European Journal of Cell Biq|f>gy
Volume 13 • Number 1 > June 1&7#
m
III
C O N T E N T S V O L U M E 13
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 R a n k e n v o n C u c u r b i t a m a x i m a L .
I n v e s t i g a t i o n s o n cell 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
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 .
233
observations
251
A M E L U N X E N , F., siehe S P I E S S , E .
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. A u t o r a d i o g r a p h i e v o n Dünnschnitten
und biochemische Analyse isolierter D i c t y o s o m e n der R a n k e n
von C u c u r b i t a m a x i m a L .
260
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.
Variations ultrastructurales au sein des cellules epitheliales mesenteriques
d'Aeshna cyanea (Insecte, Odonate) en fonction de l a prise de nourriture
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
(Insecte, O d o n a t a )
451
cyanea
BARANOWSKI, Z .
Three-dimensional analysis o f movement i n Physarum p o l y c e p h a l u m plasmodia
Dreidimensionale Analyse der Bewegung bei Physarum
118
polycephalum-Plasmodien
B R U N K , U . T . , siehe S C H E L L E N S , J . P . M .
93
C H E S C O E , D . , siehe D U C K E T T , J . G .
322
C O O K E , P., A . M . P O I S N E R
M i c r o f i l a m e n t s i n bovine adrenal chromaffin cells
M i k r o f i l a m e n t e i n d e n chromaffinen
Zellen der
442
Rinder-Nebenniere
D A H L , G . , siehe S C H U D T , C H .
211
D E I C H G R Ä B E R , G . , siehe S C H N E P F , E .
341
D E U M L I N G , B . , J . SINCLAIR, J . N . TIMMIS, J . INGLE
Demonstration of satellite D N A components i n several plant species w i t h
the Ag -Cs2S04 gradient technique
+
V o r k o m m e n v o n Satelliten-DNS i neinigen Pflanzenarten bei
d e r Ag -Cs2SÖ
Gradiententechnik
224
Anwendung
+
4
DEXHEIMER, J .
Etüde de la secretion de mucilage par les cellules des glandes digestives de Drosera
(D. r o t u n d i f o l i a L . ; et D . capensis L.).
A p p l i c a t i o n de quelques techniques cytochimiques
307
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 .
D I E T E R I C H , C . E . , siehe D I E T E R I C H , H . J .
132
57
CONTENTS
IV
V O L U M E 13
D I E T E R I C H , H . J., K . A . ROSENBAUER, C . E . D I E T E R I C H
Das Blutgefäßsystem des Pecten oculi beim H a u s s p e r l i n g (Passer domesticus).
A r c h i t e k t o n i k u n d U l t r a s t r u k t u r nach licht-, transmissions- u n d rasterelektronenmikroskopischen Untersuchungen
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
electron microscopy
57
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 m i c r o a n a l y t i c a l 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
Untersuchung
der Spermatogenese i mL e b e r m o o s Phaeoceros laevis (L.) Prosk.
ELDER, J. H . , J. MORRE, T H . W .KEENAN
(Short C o m m u n i c a t i o n )
D i s t r i b u t i o n of glycoproteins a m o n g subcellular fractions f r o m rat liver
279
Glycoprotein-Muster v o n Zellfraktionen aus Rattenleber
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 l o c a l i z a t i o n of o u a b a i n
sensitive p-nitrophenyl Phosphatase activity i n the salt-secreting l a c r y m a l glands
of the marine turtle C h e l o n i a mydas
Rasterelektronenmikroskopische Darstellung derInterzellularspalten u n d Lokalisation
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 nd e n lacrimalen Salzdrüsen d e r Meeresschildkröte C h e l o n i a m y d a s
1
F R A N K E , W . W . , T H . W . K E E N A N , J. STADLER, R . G E N Z , E . - D . JARASCH, J. K A R T E N B E C K
N u c l e a r membranes f r o m m a m m a l i a n liver. V I I . Characteristics of highly p u r i f i e d
nuclear membranes i n comparison w i t h other membranes
28
Zusammensetzung hochgereinigter Kernmembranfraktionen ausder Rattenleber
i m Vergleich mit anderen
Membranen
F R A N K E , W . W . , U . SCHEER, M . F . T R E N D E L E N B U R G , H . SPRING, H . Z E N T G R A F
Absence of nucleosomes i n 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
A t y p i c a l cycle of the nucleolus i n spermatocytes of the insect L o c u s t a m i g r a t o r i a
Besonderheiten des Nucleolus-Zyklus
171
inSpermatocyten der Wanderheuschrecke
Locusta migratoria
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 C o m m u n i c a t i o n )
N u c l e o l a r development i n induced binucleate cells of A l l i u m cepa L .
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
G O E R T E N M I L L E R J R . , C . C , siehe E L L I S , R . A .
G R A T Z L , M . , siehe S C H U D T , C H .
163
Zwiebelzellen
1
211
V
CONTENTS VOLUME 13
G R A T Z L , M . , D . SCHWAB
T h e effect of m i c r o t u b u l a r inhibitors o n secretion f r o m liver into b l o o d plasma and bile
D i e W i r k u n g mikrotubulärer
Hemmstoffe
199
auf d i e S e k r e t i o n d e r L e b e r
in das Blutplasma u n ddie Galle
G U N N I N G , B . E . S., siehe R O B A R D S , A . W .
85
HAUSMANN, K., K . - H . MOCIKAT
Direct evidence for a surface coat o n the plasma membrane
of the ciliate L i t o n o t u s duplostriatus
469
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
des Ciliaten L i t o n o t u s duplostriatus
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 P o l y t r i c h u m
(Polytrichales, M u s c i )
Ultrastruktur der wasserleitenden Zellen v o n Polytrichum nach
HERLAN, G., F. WUNDERLICH
354
Gefrierätzung
(Short C o m m u n i c a t i o n )
Isolation of a nuclear protein matrix f r o m Tetrahymena
macronuclei
291
Isolation einer 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 macronuclei
HINSSEN, H . , J . D ' H A E S E
Synthetic fibrils f r o m Physarum actomyosin - seif assembly, Organization
a n d contraction
132
Synthetische Fibrillen a u s isoliertem Schleimpilzactomyosin - Entstehung, Struktur
und Kontraktion
I N G L E , J . , siehe D E U M L I N G , B .
224
J A R A S C H , E . - D . , siehe F R A N K E , W . W .
28
J O H N S O N , R . P. C , siehe H E B A N T , C .
354
K A R T E N B E C K , J . , siehe F R A N K E , W . W .
28
K E E N A N , T H . W . , siehe E L D E R , J . H .
279
K E E N A N , T H . W . , siehe F R A N K E , W . W .
28
KLOPPSTECH, K . , H . G . SCHWEIGER
In vitro translation of p o l y ( A ) R N A f r o m Acetabularia
394
In vitro Translation v o n Poly ( A ) R N A aus Acetabularia
L I N D G R E N , A . , siehe S C H E L L E N S , J . P . M .
93
L J U B E S I C , N . , siehe S C H N E P F , E .
341
M E Y E R - R O C H O W , V . B . (Short C o m m u n i c a t i o n )
A tri-directional m i c r o v i l l u s orientation i n the monocellular, distal r h a b d o m
of a nocturnal beetle
In drei verschiedenen R i c h t u n g e n angeordnete M i k r o v i l l i i m
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 .
476
monozellulären,
469
VI
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
T r a n s i t i o n elements between endoplasmic reticulum and G o l g i apparatus
in plant cells
Ubergangsstrukturen zwischen endoplasmatischem Retikulum und
in pflanzlichen
297
Golgi-Apparat
Zellen
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 . PARISH, H . M O O R
T h e elasticity of the yeast cell tonoplast related to its ultrastructure and chemical
c o m p o s i t i o n . I. Induced swelling and s h r i n k i n g ; a freeze-etch membrane study
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 .Induziertes D e h n e n u n d Schrumpfen; M e m b r a n u n t e r s u c h u n g e n
mit der
Gefrierätztechnik
364
NIEDERMEYER, W.
T h e elasticity of the yeast cell tonoplast related to its ultrastructure and chemical
c o m p o s i t i o n . II. C h e m i c a l a n d cytochemical investigations
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
Zusammensetzung. I L Chemische und cytochemische Untersuchungen
NEHLS, R . , G . SCHAFFNER
(Short C o m m u n i c a t i o n )
Specific negative staining of proteins i n situ w i t h i r o n tannin
Proteinspezifischer
380
285
N e g a t i v k o n t r a s t in situ 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
PIGON, A .
C e l l surface a 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 culture
Zelloberfläche
107
und Medium-conditioning in Acanthamoeba-Kulturen
PIPAN, N .
Death a n d phagocytosis of epithelial cells i n developing mouse kidney
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
435
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 . SOBOTA
C a l c i u m dependent deposits at the plasma membrane d u r i n g development
of the oocyte of G a l l e r i a mellonella
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 Entwicklung von Galleria mellonella
R E I N H A R T Z , A . , siehe F R I E D L A N D E R , M .
182
171
VII
C O N T E N T S V O L U M E 13
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 w a l l - d e g r a d i n g enzymes
Isolierung der E n d o d e r m i s
m i t Hilfe
Zellwand-abbauender
85
Enzyme
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 o n r u f f l i n g activity, pinocytosis and proliferation
of i n v i t r o cultivated h u m a n glia cells
D e r Einfluß d e s S e r u m s auf Ruffling-Aktivität,
von kultivierten menschlichen
Pinocytose und
93
Zellvermehrung
Glia-Zellen
S C H N E P F , E., G . DEICHGRÄBER, N . LJUBESIC
T h e effects of colchicine, ethionine, and deuterium oxide o n microtubules
in y o u n g Sphagnum leaflets. A quantitative study
D i e W i r k u n g v o n C o l c h i c i n , Äthionin
in j u n g e n
Sphagnum-Blättchen
341
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
SCHUDT, C H . , G .DAHL, M . GRATZL
C a l c i u m - i n d u c e d fusion o f plasma membranes isolated f r o m myoblasts
g r o w n i n culture
211
C a l c i u m - i n d u z i e r t e Fusion v o n P l a s m a m e m b r a n e n , isoliert aus M y o b l a s t e n i n K u l t u r
SCHUDT, C H . , D .PETTE
Influence of monosaccharides, m e d i u m factors and enzymatic m o d i f i c a t i o n
on f u s i o n of myoblasts i n vitro
Einflüsse
auf
von Monosacchariden, M e d i u m f a k t o r e n und enzymatischer
74
Modifikation
die Fusion von Myoblasten i n vitro
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
S P I E S S , E . , siehe A M E L U N X E N , F .
233
S P I E S S , 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. N a c h w e i s der Zellwandsynthese u n d
der Synthese von Cellulose, Hemicellulose und Pektin i n den R a n k e n
von C u c u r b i t a 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 . 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 .
S T A D L E R , J . , siehe F R A N K E , W . W .
S T I E M E R L I N G , R . , siehe S T O C K E M , W .
251
401
28
158
C O N T E N T S V O L U M E 13
VIII
STOCKEM, W., R . STIEMERLING
Intracellular segregation of endocytotically ingested substances
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
158
Substanzen
STOCKERT, J. C .
M e t a c h r o m a t i c staining of nucleolar differentiations by toluidine blue-molybdate
in C h i r o n o m u s salivary glands
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
191
T H I O T I A N G N I O , E . , siehe A M E L U N X E N , F .
233
T H I O T I A N G N I O , E . , siehe A M E L U N X E N , F .
260
T H I O T I A N G N I O , E . , siehe S P I E S S , E .
251
T I M M I S , J . N . , siehe D E U M L I N G , B .
224
T R E N D E L E N B U R G , M . F . , siehe F R A N K E , W . W .
401
WOLBURG, H .
Differences i n distributions of 3 H-proline and 3 H-uridine-labelled Compounds in
the fish visual System d u r i n g ouabain-induced W a l l e r i a n degeneration
13
Unterschiedliche Verteilung 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 Verbindungen
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
Wallerschen Degeneration
W U N D E R L I C H , F . , siehe H E R L A N , G .
291
Z E N T G R A F , H . , siehe F R A N K E , W . W .
401
BOOK REVIEWS
B O S C H K E , F. (Hrsg.): Photochemistry
169
D O S E , K . , H . R A U C H F U S S : Chemische E v o l u t i o n und der U r s p r u n g lebender Systeme
170
D R E W S , U . : Cholinesterase i n E m b r y o n i c Development
482
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 ( H r s g . ) : Q u a n t i t a t i v e S c a n n i n g
Electron M i c r o s c o p y
M O D I S , L . : T o p o - o p t i c a l Investigations of Mucopolysaccharides ( A c i d G l y c o s a m i n o g l y c a n s )
ORCI, L., A. PERRELET:
Freeze-Etch H i s t o l o g y
168
482
170
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 ) a n d D I E T E R SCHWAB
l
Fachbereich Theoretische M e d i z i n der Universität des Saarlandes,
Homburg/Saar, Germany
Received A p r i l 10, 1976
Abstract
Liver - m i c r o t u b u l a r inhibitors - secretion - p l a s m a - bile
Injection of either colchicine o r vinblastine into rats in v i v o reduced the levels of coagulation factors V and V I I and triglycerides i n b l o o d p l a s m a . In contrast, lumicolchicine, a structural
isomer of colchicine w h i c h does not disrupt microtubules, h a d n o effect o n the levels of
coagulation factors o r triglycerides i n b l o o d plasma. T h e r e d u c t i o n i n the levels of proteins
and triglycerides i n b l o o d is accompanied by an a c c u m u l a t i o n of vesicles w i t h i n the hepatocyte, as seen under a light microscope. These vesicles have been identified as G o l g i - d e r i v e d
vesicles by electron microscopy. G o l g i - d e r i v e d secretory vesicles isolated f r o m rat liver were
f o u n d to contain more protein, coagulation factor V a n d triglycerides after injection of
colchicine o r vinblastine. These findings suggest an involvement of microtubules in secretion
f r o m liver into b l o o d plasma. O n the other h a n d , excretion of bilirubin-glucuronides a n d
f l u i d into bile w a s not affected by these chemicals. T h i s indicates that glucuronides are not
transferred to the extracellular space by vesicular transport.
Introduction
T h e interference
proteins
o f 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 secretion
f r o m the hepatocyte
of proteins a n d lipo-
into the b l o o d p l a s m a studied i n different
laboratories
[11, 1 2 , 1 9 , 2 2 , 2 3 ] suggest a n i n v o l v e m e n t o f t h e m i c r o t u b u l a r S y s t e m 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 triglyceride level i n rats a n d c o n c o m i t a n t l y
) A s s . Prof. D R . M . G R A T Z L , Department
Saarland, D-6650 H o m b u r g / S a a r , G e r m a n y .
l
of
Physiological
Chemistry,
University
of the
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 distribution 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 redistribution 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 lumicolchicine 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. T o 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. T o 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 D a w l e y rats (180 to 220 g) were used throughout. T h e 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 m l 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 Eli L i l l y G m b H , Giessen, G e r m a n y ) . T h e remaining
replacement Solutions contained only sodium chloride. After removal of cells by centrifugation
plasma was assayed for P r o t h r o m b i n time, factor V , factor V I I and triglycerides:
M i c r o t u b u l a r inhibitors and secretion f r o m liver
201
P r o t h r o m b i n time was determined by a d d i n g to 0.1 m l of p r e w a r m e d plasma 0.2 m l of 1 : 1
m i x t u r e of 25 m M C a C U and thromboplastin ( H o f f m a n n - L a Roche, Basel, Switzerland).
Factor V activity was tested by m i x i n g in p r e w a r m e d test tubes 0.1 m l of a Solution of plasma
deficient in factor V (Behring W e r k e A G , M a r b u r g , Germany) w i t h 0.1 m l plasma (diluted
1 : 20 i n 0.05 M diethyl barbiturate-acetate buffer, p H 7.6) incubating for exactly 30 sec. at
3 7 ° C a n d then a d d i n g 0.2 m l p r e w a r m e d calcium thromboplastin Solution (Behring W e r k e A G ,
M a r b u r g , G e r m a n y ) . Factor V H - a c t i v i t y was determined i n a similar w a y using factor V I I deficient plasma (obtained f r o m M e r z + Dade A G , B e r n , Switzerland) and a 1 : 10 d i l u t i o n
of the plasma to be tested in a Solution containing 2.8 X 10" M s o d i u m barbital, 1.25 M
X 10" M s o d i u m 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 a n d thromboplastin ( M e r z + D a d e A G , Basel, Switzerland) were added. T h e
time of clot f o r m a t i o n after the a d d i t i o n of C a
was measured i n 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 L e m g o - B r a k e , Germany) and compared to
a Standard curve prepared by testing dilutions of n o r m a l p o o l e d plasma (mixture of five
plasmas) and p l o t t i n g their clotting times o n log-log paper. 100 units of coagulation factors
were taken to be the amount present in 1 m l of pooled plasma.
2
1
2 +
Triglycerides were assayed by the enzymatic determination of glycerol obtained after alkaline
hydrolysis [5].
Bile was collected i n cooled and darkened Polyethylene vessels containing 0.2 m l 10 m M
E D T A . T h e vessels were changed every hour. A z o p i g m e n t s derived f r o m conjugated bile
pigments were analyzed by c o u p l i n g w i t h the d i a z o n i u m salt of ethyl anthranilate [9].
G o l g i - f r a c t i o n s were isolated f r o m rat liver homogenates of control rats and of drugireated rats four hours after intraperitoneal injection of 0.5 mg/100 g body weight colchicine
or 1 mg/100 g body weight vinblastine. A s a slight m o d i f i c a t i o n of the isolation procedure
[6] we buffered all Solutions with 0.01 M cacodylate at p H 7.4. L u m i c o l c h i c i n e was prepared
by Irradiation of colchicine w i t h ultraviolet light [24]. Protein was determined as described
[13] using crystalline bovine serum a l b u m i n as a Standard. A l l chemicals not specified were
of the purest grade commercially available.
For electron microscopic investigations, liver slices of both the control rats and those
injected four hours previously w i t h m i c r o t u b u l a r inhibitors were f i x e d at r o o m temperature
by treatment w i t h 2 % glutaraldehyde i n 0.25 M sucrose, 0.01 M cacodylate at p H 7.4 for
60 min. A f t e r postfixation in 1 % o s m i u m tetroxide i n 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 H e p a t o cyte. - N N u c l e u s . - E Erythrocytes. - a. Hepatocytes of c o n t r o l 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 i p i d droplets
(arrows) i n 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 v a r y i n g size assembled i n Clusters can be demonstrated throughout the cytoplasm. N N u c l e u s . - M M i t o c h o n d r i a . - E R E n d o p l a s m i c reticulum. - g G l y c o g e n . - B Bile capillary. 10 500 X . - b. In higher magnification 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 G l y c o g e n . - 50 000 X .
202
M A N F R E D G R A T Z L and
DIETER
SCHWAB
M i c r o t u b u l a r inhibitors and secretion f r o m liver
Legend see page 201.
F i g . 3. Hepatocytes of colchicine-treated rats. Clusters of secretory vesicles ( a r r o w s ) are scattered
throughout the cytoplasm. - N N u c l e u s . - E R E n d o p l a s m i c reticulum. - g G l y c o g e n . - 10 500 X .
M i c r o t u b u l a r inhibitors and secretion f r o m liver
205
c x a m i n e d w i t h a Siemens E l m i s k o p 101. For light microscopy epon embedded material was
cut into sections of 1 u m and stained w i t h methylene blue/azur according to R I C H A R D S O N .
Photographs were taken w i t h a Zeiss Photomicroscope II o n A g f a p a n 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 colchicinetreated rats, but was most prominent after treatment with vinblastine. These observations 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]. T o 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.
O
o
50
PI
2
>
M i c r o t u b u l a r inhibitors a n d secretion f r o m 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). T o 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 inactivation of this protein during the incubation in alkaline medium.
T a b . I. Effect of colchicine o r vinblastine (4 hours, intraperitoneally) o n the recovery
G o l g i I fraction and i n t r a l u m i n a l Compounds. Average of three experiments.
Intraluminal
Golgi 1
Triglycerides
JA M o l / g liver
Protein
mg/g liver
Protein
mg/g liver
Factor V
U / g liver
Triglycerides
u. M o l / g liver
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
o Control
^
£
T
Colchicine
Vinblastine
3.0
F i g . 8. Effect of colchicine
or vinblastine on bile f l o w
and bilirubin-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).
of
2.0
Vinblastine
1.0
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]. T o 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 administration 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. T o 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 . GRATZL, unpublished observations). Changes in the array of intramembranous 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, freezecleaving of hepatic tissue provided no evidence for the inhibitory effect of antimicrotubular 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
209
M i c r o t u b u l a r inhibitors and secretion from liver
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 homogenates 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 nonvesicular 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. T h e 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 M e e t i n g of the Federation of European
Biochemical Societies, Paris, July 20-25, 1975 and the "International Symposium o n microtubules a n d m i c r o t u b u l a r i n h i b i t o r s " , Beerse (Belgium) September 2 - 5 , 1975.
T h i s 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 i n m a m m a l i a n cells. J . C e l 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 :
Golgi
fractions
prepared f r o m rat liver homogenates. II. Biochemical characterization. J . C e l l B i o l . 59, 73-88
(1973).
[3] B E R R Y , C , a n d T . H A L L I N A N : " C o u p l e d t r a n s g l u c u r o n i d a t i o n " : A new tool for studying
the latency of U D P - g l u c u r o n y l transferase. F E B S 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 a n d a l b u m i n by
isolated rat hepatocytes. B i o c h i m . 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 :
T r i g l y c e r i d e and
G l y c e r i d e . In:
H . U . BERGMEYER
Methoden der enzymatischen Analyse, p. 1871-1878, 3. Auflage. V e r l a g C h e m i e ,
Bergstraße 1974.
(ed.):
Weinheim/
210
[6]
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
E H R E N R E I C H , J. H . , J. J. M . B E R G E R O N ,
P . SIEKEVITZ,
and
prepared f r o m rat liver homogenates. I. Isolation procedure
t i o n . J . C e l l B i o l . 59, 45-72 (1973).
[7]
G . E. P A L A D E :
Golgi
and morphological
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
fractions
characteriza-
Golgi
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 , a n d J . L . E . E R I C S S O N : Studies
o n the synthesis
and intra-
cellular transport of lipoprotein particles i n rat liver. J . C e l l B i o l . 64, 356-377 (1975).
[9]
HEIRWEGH, K . P. M . , G . P. V A NHESS,
P. LEROY,
F . P . V A N R O Y , and
geneity of bile pigment conjugates as revealed by chromatography
azopigments. Biochem. J . 120, 877-890 (1970).
F . H . JANSEN:
of their ethyl
Hetero-
anthranilate
[10] K A R U P , W . , a n d J . A . L A R S E N : T h e effect of slight hyperthermia o n liver f u n c t i o n as
measured b y the elimination rate of ethanol, the hepatic uptake a n d excretion of indocyanine
green a n d bile f o r m a t i o n . A c t a P h y s i o l . Scand. 84, 396-407 (1972).
[11]
L E M A R C H A N D , Y.,
C . PATZELT,
F . ASSIMACOPOULOS-JEANNET,
E.G.LOTEN,
and
B . JEAN-
R E N A U D : Evidence f o r a role o f the m i c r o t u b u l a r System i n the secretion of n e w l y synthesized
a l b u m i n a n d other proteins by the liver. J . C l i n . Invest. 53, 1515-1517 (1974).
C . ROUILLER,
and
B . J E A N R E N A U D : A role f o r the m i c r o t u b u l a r System i n the release of very l o w density
p r o t e i n b y perfused mouse livers. J . B i o l . C h e m . 248, 6862-6870 (1973).
[12]
lipo-
[13]
L EM A R C H A N D , Y.,
A .SINGH,
F . ASSIMACOPOULOS-JEANNET,
LOWRY, O . H . , N . J . ROSEBROUGH,
A . L . FARR,
and
L . ORCI,
R. J. RANDALL:
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 .VENTILLA,
and M . L . S H E L A N S K I :
Vinblastine induced precipitations
of
m i c r o t u b u l e proteins. Science 165, 498-499 (1969).
[15] M I L L E R , L . L . , a n d W . F . B A L E : Synthesis of a l l protein fractions except g a m m a globulins
by the liver. 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 . DALLNER:
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. BERLIN:
Effects
of
Phagocytosis
and
colchicine
on the d i s t r i b u t i o n of lectin-binding sites o n cell surfaces. Proc. N a t l . A c a d . Sei. ( U S A ) 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 : Colchicine-binding
p r o t e i n o f the liver. Its characterization
(1975).
[19]
REDMAN, M . ,
D. BANERJEE,
a n d relation to microtubules. J . C e l l B i o l . 66, 609-620
K . HOWELL,
and
G . E. P A L A D E :
Colchicine
inhibition
of
p l a s m a 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 . : Regulation of the metabolism of proteins i n v o l v e d i n b l o o d coagulation.
In: M . A . R O T H S C H I L D a n d T . W A L D M A N N (eds.): Plasma Protein M e t a b o l i s m , p p . 339-441.
A c a d e m i c Press, N e w Y o r k a n d L o n d o n 1970.
[21] S T A D L E R , J . , a n d W . W . F R A N K E : Characterization of the colchicine b i n d i n g of membrane
fractions f r o m rat a n d mouse liver. J . C e l l B i o l . 60, 297-303 (1974).
[22] S T E I N , O . , L . S A N G E R , a n d Y . S T E I N : Colchicine induced i n h i b i t i o n of l i p o p r o t e i n a n d
p r o t e i n secretion into the serum and lack of interference w i t h secretion of biliary p h o s p h o l i p i d s
a n d cholesterol by rat liver i n v i v o . J . C e l l B i o l . 62, 90-103 (1974).
[23] S T E I N , O . , a n d Y . S T E I N : Colchicine-induced i n h i b i t i o n of very l o w density lipoprotein
release b y rat livers i n v i v o . B i o c h i m . Biophys. A c t a 306, 142-147 (1973).
[24]
WILSON, L.,
J . R. B A M B U R G ,
S. B . M I Z E L ,
L. M . GRISHAM,
and
K . M . CRESWELL:
Inter-
action of drugs w i t h microtubules proteins. F e d . 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. SPETH:
Direct
i m p a i r e m e n t i n the m o b i l i t y of membrane components.
1136-1139 (1973).
evidence
Science
for
colchicine-induced
(Washington
D . C.) 182,