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Degranulation of endothelial specific granules of the toad aorta after treatment with compound 4880.

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T H E ANATOMICAL RECORD 203:197-204 (1982)
Degranulation of Endothelial Specific
Granules of the Toad Aorta After Treatment With
Compound 48/80
Department of Anatomy. Uniiieriity of Occiipational and Enuirunmental
Health, Sthool of Medicin<,.Kitukyu\hu, Japan 8 O ï
Incubation of isolated toad aortas in Ringer solution containing
compound 48/80, a histamine liberator, resulted in marked degranulation of endothelial specific granules.
Since incubation of these vessels in Ringer solution only did not show significant morphologic changes in these granules, these findings suggest that the degranulation was induced by histamine release from the granules, as in the case for
mast cel1 degranulation, and that endothelial specific granules are a storage site of
histamine in the toad aorta.
The present morphologic data were supported by preliminary chromatography,
which showed appreciable concentrations of histamine in the granule-containing
pellets of subcellular fractions of homogenized toad aortas.
Since the first description of endothelial specific granules by Weibel and Palade (1964),the
existence of similar cytoplasmic components
in endothelial cells of various blood and lymphatic vessels has been described by many
workers (Zelickson, 1964; Fuchs and Weibel,
1966; Kühnel, 1966; Fujimoto, 1967; Oehmke,
1968; Burri and Weibel, 1968; Piezzi e t al.,
1969; Lemeunier et al., 1969; Bertini and
Santolaya, 1970; Matsuda and Sugiura, 1970;
Steinsiepe and Weibel, 1970; Tabuchi and
Yamamoto, 1974).
Several histochemical and biochemica1 approaches have been made on these granules to
elucidate their exact nature. Lemeunier e t al.
(1969) have denied the possibility that these
granules might be lysosomal bodies, since they
did not show a positive reaction for acid phosphatase activities. Burri and Weibel (1968)
have suggested that these granules might contain "a procoagulative substance," since epinephrine-perfused rabbit aortas released a coagulation-activating substance int0 t h e
perfusate concomitantly with a marked decrease in number of the endothelial specific
Recently, we observed a remarkable increase
in number of endothelial specific granules similar in ultrastructure to those described by
others in the postnatal rabbit umbilical veins
between 2 and 10 days after birth (Takeshige
and Fujimoto, 1977;Fujimoto et al., 1978).The
0003-276X/82/2032-0197$02.50 C 1982 ALAN R. LISS. INC.
increase was much more pronounced in regions
where vascular obliteration was actively progressing. Since concentrations of these granules were not found in the late prenatal vessels
except for a few immature forms near Golgi regions and since almost al1 granules have disappeared around 30 postnatal days, such a
temporary increase in number of granules in a
limited postnatal stage suggests that they
play an important role in the postnatal obliteration of this vessel.
I t is wel1 known that endothelial contraction
can be induced by biogenic amines such as
histamine, serotonin, and bradykinin in some
vertebrates, including frogs (Majno et al.,
1967, 1969), and roles of histamine in the
regulation of vascular tonus have been proposed by many workers. Furthermore, appreciable concentrations of histamine were assayed in the homogenates of rat and rabbit
blood vessels by chromatographic techniques
(Howland and Spector, 1972).
On these grounds, we tried incubation of
toad aortas in Ringer solution containing compound 48180. The purpose of this experiment
was to examine the ultrastructural effects of
this histamine liberator on the morphology of
endothelial specific granules and to throw
some light on our hypothesis that endothelial
Keceived July 20. 19x1; accepted Fehruary 2 4 . 1982.
specific granules in various vertebrates might
be histamine storage particles.
Abdominal aortas of the toads, Rana catesbiana, were isolated and incubated in Ringer
solution containing compound 48/80 (1, 2, 10,
100, and 500 pgiml Ringer) for 5 min. For control experiments, the vessels were incubated in
Ringer solution without the drug. After incubation, materials were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer for 2 hr,
postfixed in 1%osmium tetroxide in the same
buffer for 1 hr, dehydrated in graded concentrations of acetone, and embedded in epoxy
resins. Sections were made on a Sorvall MT-1
ultramicrotome, stained both with uranyl
acetate and lead acetate, and observed either
with a Hitachi 12-A or a Hitachi 500 electron
The preliminary high-performance liquid
chromatography (HPLC)was done to correlate
with the present morphologic studies. The
toad aortas in 0.01 M tris-HC1 buffer containing 0.25 M sucrose (pH 7.4) were homogenized
and then centrifuged a t 80,000g for 10 min.
One-half of the precipitates was prepared for
electron microscopic samples t o confirm that
they were rich in the intact endothelial specific
granules, together with contaminant mitochondria, and the other half for chromatographic analyses was homogenized again in 0.4 M
perchloric acid and centrifuged at 10,000g for
10 min. After basic amines were extracted
from the supernatants by a column chromatograph (Amberlite CG 50, 10mm X 10 mm),
they were condensed by a freeze-drying
method and reacted with o-phthalaldehyde
and 2-mercaptoethanol (OPT-2ME)for the formation of histamine fluorescence. The sample
solution (pH 5.0) was injected int0 a Hitachi
638-50 HPLC (a separation column was filled
with Unisil P H ) equipped with a Hitachi
650-10 LC fluorometric detector (excitation
355 nm, emission 440 nm).
Endothelia incubated in Ringer
solution only
Incubation of the isolated vessels resulted in
a marked vascular contraction, and nuclear regions of the endothelial cells were protruded
int0 the vascular lumen (Fig. 1). However, we
found basically no inconsistencies with the
neneral cvtodasmic features of toad arteries
reported by &hers (Stehbens, 1965; Piezzi e t
al., 1969; Steinsiepe and Weibel, 1970) in our
materials. Each endothelial cell contained
abundant osmiophilic granules, which were
round or rodlike in shape and bounded with a
single limiting membrane (Fig. 2). Concentrations of these granules greatly varied from cell
to cell. The cytoplasm of some endothelial cells
contained a large number of the specific granules but others contained only a few. Most
granules had a fine granular matrix (Fig. 2),
but a few clearly showed microtubular structures approximately of i00Ä inner diameter in
their interiors. Incubation only in Ringer solution did not result in significant ultrastructural changes in these granules.
Endothelia incubated i n Ringer solution
containing compound 48/80
In specimens treated with lower doses of the
drug (1pg and 2 pg), the outline of the apical endothelial cell surface became irregular owing to
the occurrence of many short cytoplasmic projections (Fig. 3). The ultrastructure of almost
al1 granules was not much different from that
of the controls described above. With a few exceptions, altered granules with a somewhat
swollen appearance and decreased electron
density were observed (Fig. 3). In these granules, microtubular structures were frequently
present. However, such altered granules were
always “intracellular” and we did not find any
contact with or opening to the apical cell membrane in materials treated with low doses of
compound 48/80.
In specimens treated with 10 pg of the drug,
the increase in number of altered intracellular
granules as described above was apparent
(Fig. 4). Many granules contained fibrillar
masses of low electron density with a widened
space separating the granular matrix from the
limiting membrane (peripheral halo). With
progressing degranulation, fusion of the
adjacent altered granules and contact with or
close apposition to the apical cell membrane
were seen (Fig. 5). One end of such fused granules was often opened t o the extracellular
space with the extrusion of the granular
In specimens treated with 100 pg and 500 pg
of the drug, the general morphology of the degranulation process was fundamentally the
same as t hat of specimens treated with 10 pg,
but the degree of alteration was more pronounced. In an extreme case of a successive
fusion of adjacent altered granules and their
extrusion int0 the extracellular space, a
greater part of the cell cytoplasm was occupied
- large
- membrane-bound anastomosing
Fig. 1. The endothelium contains abundant specific grandes. Incubation in Ringer solution only does not result in significant degranulation processes of the granules a s shown in this figure. X 6,000.
cavities containing remnants of altered
granular contents (Fig. 6). In addition, fusion
of the altered grandes with the basal cel1 membrane of the endothelial cells and extrusion of
the granular masses int0 the subendothelial
connective tissue were observed (Fig. 7).
Chromatographic separation of histamine
in the endothelial grandes
A half of the precipitate was observed with
the electron microscope and it was confirmed
that they were rich in the endothelial specific
After the other half of these granule-containing pellets were reacted with OPT-ZME for the
formation of histamine fluorescence, the sample solution was injected int0 the HPLC equipped with the fluorometric detector. Chromatographic separation revealed a fluorescent peak
Fig. 2. The grandes are bounded with a single iimiting
membrane and have eiectron-dense fine granuiar matrix.
Incubated in Ringer soiution oniy. X 32.000.
Fig. 3. A swollen g r a n d e ( S G )with a wide peripheral halo and decreasing electron density is observed. Microtuhular
structures exist in the matrix of this altered grande. ï r e a t e d with 2 pg of compound 48/80. X 19,000.
that had the Same retention time as that of the
standard histamine solution (Fig. 8). By light
microscopy of a greater sampling of the nonincubated vessels, we seldom encountered mast
cells in the adventitia (Fig. 9).
Since Weibel and Palade (1964) described
membrane-bound endotheliai specific granules
in dog arteries, similar cytoplasmic components have been found in various blood and
lymphatic vessels. Although detailed descriptions of their ultrastructural features have
been made by previous morphologists, their
exact nature and functional significance
Fig. 5 . Successive fusions of adjacent altered granules
result in the formation of many large vacuoles (FG)in the cytoplasm. One end of such fused g a n u l e s is opened to the extracellular space with theextrusionof thegranular contents
(arrow).‘ïreated with 10 pg of compound 48/80. X 26.000.
Fin. 4. Altered manules a s shown in Firnre 3 increase in
Fig. 6. A greater part of the cytoplasm is occupied hy
large memhrane-hounded anastomosing cavities containing
remnants of altered granules. Treated with 500 /cg of com-
20 1
remain uncertain to the present time. The possibility that they contain acid phosphatase has
been denied by Lemeunier et al. (1969),and the
absence of catalase activity with the diaminobenzidine reaction suggests that they are different in nature from such organelles as peroxisomes in liver cells (Tabuchi and Yamamoto,
Burri and Weibel (1968) observed that epinephrine-perfused rabbit aortas deliver “a coagulation activating substance” in the perfusate with a marked loss of the specific
granules and suggested that they might contain some biologically active substances that
have an important role in the regulation of vascular tonus. A biochemica1 approach to elucidate the nature of endothelial specific granules
was tried by Bertini and Santolaya (1970).
They revealed that subcellular fractions of
these granules together with contaminant
mitochondria from homogenized toad aortas
are rich in “hypertensive activity.” These data
may indicate that endothelial specific granules
play some role in the regulation of blood
In our previous studies (Takeshige and Fujimoto, 1977; Fujimoto et al., 1978), postnatal
rabbit umbilical veins showed remarkable increase in number of endothelial specific granules originating from the Golgi apparatus.
Since concentrations of these granules were extremely rare in prenatal vessels, this finding
suggests that the increase might be related to
physiologic obliteration of this fetal circulation after birth.
I t is wel1 known that vascular endothelial
and medial cells in some vertebrates contract
under the influence of histamine-type mediators (Majno et al., 1969). In dogs, intravenous
injection of compound 48/80 causes a rise in
Fig. 7. The contents of altered granules ( S G )are exocytosed from the basal part of the endothelial cells (arrow).
Treated with 100 pg of compound 48/80. X 17,000.
Fig. 8. A chromatographic separation of OF‘T-2ME-histamine complex is shown. The vertical and horizontal axes
represent the fluorescent intensity and retention time, respectively. H. Histamine fluorescent peak of the sample
solution with the same retention time a s that of standard
histamine solution.
Fig. 9. A light micrograph of 1 pm section of the toad
aorta stained with toluidine blue. Mast cells were not ob.
served in this section. although two profiles of pigment cells
(P)were observed in the adventitia (A). I, Intima: M, Media.
x 200.
the pressure of the portal vein (Paton, 1951;
Rowley, 1964).If histamine-type mediators are
important in the regulation of vascular tonus
or blood pressure, knowledge of its disposition in various vessels might be of special
Compound 48/80, a condensation product of
p-methoxyphenylethylmethylamine and formaldehyde, is the most specific and least toxic
agent in various histamine liberators, and ultrastructural effects of this drug on mast cell
degranulation have been observed by several
workers (Bloom and Haegermark, 1965; Horsfield, 1965; Singleton and Clark, 1965;
Yamasaki et al., 1970; Röhlich et al., 1971).
I t is of interest that endothelial specific
granules of toad aortas showed several dynamic changes after treatment with this drug
in the present experiments. The formation of
the peripheral halo with a decreased electron
density, the fusion of such altered granules
with adjacent ones or with the apical cell membrane, and the extracellular release of the
granular contents were findings similar to
those observed in mast cell degranulation processes accompanying histamine release by the
Same drug (Bloom and Haegermark, 1965;
Horsfield, 1965; Röhlich et al., 1971).
In the case of mast cells, degranulation without accompanying histamine release can als0
be induced by sonic oscillation, lysis in distilled water, and incubation in the buffer solution
(Bloom and Haegermark, 1965). However, in
the present experiments, morphologic changes
of the endothelial cell granules were extremely
rare in specimens incubated only in Ringer
solution or those treated with smaller doses of
the drug, although a few granules appeared
slightly swollen. On the contrary, in the cells
treated with higher doses of the drug, the degranulation became more pronounced. These
findings strongly suggest that the degranulation may represent the liberation of histamine
from the endothelial specific granules.
Considerably high concentrations of histamine in the granule-containing pellets of the
homogenized toad aortas were determined by
the present chromatographic analyses. One
question that wil1 arise is whether this value
might be affected by contamination of mast
cell granules in the pellets. However, our chromatography has als0 demonstrated an appreciable concentration of histamine in the perfusate from compound 48180 - perfused toad
aortas (unpublished data). This would give a
validity to the tentative conclusion that the
histamine concentration of the pellets came
mainly from endothelial specific granules. Al-
though detailed descriptions of these chromatographic studies including those of the postnatal rabbit umbilical veins wil1 appear in a
separate article, the present preliminary chromatographic data may give further weight to
the morphologic data that suggest that endothelial specific granules may be a storage site
of histamine. More detailed morphologic and
biochemica1 studies are necessary to elucidate
the nature and functional role of endothelial
specific granules from the standpoint of comparative anatomy; such studies are now in progress in our laboratory.
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endothelial, treatment, compounds, specific, granules, aorta, 4880, toad, degranulation
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