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The effect of colcemid on the structure and secretory activity of ameloblasts in the rat incisor as shown by radioautography after injection of 3H-proline.

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The Effect of Colcemid on the Structure and Secretory
Activity of Ameloblasts in the Rat Incisor as Shown
by Radioautography after Injection of 3H-Proline
A. KARIM AND H. WARSHAWSKY
Department of Anatomy, Faculty of Medicine, McGill Uniuersity,
Montreal, Quebec, Canada H3A 2B2
ABSTRACT
Enamel secretion by ameloblasts was investigated in the incisors of 100 gm normal and colcemid-injected male rats. Morphological studies
were done on rats given a single intraperitoneal injection of 0.1 mg (1.25 mM)
of colcemid and sacrificed 1 to 4 hours after injection. Protein synthesis and secretion were investigated with radioautography in normal and colcemid-treated
rats injected with 3H-proline and sacrificed a t intervals between 0.5 and 3.5
hours after injection. Colcemid was injected 0.5 hours prior to 3H-proline in
each experimental rat. Electron microscopic examination revealed several morphological alterations between 1 and 4 hours after injection of colcemid. These
changes included fragmentation of the normally elongated rough endoplasmic
reticulum into shorter profiles; a disorganization of the normally tubular configuration of the Golgi apparatus into a number of separate but intact stacks of
Golgi saccules; the disappearance of secretion granules and profiles of smooth
endoplasmic reticulum from Tomes' processes; and the accumulation of secretion granules at the mature face of the Golgi stacks, as well as in the infranuclear cytoplasm where they are normally not found. Radioautography revealed
that protein synthesis by the rough endoplasmic reticulum had continued in
colcemid-altered ameloblasts. Labeled secretion granules were found at the
mature surface of the Golgi stacks and in the infranuclear cytoplasm, however
they did not migrate into Tomes' processes. Consequently, labeled enamel matrix did not appear extracellularly a t the same time as in normal controls.
Quantitative radioautography in the light microscope revealed that the effect
of colcemid, although reversed within 4 hours, had temporarily inhibited normal migration and exocytosis of secretion granules.
As early as the 1930's it was known that colchicine (or its derivative colcemid) will arrest
mitotic cells at metaphase (Dustin, '36; Ludford, '36; Brunes, '36; Bucher, '39). This inhibition on mitosis was attributed to a disruptive
effect on spindle microtubule formation. It
has also been shown that colchicine (and colcemid) affected other cellular functions and in
most studies microtubules were implicated as
the organelle affected by the drug. In particular, studies have indicated that microtubules
are associated with several types of intracellular movement (Porter, '66; Buckley and
Porter, '67) including chromosome movement
(Inoue and Sato, '67; Behnke and Forer, '671,
the transport and release of secretory products (Lacey e t al., '68; Williams and Wolff,
ANAT. REC. (1979) 195: 587-610.
'72), and cytoplasmic streaming (Porter, '66;
Nachmias et al., '70), and that these diverse
functions are inhibited by colchicine and similar alkaloids. Studies of the structure of
ameloblasts which secrete the inner enamel
layer have shown that secretion granules are
produced in the Golgi apparatus (Weinstock
and Leblond, '71) which is a tubular structure
located in the supranuclear portion of the cell
(Kallenbach e t al., '63). Thus, secretion granules are found in the vicinity of the Golgi apparatus and also in the core of Tomes' process,
the apical extension of the cell which projects
into the forming enamel (Warshawsky, '66,
'68; Weinstock and Leblond, '71). RadioautoReceived July 13, '77. Accepted June 12, '79
587
588
A. KARIM AND H. WARSHAWSKY
graphic studies have shown that the secretion
granules must move very rapidly and over a
comparatively long distance to reach the
Tomes' process where they are eventually
released (Warshawsky, '66; Weinstock and
Leblond, '71). In addition, these cells were
shown to contain numerous microtubules distributed along the lateral cell membranes and
in the core of Tomes' process. Preliminary
work demonstrated a dramatic reorganization
of cell organelles as a result of colcemid injection (Warshawsky, '71) and the present work
was undertaken to study the effect of colcemid
on the synthetic and secretory processes of
ameloblasts which produce the inner enamel
in rat incisors. 3H-proline was used as a precursor of enamel matrix protein and its distribution within the ameloblasts and enamel
was visualized with radioautography.
MATERIALS AND METHODS
Four experiments were done on a total of 26
male Sherman rats weighing 100 2 8 gm. The
experimental groups were as follows: (1)Normal structure; (2) Normal radioautographic
distribution of 3H-proline (Normal radioautography); (3) Colcemid effect on structure
(Colcemid-affected structure) ; (4) Colcemid
effect on the radioautographic distribution of
3H-proline (Colcemid-affected radioautography).
Experiment 1 - Normal structure
Two rats were injected intraperitoneally
with 0.2 ml of physiological saline (sham
injection). They were sacrificed by perfusion
a t room temperature via the left ventricle 1
and 2 hours after injection. The rats were first
perfused for 15minutes with 3%formaldehyde
in phosphate buffer followed by an additional
15 minute perfusion with phosphate buffered
2.5% glutaraldehyde.' The upper and lower
jaws were dissected, cleaned of soft tissue and
decalcified for 2 weeks in disodium EDTA
(Warshawsky and Moore, '67). After decalcification the incisors were cut into 1 mm
thick cross-sectional segments and washed in
several changes of phosphate buffer. The segments were then postfixed in 1%osmium
tetroxide at 4°C for 4 hours. Dehydration,
infiltration and embedding in Epon were done
according to Luft ('61) except that a 5:5 mixture of Epon solution A and B, and acetone instead of ethanol, were used. The Epon embedded tissues were polymerized a t 60°C for two
days.
For electron microscopic examination thin
sections (pale gold or silver interference color)
were prepared from blocks in the region of
inner enamel secretion (Warshawsky and
Smith, '74). The sections were stained with 4%
uranyl acetate for 10 minutes and then with
lead citrate (Reynolds, '63) for 15 minutes.
They were examined with a Siemens Elmiskop
1 operated a t 50 kv.
Experiment 2 - Normal radioautography
Eight rats were injected intraperitoneally
with 0.2 ml of normal saline (sham injection).
This was followed 0.5 hours later by a single
injection of 10 FCi/gm body weight of 2,3-3HL-proline (New England Nuclear, NET-323;
specific activity 39.7 Ci/mM). The schedule of
sacrifice was timed to give 1, 2, 3 and 4 hours
after sham injection of saline and 0.5, 1.5, 2.5
and 3.5 hours after injection of 3H-proline.
The tissue was processed for light (Kopriwa
and Leblond, '62) and electron microscope
(Kopriwa, '73) radioautography.
Experiment 3 - Colcemid-affected
structure
A group of 8 rats was injected intraperitoneally with 0.1 mg of colcemid in 0.2 ml of
normal saline and sacrificed by perfusion a t I,
2 , 3 and 4 hours after injection. The teeth were
'Rationale for double fixation: Many chemical agents have been
used to fix tissues but only a few give excellent fixation for electron
microscopic investigations. Warshawsky and Moore ('67) described a
technique for t h e fixation and decalcification of rat incisors for electron microscopy. In this technique fixation was accomplished by perfusion with slightly hypertonic neutral phosphate-buffered 2.5% glutaraldehyde which was then followed by postosmication in 1%osmic
acid in veronal-acetate buffer. This method gave excellent preservation of cellular structure in r a t incisors which had been decalcified
in disodium EDTA between t h e two fixations.
However, glutaraldehyde fixation has certain disadvantages for
radioautographic studies of protein synthesis from injected radioactive amino acids (Peters and Ashley, '67). In their investigation,
liver slices were incubated for two minutes in the presence of labeled leucine and puromycin which permits absorption of leucine
into the cell but inhibits incorporation into protein. Quantitative
analysis and radioautographic techniques showed t h a t glutaraldehyde bound, in a non-peptide form, 30 times, and nsmic acid 6 times
as much free amino acid as did formaldehyde. I t was also calculated
t h a t in radioautographs prepared after fixation with glutaraldehyde, osmic acid, and formaldehyde, 63%. 25%, and 4% respectively,
of t h e grains were due to non-specific binding of free labeled amino
acids. Preservation of cellular structure by formaldehyde fixation,
however, was not as good as glutaraldehyde fixation.
Therefore, in order to obtain good preservation of tissues and a t
t h e same time prevent t h e above artefact in radioautography, a pilot
experiment using Wproline (H. Warshawsky, unpublished data)
was done to analyze quantitatively t h e grains in radioautographs
after double perfusion fixation (3%formaldehyde solution followed
by 2.5% glutaraldehyde solution) and 3% formaldehyde solution
alone. Results revealed t h a t there was no significant difference in
t h e grain count over tissues fixed by either method, and t h a t fixation by t h e double perfusion technique was much improved over
formaldehyde fixation alone. (The authors gratefully acknowledge
the assistance of Dr. Ithamar Vugman in these experiments.)
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
prepared for light and electron microscopy as
described above.
Experiment 4 - Colcemid-affected
radioau tography
This experiment was designed to study by
radioautography the ability of the secretory
ameloblasts to synthesize and secrete enamel
matrix protein while affected by colcemid.
Eight rats were injected intraperitoneally
with 0.1 mg of colcemid in 0.2 ml of normal
saline and 0.5 hours later they were injected
with 10 pCi/gm of body weight of 3H-proline.
The animals were sacrificed by perfusion 1, 2,
3 and 4 hours after colcemid injection, which
gave intervals of 0.5, 1.5, 2.5 and 3.5 hours
after 3H-proline injection. The tissue was
processed for light and electron microscope radioautography .
A quantitative analysis was made from
counts over sections exposed for 3 months and
processed for light microscope radioautography. Briefly, the grains were counted a t 1,000
x magnification in relation to an ocular grid
scored in 10 p m by 10 p m squares. The
squares of the grid were placed over the cells
and the enamel matrix and grains were
counted in successive rectangles (50 p m by 10
pm) until the entire height of the cells and the
thickness of the matrix were examined. Eight
(and in some cases, 6) 1 p m thick non-serial
Epon sections were counted for each time interval. For each section the grains counted
within all the successive rectangles were averaged. This value was averaged for the 6 or 8
sections counted. From these counts a compartmental analysis was performed on the
total number of grains counted over the cells
and the matrix a t each time interval after 3Hproline injection in both normal and colcemidaffected groups. The counts in the individual
rectangles were grouped into four compartments (figs. 22-25). The percentage of grains
over each compartment out of t h e total grains
counted over the cells and the matrix was calculated and compared in the graphs shown in
figures 22-25.
RESULTS
Experiment 1 - Normal structure (fig. l a )
The structure of normal inner enamel
secretory ameloblasts
The inner enamel secretory ameloblast is a
tall columnar cell about 60-70 p m in height.
However, the exact height of these cells is difficult to measure because of uncontrollable
589
variation in the plane of section. The base, or
proximal end of the cell is adjacent to the
stratum intermedium and the apex, or distal
end, called Tomes’ process, projects into the
enamel matrix (fig. 2). These cells show both
proximal and distal cell web-junctional complex systems. For descriptive purposes the cell
is divided into a number of morphologically
homogeneous compartments (fig. l a ; Warshawsky, ’68). In the basal bulge, which is
located proximal to the proximal cell web (fig.
41, the cytoplasm contains some profiles of
rough endoplasmic reticulum, bundles of
dense fibrils, coated vesicles and the occasional secretion granules. Mitochondria are
sometimes found in this compartment. However, the mitochondria1 compartment, situated between the proximal cell web and the
nucleus, contains almost all of the cell’s mitochondria (fig. 4). Few profiles of rough endoplasmic reticulum, polysomes in the form of
rosettes, occasional secretion granules, microtubules and bundles of dense fibrils are present. The nuclear compartment shows high and
low level nuclei (fig. 2). Some profiles of rough
endoplasmic reticulum, free ribosomes and
small bundles of dense fibrils are found in the
cytoplasm between the nucleus and the lateral cell membrane. In the supranuclear compartment the Golgi complex is made up of numerous stacks of elongated flattened saccules
arranged to form the walls of a long tubule
which extends for some distance parallel to
the long axis of the cell (fig. 7). Like the nuclei, the apparatus is at varying levels within
the cell. Located in the Golgi region are coated
vesicles, many secretion granules, few heterogenous granules and multivesicular bodies.
The rough endoplasmic reticulum in the form
of long profiles predominates in the area above
the Golgi apparatus. The proximal portion of
Tomes’ process contains many microtubules
which are oriented mainly along the long axis
of the process (fig. 9).Secretion granules and
free polysomes are present. In the interdigitating portion of the process there is an
abundance of secretion granules and microtubules (fig. 9).Many coated vesicles are seen
opening onto t h e cell membrane around
Tomes’ process.
Experiment 2 - Normal radioautography
Qualitative light microscope radioautography
of normal ameloblasts
At 0.5 hours after 3H-proline injection (fig.
590
A. KARIM AND H. WARSHAWSKY
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
14) there was an intense band of reaction
(silver grains) over the supranuclear compartment. Another intense reaction band was
present over both portions of Tomes’ processes
and the adjacent enamel matrix (fig. 14). The
reaction over the nuclear compartment was
weak, and there were practically no grains
over the infranuclear cytoplasm.
At 1.5 hours after injection of 3H-proline
(fig. 16)a reaction, less intense than that seen
a t 0.5 hours, was observed over the supranuclear cytoplasm. However, there was a more
intense reaction over Tomes’ processes, and
the silver grains over the matrix were distributed in a gradient, the least intense reaction
being near the dentino-enamel junction. The
reaction over the nuclear and infranuclear
compartments did not change from that seen
at 0.5 hours.
At 2.5 hours (fig. 18) and 3.5 hours (fig. 20)
after 3H-prolineinjection, the most apparent
change was a reduction in reaction intensity
over all portions of the cells. Most of the silver
grains were now located over the enamel matrix above Tomes’ processes. The gradient pattern of the grains over the enamel matrix was
more apparent.
Experiment 3 - Colcemid-affected
structure (fig. l b )
The structure of colcemid-affected
ameloblasts
The ultrastructural observations of the
ameloblasts from colcemid treateu animals
showed marked differences from the normal
cells. Although the overall shape of the cells
was unaltered (compare figs. 2 and 3) there
was an abnormal arrangement and distribution of the organelles within them.
One hour after colcemid injection there was
an accumulation of secretion granules in the
infranuclear compartment (figs. 5, 6). Also a
gap appeared between the nucleus and the
Fig. 1 A diagrammatic comparison between normal
(a) and colcemid-affected (b) ameloblasts a t 1 hour after
injection. The distribution of organelles in the various
compartments of t h e normal ameloblast is shown in figure
l a . The effect of colcemid is illustrated in figure lb. Note
the accumulation of secretion granules in t h e supranuclear and infranuclear compartments and their absence in
Tomes’ process in the colcemid-affected cell. In t h e latter
t h e rough endoplasmic reticulum is fragmented and some
of the organelles (nucleus and Golgi apparatus) are displaced distally, apparently accounting for t h e gap t h a t appears between t h e mitochondria and the nucleus. The
proximal and distal cell webs (pcw, dcw) are lacking in the
colcemid-affected cell, but bundles of fine filaments are
found in Tomes’ process.
591
group of mitochondria (figs. 3, 5, 6 ) . This gap
contained some rough endoplasmic reticulum
and secretion granules.
In the supranuclear cytoplasm the number
of separate profiles of rough endoplasmic reticulum had increased indicating either a disruption of the long profiles or an increase in
the cisternal fenestrations (fig. 8 ) . The tubular Golgi apparatus was disrupted into separate stacks of saccules which were displaced to
abnormal positions. There was also an accumulation of secretion granules in the supranuclear cytoplasm particularly related to the
mature face of the separated Golgi saccules
(fig. 8). The distal cell web had completely disappeared and Tomes’ processes, while being
devoid of secretion granules, contained bundles of fine filaments (fig. 10). In addition,
profiles of rough endoplasmic reticulum were
found in Tomes’ processes of cells in which the
distal web was absent (fig. 11).
Two hours after injection of colcemid the
number of secretion granules in the infranuclear cytoplasm had increased. Many secretion granules were also seen in the cytoplasm
between the nucleus and the lateral cell membrane. There was a decreased number of secretion granules in the supranuclear cytoplasm
and a t this time few secretion granules were
seen within Tomes’ processes. The rough endoplasmic reticulum was still fragmented.
By 3 hours after injection of colcemid the
gap between the nucleus and the mitochondria was still present, but the number of secretion granules accumulated here had decreased. The Golgi apparatus had assumed its
normal shape and position in the supranuclear
cytoplasm of most ameloblasts. The distal cell
web was reconstituted a t this time. The rough
endoplasmic reticulum resumed its normal
configuration into long profiles, and Tomes’
processes were becoming increasingly packed
with secretion granules. In those cells where
the distal cell web was still obviously absent,
profiles of rough endoplasmic reticulum were
seen in the proximal portions of Tomes’ processes.
Four hours after injection of colcemid the
cells showed all the features of normal secretory ameloblasts. In the infranuclear cytoplasm the nucleus was very close to the
mitochondria and the number of secretion
granules and profiles of rough endoplasmic reticulum had greatly decreased. The cytoplasm
surrounding the nucleus was virtually devoid
of secretion granules. In all the cells examined
592
A. KARIM A N D H. WARSHAWSKY
the Golgi apparatus was observed in its normal position in the supranuclear cytoplasm
and was again seen t o be made up of stacks of
saccules forming the wall of a long tubular
structure. Tomes’ processes were packed with
secretion granules and many coated vesicles
were seen opening onto the surface of the
plasma membrane around t h e processes.
These coated vesicles were not seen in the
processes of cells from animals killed 1 and 2
hours after injection of colcemid.
Between 1.5 and 4 hours after injection of
colcemid material resembling enamel matrix
in electron density was seen extracellularly in
various abnormal locations (figs. 12, 13).
Experiment 4 - Colcemid-affected
radioautography
Qualitative light microscopic radioautography of colcemid-affected ameloblasts
One hour after injection of colcemid and 0.5
hours after 3H-proline injection (fig. 151, the
silver grains were almost evenly distributed
over all portions of the cell. Thus, no clear
reaction bands were present. Indeed, there
were few grains over Tomes’ processes. The
reaction over the nuclear and infranuclear
compartments was more pronounced than
over the normal a t this time.
Two hours after injection of colcemid and
1.5 hours after 3H-proline injection (fig. 17)
the grains over the nuclear and infranuclear
compartments had increased. However, there
were some silver grains over Tomes’ processes
and the enamel matrix at this time.
At 3 hours after colcemid and 2.5 hours
after 3H-prolineinjections (fig. 191, the number of grains over the supranuclear, nuclear
and infranuclear compartments had decreased, although the reaction was still more
intense than the normal a t this time interval.
With this cellular decrease there was a concomitant increase in the number of grains
over the enamel matrix above Tomes’ processes.
By 4 hours after injection of colcemid and
3.5 hours after 3H-proline injection (fig. 211,
there was a weak reaction over all portions of
the cells but there was a marked increase in
the number of grains over the enamel matrix.
Quantitative analysis of the light
microscope radioautographs
The grain counts were analyzed with respect to their distribution over the different
compartments of the cells and the enamel matrix. Figures 22-25 show this distribution by
comparing the percentage of grains over the
normal and colcemid-affected groups in each
compartment a t the various time intervals
after injection of 3H-proline. Figure 22 shows
the distribution of grains over the infranuclear and nuclear compartments. Between 0.5
and 1.5 hours the labeling in the normal group
decreased sharply (from 25%to 9%)and thereafter remained stable. The labeling in the colcemid-affected group accounted for 34%of the
total grains a t the 0.5 hour interval. This increased to about 40%a t 1.5 hours and then declined gradually towards the normal level.
The distribution of grains over the supranuclear compartment (fig. 23) followed the same
pattern in both groups. Figure 24 shows the
distribution of grains over Tomes’ processes
and the prongs of enamel matrix. The labeling
in the normal group increased from 35%at 0.5
hours to 45%a t the 1.5 hour interval. By 2.5
hours the labeling was not significantly different from that of the colcemid-affected
group which increased from about 13%at 0.5
hours to 42%at the 2.5 hour interval. The percentage of the grains over the enamel matrix
above Tomes’ processes is shown in figure 25.
By 1.5 hours after injection, 28%of the grains
were over the matrix in the normal, while
there was less than 10%in the colcemid-affected group. By 2.5 hours 13%of the grains
were over the matrix in the colcemid-affected
group. This was much less than that over the
normal at the same time interval. However, by
3.5 hours the percentage of grains over the
matrix in both groups was not significantly
different. Thus, by 1.5 hours after injection
there was a sharp increase in the percentage
of grains over the matrix in the normal group.
Thereafter there was a gradual increase to 3.5
hours. On the other hand, there was a gradual
increase in the percentage of grains between
0.5 and 2.5 hours in the colcemid-affected
group, while there was a sharp increase t o the
normal level between 2.5 and 3.5 hours.
DISCUSSION
Effect of colchicine on cellular
microtu bules
A correlation was first made between microtubule orientation and direction of cytoplasmic flow by Ledbetter and Porter (‘63). It was
later suggested that the inhibitory effect of
colchicine (its derivative colcemid and similar
alkaloids) is apparently due to its ability to
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
disrupt microtubules (Porter, '66). This phenomenon was then demonstrated for proximodistal transport of neurosecretory substances
(Karlsson and Sjostrand, '69; Kreutzberg, '69;
James e t al., '70; Dahlstrom, '71; Hokfelt and
Dahlstrom, '71; Karlsson et al., '71; Fink e t
al., '73; Marchisio et al., '73; Gremo and
Marchisio, '75) and movement of pigment
granules in fish melanophores (Wikswo and
Novales, '69, '72; Schliva and Bereiter-Hahn,
'73). Finally, the action of colchicine on microtubules was correlated with an inhibitory effect on insulin secretion from the beta cells of
the islets of Langerhans (Lacey et al., '681,
histamine release from leukocytes (Levy and
Carlton, '691, and thyroid secretion (Williams
and Wolff, '72). Therefore, it can be concluded
that colchicine, or its derivative colcemid, alters microtubule stability and it can be speculated that the alteration in the structure and
function observed in the cell is related to the
disrupted functions mediated to some extent
by microtubules.
Morphological alterations i n ameloblasts
due to a single injection of colcemid
In the present study morphological changes
were seen in the ameloblasts of inner enamel
secretion after a single intraperitoneal injection of colcemid. Between 1 and 1.5 hours following injection of colcemid, while the overall
architecture of the cells remained unchanged,
profound alterations were observed in the organization of the organelles. In figure 1 these
changes are diagrammatically shown and
compared to the normal situation.
In the normal ameloblasts, few secretion
granules were seen in the supranuclear cytoplasm. They appeared to accumulate within
Tomes' processes from where they were extruded and added to the extracellular enamel
matrix. However, in the colcemid-affected
ameloblasts secretion granules continued to
form a t the mature face of the individual
stacks of Golgi saccules, but these granules
did not migrate from that position as they normally would. As a result, the granules present
in Tomes' processes before the injection of colcemid were secreted from the cells but more
granules did not migrate into the processes to
take their place. The granules which were
formed were, however, still capable of being
secreted to the outside, and they in fact did so
a t various abnormal locations, such as the cell
base (fig. 13) or supranuclear region (fig. 12).
The morphological defects created seemed
593
to be of two kinds. First, a disruption of the
supporting system which holds the internal
cell structures in their normal position, thus
disrupting the apical and basal cell webs,
allowing the tubular Golgi apparatus to drift
into separate stacks of saccules, allowing the
nucleus t o migrate, and the endoplasmic reticulum to move into Tomes' processes. The second defect seemed to be a disruption of the
normal streaming processes which either directed or physically carried secretion products
into Tomes' processes. The absence of granules
in these processes prevented enamel from
being secreted to the normal extracellular position.
Radioautographic studies
Qualitative analysis
An examination of radioautographs of normal ameloblasts revealed that radioactivity
first accumulated in the supranuclear and
distal cytoplasm a t 0.5 hours after injection
of 3H-proline (fig. 14). With a decrease in
radioactivity within the cell, the enamel matrix became heavily labeled (figs. 16, 18 and
20). This indicated that enamel proteins were
synthesized within the cells and added to the
extracellular matrix via Tomes' processes.
However, from the radioautographs of colcemid-affected ameloblasts, it was seen that
although the cells continued to produce proteins these took a longer time t o be exported
out of the cells. At 1 and 2 hours after colcemid injection (figs. 15 and 17, respectively),
that is, 0.5 and 1.5 hours after 3H-proline
injection, most of the labeling was over the
different compartments of the cells. By 4
hours after injection of colcemid (fig. 211, the
labeling pattern was similar to that of the normal at the same time interval (fig. 20).
Thus one of the visible effects of colcemid,
as revealed by these radioautographic studies,
was the transient accumulation of radioactivity within the cells. This indicated that the
cells were capable of synthesizing proteins
which were temporarily inhibited from being
secreted extracellularly.
Quantitative analysis
The results of the grain counts expressed as
percentage of grains over the different portions of the cells, indicated that the drug temporarily inhibited the secretion of proteins out
of these cells (figs. 22-25). This inhibition of
secretion is reflected by the accumulation of
radioactivity within the cells. These results
594
A. KARIM AND H. WARSHAWSKY
('75) who studied the
effect Of colchicine On the secretion Of dentin
and
in rat
and with those Of
Moe and Mikkelson ('77) who used vinblastine
in the
ameloblasts
of rat incisors. The results
. ~
~ .
also agree with other investigators working
with other systems (Lacey et al., '68; Levy and
Carlton, '69; Williams and Wolff, '72).
The present work demonstrated that colcemid had temporarily inhibited matrix secretion in the ameloblasts which produce inner
enamel. It is suggested that this effect, which
was reversible, is presumably due to the altered aggregation of cytoplasmic microtubules. It is further suggested that the functional role of microtubuies in the secretory
ameloblasts is mainly related to intracellular
stability and transport of secretion granules.
a
m e e with
of Kudo
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ACKNOWLEDGMENTS
This work was supported by a grant from
the Medical Research Council of Canada.
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EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
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Abbreviations
ae, abnormally located enamel
bb, basal bulge
Colc., colcemid
cv. coated vesicle
D, dentin
dcw, distal cell web
En, enamel
f, filaments
G, Golgi apparatus
H, high level nucleus
in, infranuclear compartment
L, low level nucleus
m, mitochondria
mt, microtubule
mvb, multivesicular body
n, nucleus
pcw, proximal cell web
pt, proximal portion of Tomes’ process
s, supranuclear compartment
sg, secretion granule
SI, stratum intermedium
T, Tomes’ process
PLATE 1
EXPLANATION OF FIGURES
2
Normal ameloblasts of inner enamel secretion. Light mirrograph of a I - p m thick Epon section of ameloblasts of inner enamel secretion. Cross section of the incisor. Stained with toluidine blue. x 750.
The base of the ameloblast is related to the stratum intermedium (SI).
The infranuclear compartment
(in) contains the dark staining mitochondria. The nuclei are arranged into high (HI and low (L) levels.
The long supranuclear compartment is separated from the proximal portions of Tomes’ process ipt) by the
distal cell web (dcw). The interdigitating portionp :it Tomes‘ processes are embedded in the forming layer
of inner enamel (En).
3 Colcemid-affected ameloblasts of inner enamel secretion I i & t micrograph of a I - p m thick Epon section
of ameloblasts of inner enamel secretion. Cross sectioned incisor, one hour after injection of colcemid.
Stained with toluidine blue. x 750.
An enlarged gap is present between the low level nuclei (L) and the clump of infranuclear mitochondria. Dark stained material scattered in the supranuclear cytoplasm represents the disaggregated Golgi
complexes. The distal cell web is absent and Tomes’ processes are pale stained in contrast to the dark
cores which are normally present.
4
Normal infranuclear compartment. Electron micrograph. x 10,000.
The mitochondrial compartment contains numerous mitochondria im) which are close to t h e nucleus
(n). The proximal cell web (pcw) is evident and separates t h e mitochondrial compartment from the basal
bulge ibb).
5 Colcemid-affected infranuclear compartment. Electron micrograph. x 10,000.
One hour after injection of colcemid there is an accumulation of secretion granules (sg) and an increased number of profiles of rough endoplasmic reticulum occupying t h e gap that i p created between the
nucleus (n) and the mitochondria (m).
596
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
A. Karim and H. Warshawsky
PLATE 1
597
PLATE 2
EXPLANATION OF FIGURES
6 Colcemid-affected infranuclear compartment. Electron micrograph. x 15,000.
Some ameloblasts a t one hour after injection of colcemid showed a marked accumulation of secretion granules (sg) in the proximal part of the mitochondria1 compartment and in the basal bulge (bb).
7 Normal supranuclear compartment. Electron micrograph. x 20,000.
In the normal ameloblast stacks of saccules make up t h e tubular configuration of
the Golgi apparatus (GI. A few secretion granules are present within the Golgi
tubule. Long profiles of rough endoplasmic reticulum occupy the cytoplasm between
the Golgi saccules and the lateral cell membrane.
8 Colcemid-affected supranuclear compartment. Electron micrograph. x 15,000.
At one hour after injection of colcemid there is an accumulation of large numbers
of secretion granules (sg) a t the mature faces of t h e separated stacks of Golgi saccules (GI. The rough endoplasmic reticulum appears fragmented.
598
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
A. Karim and H. Warshawsky
PLATE 2
599
PLATE 3
EXPLANATION OF FIGURE
9 Normal Tomes’ processes. Electron micrograph. x 25,000.
The proximal portion of Tomes’ process is situated between the distal cell web
(dcw) and the tip of the enamel prong. The interdigitating portion is inserted into
the enamel. Microtubules (mt), secretion granules (sg), coated vesicles (cv) and
multivesicular bodies (mvb) are seen within both portions of the process.
600
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
A. Karim and H. Warshawsky
PLATE 3
601
PLATE 4
EXPLANATION OF FIGURES
10 Colcemid-affected Tomes’ process. Electron micrograph. X 10,000.
At one hour after injection of colcemid the processes are devoid of secretion
granules but show bundles of fine filament (0. Note t h e exaggerated space between the membrane of Tomes’ process and t h e enamel matrix (En).
11 Colcemid-affected Tomes’ process. Electron micrograph. x 20,000.
With the disruption of the distal cell web the profiles of rough endoplasmic reticulum have migrated into the interdigitating portions of Tomes’ processes 1.5 hours
after colcemid injection.
12 Colcemid-affected supranuclear compartment. Electron micrograph. x 20,000.
By 1.5 hours after colcemid injection electron dense material resembling
unmineralized enamel matrix is seen in t h e intercellular space between ameloblasts (ad.
13 Colcemid-affected infranuclear compartment. Electron micrograph. x 30,000.
By 4 hours after colcemid injection some secretion granules (sg) are still seen in
the basal bulge, but masses of abnormally situated enamel matrix-like material is
present extracellularly (ae).
602
EFFECT OF COIL’EMID ON SECRETORY AMELOBLASTS
A Karirn and H Warshawsk)
PLATE 4
603
PLATE 5
EXPLANATION OF FIGURES
14 Normal radioautography, 0.5 hours after 3H-proline. Iron hematoxylin. X 720.
At 0.5 hours after %-proline injection two reaction bands are visible. One is over
the supranuclear compartment in the area of the Golgi apparatus, and the other
over Tomes’ processes and the enamel matrix.
15 Colcemid-affected radioautography, 1 hour after colcemid, 0.5 hours after %proline. Iron hematoxylin. X 720.
The reaction over Tomes’ processes is light. Most of the silver grains are scattered throughout the various parts of the cells with an evident accumulation in
the infranuclear compartment (in).
16 Normal radioautography, 1.5 hours after 3H-proline injection. Iron hematoxylin.
x 720.
The reaction over Tomes’ processes and the enamel matrix has increased and
that over the supranuclear compartment has decreased. The enamel matrix above
the interdigitating portions of Tomes’ processes is heavily labeled.
17 Colcemid-affected radioautography, 2 hours after colcemid, 1.5 hours after 3Hproline. Iron hematoxylin. x 720.
The reaction over Tomes’ processes remains lower than over the normal, but
there is still a large number of silver grains over the cells with an increased number over the infranuclear compartment (in).
604
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
A. Karim and H Warshawsky
PLATE 5
PLATE 6
EXPLANATION OF FIGUKES
Iron hematoxylin. X 720.
18 Normal radioautography, 2.5 hours after 3HH-proline.
The reaction over the cells has diminished as compared to the previous time interval tfig. 16). The heaviest reaction is seen over the enamel matrix (En! above
Tomes’ processes and over the prongs of enamel between the processes.
19 Colcemid-affected radioautography, 3 hours after colcemid, and 2.5 hours after JHproline. Iron hematoxylin. x 720.
There is an increased reaction over the enamel matrix (En) above Tomes’ processes as compared to the previous time interval (fig. 17). Although the reaction
over t h e various parts of the ameloblasts is still heavy, it is beginning to decrease.
20 Normal radioautography, 3.5 hours after H-proline Iron hematoxylin. x 720.
The reaction is similar to that a t 2.5 hours (fig. 18!,except that the reaction over
the enamel matrix (En, above Tomes’ processes) is increased. The reaction gradient
over the enamel is apparent.
21 Colcemid-affected radioautography, 4 hours after colceniid, and 3.5 hours after
3HH-proline.
Iron hematoxylin. X 720.
The reaction pattern is similar to the normal a t the same time interval (fig. 20).
606
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
Karim and H Warshawsky
PLATE 6
A
607
PLATE I
EXPLANATION OF FIGURES
22 The percentage of silver grains over the infranuclear and nuclear compartments of the ameloblasts, from
normal (dashed-line) and colcemid-affected (solid-line) groups at various time intervals after injection of
3H-proline. At 1.5 hours less than 10%of the silver grains are over these compartments from the normal
group, while about 40%are over the same compartments of t h e cells from colcemid-affected animals. By
3.5 hours t h e percentage of silver grains over the latter declines to the normal level.
23 The percentage of silver grains over the supranuclear compartment of t h e ameloblasts. from the normal
and colcemid-affected rats a t various time intervals after injection of Wproline. The labeling pattern is
similar in both groups.
24 A comparison of t h e percentage of silver grains over Tomes’ processes and the prongs of enamel between
the normal and colcemid-affected groups a t various time intervals after injection of 3H-proline.
At 1.5 hours 2% of the silver grains are over t h e cells of the colcemid-affected group, while about 45%
are over t h e control group. By 3.5 hours the percentage of silver grains over t h e cells of both groups is
similar.
25 The percentage of silver grains over the enamel matrix of the normal animals is compared to that of the
colcemid-affected group a t various time intervals after injection of 3H-proline. Between 0.5 and 2.5 hours
there is a lag in t h e appearance of silver grains over the enamel of t h e colcemid-affected group. During
this time about 13%of the silver grains are over the enamel of the colcemid-affected cells, while about
38% are over the enamel in the normal group. However, by 3.5 hours the labeling pattern over both
groups is similar.
608
PLATE I
EFFECT OF COLCEMID ON SECRETORY AMELOBLASTS
A. G r i m and H. Warshawsky
SUPRANUCLEAR
INFRANUCLEAR AND NUCLEAR
60
I0I
50
1.5
2.5
Time after injection of 3H-Proline (hours)
3'5
TOMES' PROCESSES PLUS PRONGS
OF ENAMEL
d'Tim:':f+er
1.5
injecf ion of
ENAMEL MATRIX
2.5
3.5
H - P;oline (hours;
ABOVE TOMES'
PROCESSES
609
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effect, structure, injections, secretory, proline, radioautography, activity, colcemid, rat, incisors, show, ameloblasts
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