . J . R A N D A L L : Protein measurement

w i t h the F o l i n phenol reagent. J . B i o l . C h e m . 193, 265-275 (1951).

[14] M A R A N T Z , R . , M . V E N T I L L A , and M . L . S H E L A N S K I : Vinblast ine induced precipitations of

microtubule proteins. Science 165, 498-499 (1969).

[15] M I L L E R , L . L . , and W . F . B A L E : Synthesis of a l l protein fractions except gamma globulins by the l iver. J . E x p . M e d . 99, 125-132 (1954).

[16] N I L S S O N , R . , E. P E T E R S O N , and G . D A L L N E R : Permeability of microsomal membranes

isolated f r o m rat liver. J . C e l l B i o l . 56, 762-776 (1973).

[17] O L I V E R , J . M . , T . E . U K E N A , and R . D . B E R L I N : Effects of Phagocytosis and colchicine

o n the distr ibution of lect in-binding sites on cell surfaces. Proc. N a t l . A c a d . Sei. (USA) 71, 394-398 (1974).

[18] P A T Z E L T , C , A . S I N G H , Y . L E M A R C H A N D , L . O R C I , and B . J E A N R E N A U D : Colchic ine-b inding

protein of the liver. Its characterization and relation to microtubules. J . C e l l B i o l . 66, 609-620 (1975).

[19] R E D M A N , M . , D . B A N E R J E E , K . H O W E L L , and G . E. P A L A D E : Colchic ine inh ib i t ion of

plasma protein release f r o m rat hepatocytes. J . C e l l B i o l . 66, 42-59 (1975).

[20] R E G O E C Z I , E . : Regulat ion of the metabolism of proteins involved in b l o o d coagulation. In: M . A . R O T H S C H I L D and T . W A L D M A N N (eds.): Plasma Protein M e t a b o l i s m , pp . 339-441. A c a d e m i c Press, N e w Y o r k and L o n d o n 1970.

[21] S T A D L E R , J . , and W . W . F R A N K E : Characterization of the colchicine b inding of membrane fractions f r o m rat and mouse liver. J . Ce l l B i o l . 60, 297-303 (1974).

[22] S T E I N , O . , L . S A N G E R , and Y . S T E I N : Colchicine induced inhib i t ion of l ipoprote in and prote in secretion into the serum and lack of interference w i t h secretion of bi l iary phosphol ipids and cholesterol by rat liver i n vivo . J . Ce l l B i o l . 62, 90-103 (1974).

[23] S T E I N , O . , and Y . S T E I N : Colchicine-induced inhib i t ion of very l o w density l ipoprotein release by rat livers in vivo . B iochim. Biophys. A c t a 306, 142-147 (1973).

[24] W I L S O N , L . , J . R. B A M B U R G , S . B . M I Z E L , L . M . G R I S H A M , and K . M . C R E S W E L L : Inter­

action of drugs w i t h microtubules proteins. Fed. Proc. 33, 158-166 (1974).

[25] W U N D E R L I C H , R. , R . M Ü L L E R , and V . S P E T H : Direct evidence for colchicine-induced

impairement in the mobi l i ty of membrane components. Science (Washington D . C.) 182, 1136-1139 (1973).