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In vivo demonstration by radioautography of binding sites for insulin in liver kidney and calcified tissues of the rat.

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THE ANATOMICAL RECORD 214:130-140 (1986)
In Vivo Demonstration by Radioautography of
Binding Sites for insulin in Liver, Kidney, and
Calcified Tissues of the Rat
B. MARTINEAU-DOIZE, M.D. McKEE, H. WARSHAWSKY, AND J.J.M. BERGERON
Department of Anatomy, McGill University, Montreal, Quebec, Canada
ABSTRACT
An in vivo binding assay using radioautography was employed to
visualize insulin receptors in rat tissues. Two and one-half minutes after the intravenous injection of 1251-insulin,free hormone was separated from bound hormone by
whole body perfusion with lactated Ringer’s solution followed by perfusion with
glutaraldehyde. The localization of bound hormone, fixed in situ by perfusion with
glutaraldehyde, was determined. Nonspecific binding of labeled insulin was noted
in the proximal convoluted tubules of the kidney cortex, prebone and adjacent bone,
predentin and adjacent dentin, and enamel. Specific binding sites were observed a t
the periphery of hepatocytes, over osteoblasts, and in relation to the endothelial cells
of fenestrated capillaries within the papillary layer of the maturation zone of the
incisors.
In vivo radioautographic assays have demonstrated
the presence of specific insulin receptors in many tissues
(Bergeron et al., l977,1980a,b; van Houten et al., 1979).
However, only one in vivo study has shown specific
receptors for insulin in endothelial cells (van Houten
and Posner, 1979). On the other hand, several investigators have identified specific receptors in cultured human and bovine endothelial cells originating from
macrovessels (Peacock et al., 1982; Kaiser et al., 1982a,b;
Carson et al., 1983) and also from microvessels (Pillion
et al., 1982; King et al., 19831, as well as from microvessels of intact rat heart (Bar et al., 1982). In the present
study, nonspecific insulin binding sites were observed in
vivo in the proximal convoluted tubules of the kidney,
the collagenous matrices of prebone and bone, predentin
and dentin, and in the enamel of the rat incisor. Specific
binding sites were seen at the periphery of hepatocytes,
osteoblasts, and endothelial cells of fenestrated capillaries in the enamel organ of the rat incisor, but only in
the zone of maturation.
MATERIALS AND METHODS
Animal Procedures
Three series of experiments were carried out on 12
male Sherman rats weighing 95 +_ 8 gm (Tables 1, 2).
Under pentobarbital anesthesia, the experimental rats
were injected via the external jugular vein with 0.2 ml
of freshly prepared 1251-insulin(specific activity, respectively, of 155 pCi/pg, 135 pcilpg, and 148 pCi/pg; Table
2). The iodination of porcine insulin (Porcine zinc insulin, Connaught Laboratories, Willowdale, Ontario) was
performed by the chloramine T method as previously
described (Posner et al., 1978). Precisely 2 minutes and
30 seconds after the injection, the rats were perfused
through the left ventricle with lactated Ringer’s solution until the liver had visibly blanched (20-30 seconds).
This washing, presumably to remove the free hormone
0 1986 ALAN R. LISS, INC.
from the whole body, was followed immediately by perfusion for 10 minutes with 2.5% glutaraldehyde in 0.1
M sodium cacodylate buffer containing 0.05% CaC12 (pH
7.3).
Control animals received the same quantity of 1251insulin plus 50 or 100 pg of unlabeled insulin (Table 2).
Immediately after perfusion, kidney, liver, and tibia
samples, as well as the mandibular incisors, were taken
and immersed for 2 additional hours in the same fixative a t 4°C. In addition, portions of liver and kidney
were weighed on a n analytical balance and examined
for radioactive content (Table 1)using a Packard auto-a
spectrometer (efficiency 41.5%; Packard, Dowmers and
Grave, L).
The noncalcified tissues were washed in 0.1 M sodium
cacodylate buffer containing 0.05% CaC12, pH 7.3, and
postfixed in 2% aqueous osmium tetroxide. After dehydration in graded concentrations of acetone, the tissues
were embedded in Epon 812.
The calcified tissues were decalcified in 4.13% disodium EDTA, pH 7.3, for 16 days a t 4°C (Warshawsky
and Moore, 1967) and then washed for 24 hours in the
same buffer, before being processed as above.
Quantitative Analysis
For light microscope radioautography, twelve l-pmthick sections of every block were cut with glass knives
and placed in rows of four on each of three slides. The
sections were prestained with iron hematoxylin, dipped
in Kodak NTB2 liquid emulsion (Kopriwa and Leblond,
19621, and developed in three sets: one set was exposed
for 1week, another set for 3 weeks, and the last set for
Received October 1, 1984; accepted September 6, 1985.
Address reprint requests to Dr. H. Warshawsky, Department of
Anatomy, McGill University, 3640 University Street, Montreal, Quebec, H3A 2B2 Canada.
131
INSULIN BINDING IN RAT TISSUES
TABLE 1. Content of radioactivity present in fixed liver and kidney tissues after injection of ‘2511-insulin
Experiment
No.
1A
Experimental
Control
1B
Experimental
Control
2A
Experimental
Control
2B
Experimental
Control
3A
Experimental
Control
3B
Experimental
Control
1251-insu~in
injected
(dpm)’
422 x 106
422 x lo6
422 x 106
422 x lo6
432 x
432 x
432 x
432 x
lo6
lo6
lo6
lo6
lo6
642 x
642 x lo6
642 x
642 x
lo6
lo6
Concentration of
radioactivity
in fixed liver
(dpdgwt)’
lo6 + 10.17 x lo6
lo6 1.03 X lo6
25.07 x lo6 f 12.06 x lo6
6.89 x lo6 f 0.62 x lo6
27.75 x lo6 & 2.21 x lo6
3.59 x lo6 f 0.51 x lo6
34.04 x lo6 k 15.80 x lo6
3.53 x lo6 & 0.50 x lo6
26.63 x lo6 f 3.52 x lo6
7.36 x lo6 k 0.43 x lo6
34.47 x lo6 & 8.41 x lo6
6.33 x lo6 k 0.35 x lo6
28.36 x
8.02 X
P3
<’03’
Concentration of
radioactivity
in fixed kidney
(d~m/gwt)~
75.39 x 106
106.47 x lo6
<‘04’
62.31 x
83.64 x
<.Ooo
62.31 x
112.81 x
<.‘I4
60.22 x
108.50 x
<.Ooo
36.28 x
133.87 x
<.002
p3.4
121.51 x
142.81 X
lo6
lo6
lo6 k 13.78 x lo6
lo6 f 37.26 x lo6
lo6 f 10.32 x lo6
lo6 k 31.49 x lo6
lo6 k 9.29 x lo6
lo6 k 31.29 x lo6
lo6 f 25.85 x lo6
lo6 31.22 X lo6
<’055
<.06’
<.Oo2
<‘187
‘Disintegrations per minute.
2Disintegrations per minute per gram wet weight of tissue.
3By Student’s t-test.
4The experimental values never exceeded the control.
TABLE 2. Specific insulin-binding sites in the papillary layer of the rat mandibular incisor as assessed by quantitative
light microscope radioautography
Experiment
No.
1A
Experimental
Control
1B
Experimental
Control
2A
Experimental
Control
2B
Experimental
Control
3A
Experimental
Control
3B
Experimental
Control
Animal
(g)
*
1’5~-~nsulin
SA (pCilpg)
“Hot”
insulin’
“Cold”
insulin
(PLg)
(Pd
Grain concentration’
(grains/3,450 pm’
mean f SD)
p3
87
90
155
155
100
100
50
355.07 i 50.87
215.13 i 60.24
< .001*
88
85
155
155
100
100
50
348.88 i 63.96
217.86 + 39.79
< ,001”
90
92
135
135
100
100
50
375.83 k 67.46
201.42 35.60
< .001*
90
90
135
135
100
100
50
522.35 f 128.49
319.00 i 53.98
< .001*
103
105
148
148
100
100
100
641.16 f 129.14
215.15 29.96
< ,001”
103
103
148
148
100
100
100
528.20 i 63.05
260.40 i 44.37
< ,001“
’“Hot” insulin, lZ51-labeledinsulin; “cold” insulin, nonradioactive insulin.
20ver 20,000 grains were counted for each experiment at a magnification of x 1,000.
3By Student’s t-test.
*The experimental value exceeded the control by factors of 1.6 and 1.6 in experiment 1, 1.9 and 1.6 in experiment 2, and 2.9 and 2.0 in
experiment 3.
6 or 8 weeks. Coverslips were mounted in Epon, which were 39, 81, and 112 days, respectively, for the three
experiments.
was Dolvmerized overnight at 65°C.
F& efectron microscope radioautography silver-to-gold
Analysis of Electron Microscope Radioautographs
interference color sections were cut with a diamond knife
The radioautographs from the papillary layer of the
and prepared according to Kopriwa (1973). Fine-grain
development was carried out by the Agfa-Gevaert 30- incisor maturation zone were photographed without bias
lution-physical” technique as described by Kopriwa wherever silver grains were seen. The initial magnifi(1975). The exposure times for these radioautographs cation was 16,000x and the negatives were enlarged to
132
B. MARTINEAU-DOIZE, M.D. McKEE, H. WARSHAWSKY, AND J.J.M. BERGERON
50,000x magnification. A total of 542 silver grains were
quantitated by direct scoring of the structures underlying the grains (Bergeron et al., 1977).The silver grains
consisted either of single, compact spherical silver deposits or small clusters of two or more silver deposits. A
cluster of silver deposits was interpreted as one silver
grain when it fit within the space of a silver bromide
crystal (Kopriwa, 1975). To assign a cluster to one silver
grain and to determine its center, a transparency with a
7.0-mm circle (corresponding to the mean 140-nm diameter of a silver bromide crystal a t 50,OOOx magnification) was placed over the grain. The shortest distance
from the geometrical center of each silver grain to the
luminal endothelial cell membrane and to the abluminal membrane adjacent to the extracellular space
outside the capillary in the papillary layer was measured with a Bausch and Lomb measuring magnifier.
RESULTS
Content of Radioactivity Present in Fixed Liver and
Kidney Tissues
TABLE 3. Grain counts over osteoblasts in the proximal
metaphysis of the tibia after injection of '251-insulin
Experiment
No.
1A
Experimental
Control
1B
Experimental
Control
2A
Experimental
Control
2B
Experimental
Control
Light Microscope Radioautography
In the experimental rats, most silver grains were observed over the periphery of the hepatocytes and the
adjacent sinusoids. The control rats showed numerous
grains over the sinusoids, while only a few grains were
localized over the hepatocytes. This grain distribution
was similar to that described previously (Bergeron et
al., 1977).
Kidney
An intense radioautographic reaction was present over
the brush border of the proximal convoluted tubules
from both experimental and control rats. The reaction
intensity over cross-sectioned tubules was about the
same in both experimental and control animals. These
reactions thus represented nonspecific binding of labeled insulin to high-capacity sites and probably were
related to the resorption and degradation of the hormone from the urinary filtrate (Bergeron et al, 1980a).
Bone
A moderate radioautographic reaction was found over
the tibia1 bone of both experimental and control animals
(Figs. 1, 2). The silver grains were located over the
prebone and the adjacent bone covering the calcified
cartilage of the mixed spicules in the proximal metaphysis of the tibia. Since the concomitant administration of a n excess of unlabeled insulin together with the
labeled insulin did not produce a competitive inhibition
of the bone labeling, it was concluded that the reaction
represented nospecific binding to high-capacity sites.
Silver grains also were present over the osteoblasts
from experimental rats. Grain counts over the osteoblasts in experimental rats exceeded the counts in control rats by factors of 1.5 to 1.8. Therefore, these
osteoblast reactions represented binding of labeled insulin to specific sites (Figs. 1,2, Table 3). Chondrocytes,
Grain concentration
(grains1344 pm2
f SD)
P
8
8
85.05 f 23.15
48.50 & 15.32
<.ool
8
8
78.40 f 18.54
44.71 f 11.60
<.ool
6
6
54.31 f 13.00
34.75 f 9.85
<.ool
6
6
44.81
29.43
+ 9.80
+ 9.60
< .001
TABLE 4. Number of grains' over the predentin of the
mandibular incisor of rats iniected with '251-insulin
The radioactivity in experimental and control rats was
assessed in fixed liver and kidney tissue using the Pack- Experiment
ard auto-a spectrometer. Significant reduction of radio- No.
activity was observed in the liver samples from control
rats, while the radioactivity in control kidney samples 1A
Experimental
increased (Table 1).
Liver
Exposure
time
(weeks)
Control
1B
Experimental
Control
2A
Experimental
Control
2B
Experimental
Control
3A
Experimental
Control
3B
Experimental
Control
Grain concentration
(grains/69O Fm2
mean + SD)
P2
197.8 & 25.6
243.0 f 38.8
< .033*
168.0 f 38.8
217.2 -1: 20.7
< .0017*
115.1 + 20.6
171.5 + 27.4
< .0001*
153.0 f 32.2
232.1 f 41.8
< .0001*
243.4 + 26.3
271.5 f 33.7
< .0261*
192.8 f 32.9
249.4 + 42.0
< .0018*
'Counted in light microscope radioautographs exposed for 21 days
over 6,900 pm' of predentin on the labial surface of the dentin.
'By Student's t-test.
*The experimental values never exceeded the control.
osteocytes, and the older bone matrix from experimental
as well as from control rats were not labeled.
Dentin
Numerous silver grains were observed over the predentin and adjacent dentin of the incisors of both experimental and control rats (Figs. 3, 4).Grain counts over
the predentin demonstrated that the experimental animals always showed a lower count than the control
animals (Table 4).Since no decrease was observed in the
number of grains over the predentin of the control animals, it was concluded that the reaction represented
nonspecific binding of the labeled hormone.
Maturation Zone of the Incisor Enamel Organ: General
Description of the Maturation Zone
At the light microscope level, the maturation zone of
the incisor enamel organ was characterized by a full
thickness of enamel covered by alternating bands of
INSULIN BINDING IN RAT TISSUES
Figs. 1, 2. Zone of mixed spicules from the proximal metaphysis of
the tibia of an experimental (Fig. 11 and a control animal (Fig. 2). Light
microscope radioautographs of 1-pm-thickEpon sections exposed for 3
weeks. x600. Many silver grains are present over the unstained prebone and the darkly stained bone of the experimental rat (Fig. 1, solid
arrow) and the control (Fig. 2, solid arrow).The periphery of osteoblasts
are labeled in the experimental animal (Fig. 1, open arrows) but are
unlabeled in the control (Fig. 2, open arrows). c, capillary; cc, calcified
cartilage; ob, osteoblast.
133
Figs. 3, 4. Dentin (D), predentin (Pd), and odontoblasts (Od) of the
lower incisor from an experimental (Fig. 3) and a control animal (Fig.
4). Light microscope radioautographs of 1-pm-thick Epon sections exposed for 3 weeks. ~ 6 0 0Numerous
.
silver grains are present over the
predentin and the adjacent dentin from both experimental and control
animals.
134
B. MARTINEAU-DOIZE, M.D. McKEE, H. WARSHAWSKY, AND J.J.M. BERGERON
En
Figs. 5, 6 . Enamel organ i n the maturation zone of the lower incisor
from an experimental (Fig. 5) and a control animal (Fig. 6). Light
microscope radioautographs of 1-Km-thick Epon sections exposed for 3
weeks. X520. In Figure 5, numerous silver grains overlie the periphery
of the capillaries (c) of the papillary layer (PL). A weaker reaction is
present over the cytoplasm of the ruffle-ended ameloblasts (ram). In
Figure 6, the number of silver grains associated with the capillaries is
reduced considerably, whereas i t remains the same over the ameloblasts. En, enamel space.
ruffle-ended and of smooth-ended ameloblasts (Josephsen and Fejerskov, 1977). The proximal ends of the ameloblasts were in contact with the papillary layer cells
which formed furrows perpendicular to the long axis of
the incisor. Within the furrows capillaries were distributed at approximately two levels (Figs. 5,6).
Under the electron microscope (Fig. 7), the vessels
appeared as typical fenestrated capillaries (Garant and
Gillespie, 1969). The narrow portion of the endothelial
wall frequently was pierced with fenestrae, closed by
diaphragms. Tight junctions connecting the neighbouring endothelial cells often were associated with marginal folds. A continuous lamina densa separated the
endothelial cells from the narrow connective tissue space
between the capillaries and the continuous lamina densa
over the papillary layer cells. The papillary layer cells
were separated from each other by intercellular spaces
filled with numerous microvilli. Neighbouring papillary
layer cells were connected by desmosomes. Numerous
coated vesicles, tubular structures, and mitochondria
were observed in the cytoplasm of the papillary layer
cells (Figs. 7, 8).
Light Microscope Radioautography of the Papillary Layer
A survey on the entire enamel organ revealed a strong
radioautographic reaction only over the papillary layer
of the maturation zone from the incisors of experimental
rats (Fig. 5). The silver grains seemed to be located at
the periphery of the capillaries (Fig. 5). Radioautographs
of the same regions from the control rats showed a much
INSULIN BINDING IN RAT TISSUES
Fig. 7. Electron micrograph of a portion of a fenestrated capillary in the papillary layer of the rat incisor
enamel organ. ~50,000.Thin diaphragms are stretched across the fenestrae (arrows). L, lumen; E,
endothelial cell cytoplasm; P, papillary layer cell; LD, lamina densa; m, mitochondria; cv, coated vesicle;
mv, microvilli. Inset Electron micrograph illustrating the presence of marginal folds and tight junctions.
~50,000.
135
136
B. MARTINEAU-DOIZE, M.D. McKEE, H. WARSHAWSKY,AND J.J.M. BERGERON
Fig. 8. Electron microscope radioautograph of the papillary layer of the enamel organ from the lower
incisor of a rat 2.5 minutes after the injection of lz51-insulin. X27,OOO. Silver grains overlie the thinwalled endothelial cell (E) of the capillary. luminal boundary (LB);pericapillary space boundary (PB); P,
papillary layer cell.
137
INSULIN BINDING IN RAT TISSUES
Lumen
-2000
1600
1200
800
400
0
400 '
800
1200
1600
2000
Distance from the luminal cell membrane ( O ) ( In nrn)
Fig. 9. Distribution of silver grains on either side of the luminal cell membrane (0) of
capillaries in the enamel organ from rats at 2.5 minutes after the injection of 1251-insulin.The
dashed vertical line represents the abluminal surface of the endothelial cell. Most of the grains
are close to the luminal cell membrane.
weaker reaction (Fig. 6). Quantitative analysis of the grainsl690 ,urn2),only a weak reaction was observed over
radioautographs indicated a significant decrease (1.5-to the smooth-ended cells (66 grains/690 pm2). On the other
2.9-fold) in total number of grains per 3,450 pm2 be- hand, while a weak reaction was seen over the enamel
facing the ruffle-ended cells (68 grains/690 pm2), many
tween the experimental and control animals (Table 2).
grains were present over the enamel facing the smoothElectron Microscope Radioautography of the Papillary Layer ended ameloblasts (192 grains/690 prn2). Since the silver
Electron micrographs of the papillary layer from the grain distribution and density were identical for both
maturation zone in experimental rats suggested that the experimental and control animals and thus the remost grains were associated with endothelial cells of the actions did not represent specific binding sites for the
capillaries (Fig. 8). Quantitative analysis of the electron hormone, the reactions did indicate the ability of molemicrographs by the direct scoring method showed that cules as large as insulin to enter the enamel organ and
65% of the silver grains were either over the endothelial the enamel.
cytoplasm or over the capillary lumen close to the endoDISCUSSION
thelial cell or over the pericapillary space.
The in vivo radioautographic method, with labeled
A more detailed analysis of grain distribution was
obtained by measuring the shortest distance between biologically active peptide hormones, has been used to
the center of each silver grain and the luminal (Fig. 9) recognize and quantitate receptors for several hormones
and abluminal (Fig. 10) endothelial cell membranes. in a variety of target cells (Bergeron et al., l977,1980a,b,
These grain density histograms demonstrated that the 1981, 1983; Bergeron and Posner, 1979; Warshawsky et
majority of silver grains were situated over cytoplasmic al., 1980). This specific binding assay is based on the
application of the law of mass action to a living animal.
components close to the luminal cell membrane.
Thus, in control animals the concomitant administraLight Microscope Radioautography of the Ameloblasts and
tion of high concentrations of unlabeled insulin (excess)
Enamel in the Maturation Zone
together with 1251-labeled insulin produces a competiThe cells of the enamel organ in the presecretory and tion for binding on the specific saturable receptor sites.
secretory zones, as well as the enamel in the secretory Experimental animals receive the same amount of lazone, were unlabeled. In the maturation zone, the beled insulin only. Specific binding is defined as the
smooth-ended and the ruffle-ended ameloblasts as well difference in bound labeled hormone between experias the enamel layer facing these cells showed different mental and control animals.
Three separate experiments with 1251-insulininvolvgrain distributions (Figs. 11, 12). Whereas numerous
grains were found over the ruffle-ended ameloblasts (241 ing a totaI of six experimental and six control rats gave
138
B. MARTINEAU-DOIZE, M.D. McKEE, H. WARSHAWSKY, AND J.J.M. BERGERON
W I
I
I
I
I
I
I
Extracellular Space
"
2000
1200
Is'oo
860
'
400
0
400
800
I200
I600
2000
Distance from the abluminol cell membrane ( 0 )( i n nrn)
Fig. 10. Distribution of silver grains on either side of the abluminal cell membrane (0) of
papillary layer capillaries of the enamel organ from rats 2.5 minutes after the injection of 1251insulin. The dashed vertical line represents the position of the luminal cell membrane. Again,
most of the grains lie closer to the luminal cell membrane.
Figs. 11, 12. Ameloblasts and the adjacent enamel from the maturation zone of the lower incisor of two control rats. Light microscope
radioautographs of 1-pm-thick Epon sections exposed for 3 weeks.
x 520. The enamel (En) facing the ruffleended ameloblasts (ram, Fig.
11) is almost unlabeled, while many grains overlie the enamel facing
the smooth-ended ameloblasts (Sam, Fig. 12). On the other hand, the
smooth-ended ameloblasts show only a few silver grains, while the
ruffle-ended ameloblasts are prominently labeled.
INSULIN BINDING IN RAT TISSUES
similar results. Competitive inhibition, indicating specific binding, was seen in relation to the hepatocytes,
osteoblasts, and capillaries of the papillary layer in the
maturation zone of the continuously growing incisor. In
the latter case, only these capillaries were labeled and
electron microscope radioautography confirmed that the
silver grains were over the endothelial cells of these
fenestrated capillaries. Nonspecific binding was observed within the proximal convoluted tubules of the
kidney, the prebone and adjacent bone layer in the mixed
spicules of the tibia, the incisor predentin and adjacent
dentin, and the enamel.
Specific Binding of Insulin to Hepatocytes and Osteoblasts
Using the in vivo assay with radioautography, specific
receptors for insulin have been found in the exocrine
pancreas, the liver, the columnar epithelial cells of the
intestinal tract, the adrenal cortex and medulla, and the
circumventricular organs of the brain (Bergeron et al.,
1977, 1980a,b; van Houten et al., 1979). In the present
experiments the results obtained previously with liver
were confirmed, thus strengthening the reliability of the
labeling seen in other tissues of the same animals.
Specific binding sites at or near the surface of osteoblasts were demonstrated by quantitation in the region
of mixed spicules from the proximal metaphysis of the
tibia (Table 3). Similar binding was also seen over osteoblasts from other sites in the tibia and in calvarial bone.
The demonstration of specific receptors for insulin in
bone secretory osteoblasts suggests a direct role for this
hormone in bone formation.
Specific Receptors for Insulin in Capillaries of the Enamel
Organ
Van Houten and Posner (1979) observed specific insulin binding sites in endothelial cells of blood vessels in
the brain. This is the only in vivo demonstration of
insulin receptors in vascular endothelium. However, in
vitro studies have demonstrated the presence of specific
insulin binding sites in cultured human and bovine endothelial cells from macro- and microvessels (Kaiser et
al., 1982a,b; Peacock et al., 1982; Pillion et al., 1982;
Carson et al., 1983; King et al., 1983; Soda and Tavassoli, 1983) as well as in the endothelium of microvessels
from isolated but intact rat heart (Bar et al., 1982). The
discrepancy between the in vitro results obtained from
cultured endothelial cells and the in vivo radioautographic assay may be due to the lack of sensitivity of
the radioautographic method in detecting very low levels of insulin binding (Bergeron et al., 1980a).The presence of specific insulin receptors in concentrations high
enough to be detected indicates a high-density receptor
site. Such a site was shown to be present on the luminal
surface of fenestrated endothelial cells lining capillaries
within the papillary projections of the enamel organ
only in the maturation zone. Analysis of electron microscope radioautographs showed that at 2.5 minutes after
the intravenous injection of 1251-insulin,most of the
silver grains were located over components of the endothelial cells close to the luminal surface.
Significance of Insulin Receptors in Capillary Endothelium of
the Enamel Organ
The enamel organ in the postnatal rat incisor remains
an embryonic system throughout life because of the
139
continuous eruption of this tooth. Blood vessels never
penetrate the basement membrane on the outer surface
of the enamel organ, but the vessels are brought closer
to the ameloblasts by invaginations of the outer dental
epithelium. These invaginations are developed most
strikingly in the maturation zone of the incisor, after
the ameloblasts have completed enamel secretion. The
epithelial tissues form high and regular ridges which
alternate with the insulin-receptor-containing capillaries. In longitudinally sectioned incisors, these ridges
appear as papillae; hence, this layer is termed the “papillary layer” of the enamel organ. Its function is completely unknown, but is appears to be associated with
the ameloblast layer in processes believed to occur in
the enamel to affect its maturation. These processes
include the loss of proteins and water, and the additional
growth of the hydroxyapatite crystals. Papillary layer
cells are surrounded by abundant extracellular space
and contain large numbers of coated pits and vesicles.
This appearance is suggestive of a resorptive function.
The direction of movement, either from the enamel or
from the vessels toward the enamel, or in both directions, has never been ascertained. The papillary layer
cells are separated from the insulin-receptor-containing
capillaries by 1)the basement membrane of the enamel
organ; 2) a narrow extracellular connective tissue space;
and 3) the basement membrane of the fenestrated capillaries. Within this environment the role of insulin may
be related to endothelial functions, to enamel organ
functions, or more specifically,to the function of enamel
maturation. Whatever its significance,the occurrence of
these receptors in close proximity to the enamel organ
in maturation is the first documentation of any hormonal receptors in tooth formation.
Significance of the Nonspecific Binding Sites
Four sites of nonspecific binding of the labeled insulin
molecules were noted. The proximal convoluted tubules
of the kidney cortex showed an intense, nondisplaceable
reaction in the vicinity of the brush border. Such reactions have been described with various labeled peptide
hormones (Bergeron et al., 1980a, 1983; Warshawsky et
al., 1980) and represent the normal protein-clearing
function of the proximal convoluted tubules. The ubiquitous occurrence of this reaction is used to monitor the
efficacy of the intravenous administration of labeled
peptide hormones for in vivo radioautographic assays.
The occurrence of nonspecific binding in prebone and
adjacent calcified bone and in the predentin and adjacent dentin and in the enamel of the incisors is an
interesting observation concerning the penetration of
relatively large protein molecules into these extracellular matrices. In each case an epithelium or epitheliallike
layer of cells separates the matrices from the blood supply. Hence, in order for a molecule of insulin with a
molecular weight of 5,700 daltons to enter these matrices it would have to pass very rapidly through the cellular layers. Once in the matrices insulin molecules
appear to diffuse throughout the collagenous stroma of
prebone and predentin, and between the maturing hydroxyapatite crystallites of bone, dentin, and enamel.
Furthermore, they must be bound strongly enough to
these matrices to survive histological processing. Since
all these events occur within 2.5 minutes after injection,
it appears that the cellular layer (osteoblastsin the case
140
B. MARTINEAU-DOIZE, M.D. McKEE, H. WARSHAWSKY. AND J.J.M. BERGERON
of bone, odontoblasts in dentin and ameloblasts in
enamel) do not form barriers to the free passage of the
lZ5I-labeledprotein molecules. The results regarding the
passage of insulin across the enamel organ are particularly noteworthy. The zone of maturation is characterized by the presence of two types of ameloblasts and
their modulation from the ruffle-ended to the smoothended types at least five times during the maturation
process (Josephsen and Fejerskov, 1977). The present
results showed labeled insulin in the enamel related to
the smooth-ended cells, but the relative absence of this
labeled molecule from the cell layer. In contrast, ruffleended cells showed labeling associated with the cells,
but almost no labeling over the enamel in contact with
this cell type. These findings agree with those of Sasaki
(19841, who used horseradish peroxidase, a protein of
40,000 molecular weight. Although junctional complexes are present a t both ends of the maturation ameloblasts, the proximal complexes of both cell types
(closest to the vascular supply) are permeable to large
protein molecules. However, only the distal junctional
complexes of the smooth-ended ameloblasts allow this
protein molecule to pass into the enamel.
ACKNOWLEDGMENTS
This work was supported by grants from the Medical
Research Council of Canada. Dr. B. Martineau-Doize is
a recipient of a Medical Research Council Fellowship.
The authors are indebted to Dr. B.I. Posner, Department
of Medicine, McGill University, for generously providing the iodinated insulin.
LITERATURE CITED
Bar, R.S., A. Derose, W.G. Owen, A. Sandra, and M.L. Peacock (1982)
Insulin receptors in microvessels of the intact heart. A kinetic and
morphometric demonstration. Diabetes, 31:(Suppl. 21173.
Bergeron, J.J.M., G. Levine, R. Sikstrom, D.O'Shaughnessy, B. Kopriwa, N.J. Nadler, and B.I. Posner (1977) Polypeptide hormone
binding sites in uiuo: Initial localization of 1251-labeledinsulin to
hepatocyte plasmalemma as visualized by electron microscope radioautography. Proc. Natl. Acad. Sci. U.S.A., 745051-5055.
Bergeron, J.J.M., and B.I. Posner (1979) In uiuo studies on the initial
localization and fate of polypeptide hormone receptors by the technique of quantitative radioautography. J. Histochem. Cytochem.,
27,1512-1513,
Bergeron, J.J.M., R. Rachubinski, N. Searle, D. Borts, R. Sikstrom,
and B.I. Posner (1980a) Polypeptide hormone receptors in uiuo:
Demonstration of insulin binding to adrenal gland and gastrointestinal epithelium by quantitative radioautography. J. Histochem.
Cytochem. 282324-835.
Bergeron, J.J.M., R. Rachubinski, N. Searle, R. Sikstrom, D. Borts, P.
Bastien, and B.I. Posner (1980b) Radioautographic visualization of
in uiuo insulin binding to the exocrine pancreas. Endocrinology,
107:1069-1080.
Bergeron, J.J.M., L. Resch, R. Rachubinski, B.A. Patel, and B.I. Posner
(1983) Effect of colchicine on internalization of prolactin in female
rat liver: An in uiuo radioautographic study. J. Cell Biol., 96,875886.
Bergeron, J.J.M., S. Tchervenkov, M.F. Rouleau, M. Rosenblatt, and
D. Goltzman (1981) In uiuo demonstration of receptors in rat liver
to the amino-terminal region of parathyroid hormone. Endocrinology, 109:1552-1559.
Carson, M.P., S.W. Peterson, M.E. Moynahan, and D. Shepro (1983)
Binding, internalization and degradation of 1251-insulinby cultured bovine aortic endothelial cells: effects of serotonin. In Vitro,
19,833-840.
Garant, P.R., and R. Gillespie (1969) The presence of fenestrated capillaries in the papillary layer of the enamel organ. Anat. Rec.,
163:7 1-80.
Josephsen, K., and Fejerskov, 0. (1977) Ameloblast modulation in the
maturation zone of the rat incisor enamel organ. A light and
electron microscopic study. J. Anat., 124:45-70.
Kaiser, N., I. Voldavsky, A. Tur-Sinai, Z. Fuks, and E. Cerasi (1982a)
Binding, internalization and degradation of insulin in vascular
endothelial cells. Diabetes, 31,1077-1083.
Kaiser N., I. Vlodavsky, A. Tur-Sinai, Z. Fuks, and E. Cerasi (1982b)
Insulin binding and degradation in vascular endothelial cells: Modulation by cell growth and culture organization. Endocrinology,
113:228-234.
King, G.I., S.M. Buzney, C.R. Kahn, N. Hetu, S. Buchwald, S.G.
MacDonald, and L.I. Rand (1983) Differential responsiveness to
insulin of endothelial and support cells from micro- and macrovessels. J. Clin. Invest., 71:974-979.
Kopriwa, B.W. (1973) A reliable, standardized method for ultrastructural electron microscope radioautography. Histochemie, 37: 1-17.
Kopriwa, B.M. (1975) A comparison of various procedures for fine grain
development in electron microscope radioautography. Histochemistry, 44:201-224.
Kopriwa, B.M., and C.P. Leblond (1962) Improvement in coating technique of radioautography. J. Histochem. Cytochem., 10:269-284.
Peacock, M.L., R.S. Bar, and J. Goldsmith (1982)Interactions of insulin
with bovine endothelium. Metabolism, 31.52-56.
Pillion, D.J., J.F. Haskell, and E. Meczan (1982) Cerebral cortical
microvessels: An insulin-sensitive tissue. Biochem. Biophys. Res.
Commun., 1043386-692.
Posner, B.I., Z. Josefsberg, and J.J.M. Bergeron (1978) Intracellular
polypeptide hormone receptors: Characterization of insulin binding
sites in Golgi fractions from the liver of female rats. J. Biol. Chem.,
253:4067-4673.
Sasaki, T. (1984) Morphology and function of maturation ameloblasts
in kitten tooth germs. Z-Anat., 138:333-342.
Soda, R., and M. Tavassoli (1983) Distribution of insulin receptors in
liver cell suspensions using a minibead probe. Highest density is
on endothelial cell. Exp. Cell Res., 145t389-395.
van Houten, M., and B.I. Posner (1979) Insulin binds to brain vessels
in uiuo. Nature, 282r623-625.
van Houten, M., B.I. Posner, B.M. Kopriwa, and J.R. Brawer (1979)
Insulin-binding sites in the rat brain: in uiuo localization to the
circumventricular organs by quantitative radioautography. Endocrinology, 105:666-673.
Warshawsky, H., D. Goltzman, M.F. Rouleau, and J.J.M. Bergeron
(1980) Direct in uiuo demonstration by radioautography of specific
binding sites for calcitonin in skeletal and renal tissues of the rat.
J. Cell Biol., 85:682-694.
Warshawsky, H., and G. Moore (1967) A technique for the fixation and
decalcification of rat incisors for electron microscopy. J. Histochem.
Cytochem., 15.542-549.
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