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Induction of exocytosis from glomus cells by incubation of the carotid body of the rat with calcium and ionophore A23187.

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Induction of Exocytosis from Glomus Cells by Incubation
of the Carotid Body of the Rat with Calcium
and lonophore A231 87
MATS GRONBLAD,' KARL ERIK AKERMAN AND OLAVI ERANKO '
'Department of Anatomy and Department of Medical Chemistry, University of
Helsinki, Scltavuorenpenger 20A, 001 70 Helsinki 17, Finland
ABSTRACT
Carotid bodies from adult rats were electron microscopically
studied after incubation in glucose-containing salt solutions containing calcium andlor ionophore A23187 or neither. In the absence of the ionophore, adding
or omitting calcium had no effect on the fine structure of the glomus cells. Incubation in the medium containing both 1 mM calcium and the ionophore
caused the appearance of exocytotic membrane profiles in several glomus cells.
Exocytosis was not seen when only A23187 and endogenous calcium was present. For exocytosis to occur, calcium appeared to be essential and the event
seemed to be due to a rise in the intracellular calcium concentration caused by
the ionophore.
It has been pointed out that the glomus cells
of the carotid body share certain morphological features with neurosecretory cells (Normann, '76), neurons (Smith, '711, adrenal medullary cells (Grynszpan-Winograd, '71; Garcia
et al., '75) and paraneurons (Nagasawa, '771,
including the small intensely fluorescent
(SIF) cells in sympathetic ganglia (Matthews,
'78). The fine structure of the SIF cells closely
resembles that of the glomus cells of the
carotid body and morphological features suggest that both release their granular contents through exocytosis (Korkala et al., '73;
Eranko, '76). Exocytosis has been said to be
triggered by a rise in the free calcium concentration of the cytoplasm, which leads to a
fusion of granules with the plasma membrane
and finally to an extrusion of their contents
(Douglas, '74). With this release mechanism,
the product stored inside the secreting cell can
be transferred to its site of action without dispersion of the substance into the free cytoplasm preceding the release, preventing
waste and possibly deleterious effects of the
stored substance on the normal cell itself
(Normann, '76).
Ultrastructural features indicating exocytosis from glomus cells were reported by
Biscoe and Stehbens ('66) and Kobayashi
('68). Membrane fusion of the catecholamine
vesicles of the glomus cells have also been obANAT. REC. (1979)195: 387-396.
served in a freeze-fracture study of the carotid
body (Hansen, '77).
I t is not fully understood how an increased
intracellular calcium concentration initiates
the secretory process, although it has been
proposed that calcium acts at the inner surface of the cell membrane in the initial stage
of exocytosis by causing a fusion of the vesicle
and the plasma membranes (Normann, '76).
Calcium has been shown to aggregate isolated
chromaffin granules (Schober e t al., '77). Calcium may interact with specific proteins at
the plasma membrane and/or the granule
membrane (Thorn et al., '78). Binding sites for
divalent cations have indeed been demonstrated on adrenal medullary granules (Morris and Schober, '77) and on the catecholamine-storing vesicles in the glomus cells of
the carotid body (Hess, '77).
In the present study, the ionophore A23187,
which acts as a freely mobile carrier of divalent cations across membranes (Reed and Lardy, '721, has been employed to examine in vitro
the effect of an increased calcium concentration on the fine structural morphology of
glomus cells. A23187 electroneutrally exchanges calcium ions against protons (Reed
Received Jan. 3, '79. Accepted Apr. 24, '79.
Reprint requests: Dr. M. Gronblad, Department of Anatomy,
University of Helsinki, Siltavuorenpenger ZOA, 00170 Helsinki 17,
Finland.
387
388
M. GRONBLAD, K. E. AKERMAN AND 0. ERANKO
and Lardy, '72) and it is thus able to equilibrate the calcium ion gradient created by native calcium transport systems. Therefore,
treatment of cells and tissues with this agent
can be expected to equilibrate the calcium ion
gradient across the plasma membrane. If this
is so, the net result after incubation in a medium containing calcium and A23187 would
be an increase in the cytosolic calcium concentration (Douglas, '74). A23187 has previously
been successfully used for the initiation of
secretion in several organs (Douglas, '74; GOmez-Puyou and Gomez-Lojero, '76). Since no
direct evidence of interdependence of exocytosis and calcium has been presented concerning the carotid bodies i t appeared to be of obvious interest to study whether A23187 might
also stimulate exocytosis from glomus cells.
To our knowledge, exocytosis has not previously been demonstrated fine structurally following incubation with A23187 in any aminestoring tissues other than mast cells (Cochrane and Douglas, '74; Behrendt et al., '77).
MATERIAL AND METHODS
Tissue preparation
Adult male Sprague-Dawley rats weighing
250-300 g were used for the experiment. After
sacrificing the animal by a sharp blow on the
back of the head, the carotid bodies were dissected within 1-2 minutes and they were then
immersed in the incubation medium.
Zncu bation
The incubation time was 10-15minutes, the
temperature 37°C and the incubation volume
1 ml. The medium was ventilated by constantly bubbling air into the solution. Four
media were used, all reagents being made of
commercial products of the highest analytical
grade. (1) 140 mM NaC1, 6 mM KC1, 1.5 mM
MgC12,1mM CaCl,, 6 mM glucose and 20 mM
Hepes-Tris buffer a t pH 7.4; (2) as medium 1
but without calcium; (3) as medium 2 but
with 16 pM of the ionophore A23187 (Eli Lilly
Co.); (4) as medium 2 but with both 16 p M
A23187 and 1 mM CaCl,. Twenty carotid
bodies were examined, 5 carotid bodies being
incubated in each medium. After incubation
the carotid bodies were transferred into the
fixative.
Fixation and electron microscopy
Fixation was carried out by immersion of
the carotid bodies into 2.5% glutaraldehyde in
0.1 M phosphate buffer, pH 7.2, for 2 hours a t
4°C. The tissue samples were then rinsed overnight in the buffer and further fixed in 1%
OsO, a t pH 7.4 for 1hour. The specimens were
then dehydrated in a graded series of ethyl
alcohol and propylene oxide and embedded in
an epoxy resin mixture. Ultrathin sections of
the organ were cut with a Reichert ultramicrotome, and the sections were stained on
grids with uranyl acetate and lead citrate. A
Philips 300 electron microscope was used for
the examination and electron micrography at
60 kv.
RESULTS
Control media
The incubation of the carotid body tissue in
the control media (1or 2) did not damage the
fine structure of the organ. No ionophore was
present in these media, and the presence or absence of calcium had no detectable effect on
the electron microscopic appearance of the
glomus cells. Dense-cored vesicles were in
both instances seen in abundance randomly
distributed in the cytoplasm of the glomus
cells (fig. 1). The ultrastructure was well preserved and the mitochondria appeared normal
with clearly visible cristae. The plasma membranes of adjacent glomus cells were separated by an intercellular cleft of approximately 15-20nm, as was ordinarily seen in untreated carotid bodies, and no signs of granule
extrusion by exocytosis was found when 100
glomus (Type I) cells in each of the five carotid
bodies were examined.
No obvious alterations were observed in the
distribution or number of the dense-cored vesicles of the glomus cells after incubation in
medium 3 containing the ionophore but no calcium (fig. 2). Neither were there any differences seen in the fine structure of the
dense-cored vesicles or the plasma membrane,
and no exocytotic figures were observed.
Effect of the ionophore and
1 m M calcium
When carotid bodies were incubated in medium 4 containing both 16 pM A23187 and 1
mM calcium chloride, there were distinct
changes in the fine structural appearance of
the glomus cells, as compared with those in
the other media. The dense-cored vesicles
seemed to be reduced in number and fusions
between dense-cored vesicles and the membrane of the glomus cell were observed (fig. 3).
Different stages of exocytosis (figs. 4-6, 8) appeared on glomus cells. However, not all
CALCIUM-INDUCED EXOCYTOSIS FROM GLOMUS CELLS
glomus cells incubated in medium 4 showed
signs of exocytosis, and no glomus cells appeared to be totally depleted of their catecholamine vesicles. Exocytotic profiles were never
observed i n unincubated control carotid
bodies or in carotid bodies incubated with the
ionophore only.
Near the sites of exocytosis either a cluster
of coated vesicles (fig. 6) or more commonly, a
single coated vesicle was sometimes seen. Occasionally, the site of exocytosis revealed a
coated pit (fig. 6) a t t h e base of the membrane
invagination of the glomus cell. Electrondense material was not always seen in the intercellular space a t presumed sites of exocytosis, although such material was often accumulated a t sites of the glomus cell membrane
where the intercellular space was wider (approximately 80-150 nm) than that normally
seen in control carotid bodies (15-20 nm). Intact dense-cored vesicles were not seen in the
intercellular space. Occasionally, a densecored vesicle had fused with the plasma membrane of the glomus cell opposite to the site of
membrane invagination (fig. 4). Membrane
profiles indicative of exocytosis were seen
most distinctly in peripherally located glomus
cells (figs. 6-8).
DISCUSSION
It was seen in the present study that incubation of carotid bodies with calcium and
the calcium-transporting ionophore A23187
resulted in the appearance of ultrastructural
features suggesting exocytosis from glomus
cells. No such features were observed in unincubated carotid bodies, nor in carotid bodies
incubated with the ionophore alone. Thus, calcium seems to be essential for this event to occur. Since calcium had no effect on glomus
cells in the absence of the ionophore, it can be
argued that A23187 was needed to raise the
intracellular free calcium sufficiently to initia t e exocytosis, in accordance with the role of
calcium in other secretory cell systems (Douglas, '74). After the short incubation and constant aeration of the medium used in the present study, the glomus cells retained their
viability and the fine structure of different
cell organelles was well preserved.
In view of the observations of the present study and earlier reports of exocytosis in
the carotid body (Biscoe and Stehbens, '66;
Kobayashi, '68) and the recent observations
on exocytosis in the small intensely fluorescent (SIF) cells of sympathetic ganglia (Mat-
389
thews, '781, which resemble glomus cells
(Eranko, '76; Yates e t al., '76; McDonald, '771,
i t appears that calcium-stimulated exocytosis
also occurs in glomus cells of the carotid body.
Since exocytosis can be induced by artificial
means upon incubation of the whole carotid
body in the presence of calcium and the
ionophore A23187, a simple method is available to study the mechanism of catecholamine
secretion from glomus cells. Using this method, the effect of various parameters such as
incubation time and concentration of calcium
in the medium on the extent of fine structurally observable exocytosis should be further
examined.
Several intercellular sac-like widened areas
were observed between glomus cells when incubation was done with calcium and the
ionophore. Similar intercellular widenings
with electron-densematerial in the intercellular space were noted by Grynszpan-Winograd
('71) at sites of exocytosis between cells of the
adrenal medulla. Whether all the intercellular widenings seen in the present study represented sites of exocytosis is not known, since
they did not always contain electron-dense
material. I t is possible that the contents of the
catecholamine granules of glomus cells quickly disperse or dissolve after their discharge
from the glomus cells.
ACKNOWLEDGMENTS
We wish to thank Mrs. S. Huhtaniitty and
Mrs. T. Stjernvall for valuable technical assistance in cutting thin sections for electron
microscopy. We also wish to express our gratitude to Dr. Robert Hamill of Eli Lilly Co. for
supplying the ionophore A23187.
The present study was supported by a
grant from Finska Lakaresallskapet to M. G.
and a grant from the Sigrid Juselius Foundation to 0. E.
LITERATURE CITED
Behrendt, H., W. Goertz and C. Stang-Voss 1977 Ultrastructural changes in isolated guinea pig mast cells
caused by ionophore A23187. Naunyn-Schmiedeberg's
Arch. Pharmacol., Suppl. 11,297: R 44.
Biscoe, T. J., and W. E. Stehbens 1966 Ultrastructure of
the carotid body. J. Cell Biol., 30: 563-578.
Cochrane, D. E., and W. W. Douglas 1974 Calcium-induced
extrusion of secretory granules (exocytosis) in mast cells
exposed to 48/80 or the ionophores A23187 and X-537A.
Proc. Nat. Acad. Sci. (U.S.A.),71: 408-412.
Douglas, W. W. 1974 Involvement of calcium in exocytosis and the exocytosis-vesiculation sequence. In: Calcium
and cell regulation. R. M. S. Smellie, ed. Cambridge Univ.
Press, pp. 1-28.
ed. 1976 SIF Cells. Structure and Function
Erhko, 0..
390
M. GRONBLAD, K. E. AKERMAN AND 0. ERANKO
of the Small Intensely Fluorescent Sympathetic Cells.
DHEW Publication No. (NIH) 76-942. U. S. Government
Printing Office, Washington, D. C., 260 pp.
Garcia, A. G., S. M. Kirpekar and J. C. Prat 1975 A calcium
ionophore stimulating the secretion of catecholamines
from the cat adrenal. J. Physiol., 244: 253-262.
Gomez-Puyou, A., and C. Gomez-Lojero 1976 The use of
ionophores and channel formers in the study of the
function of biological membranes. Current Topics in
Bioenergetics, 6: 222-257.
Grynszpan-Winograd, 0. 1971 Morphological aspects of
exocytosis in the adrenal medulla. Phil. Trans. Roy. SOC.
Lond. B., 261: 291-292.
Hansen, J.T. 1977 Freeze-fracture study of the carotid
body. Am. J. Anat., 148: 295-300.
Hess, A. 1977 The calcium binding sites of dense-core
vesicles in the catecholaminergic glomus cells of t h e rat
carotid body. Brain Res., 238: 555-560.
Kobayashi, S. 1968 Fine structure of the carotid body of
the dog. Arch. Histol. Jap., 30: 95-120.
Korkala, O., 0. Eranko, S. Partanen, L. Eranko and A. Hervonen 1973 Histochemically demonstrable increase in
the catecholamine content of the carotid body in adult
rats treated with methylprednisolone or hydrocortisone.
Histochem. J.,5: 479-485.
Matthews, M. R. 1978 Ultrastructural evidence for discharge of granules by exocytosis from small-granule-containing cells of the superior cervical ganglion in the rat.
In: Peripheral Neuroendocrine Interaction. R. E. Coupland and W. G. Forssmann, eds. Springer-Verlag, Berlin
Heidelberg New York, pp. 80-85.
McDonald, D. M. 1977 Role of glomus cells as dopaminergic interneurons in the chemoreceptive function of
the carotid body. In: Advances in Biochemical Psychopharmacology. E. Costa and G. L. Gessa, eds. Raven
Press, New York, 16: 265-274.
Morris, S. J., and R. Schober 1977 Demonstration of binding sites for divalent and trivalent ions on the outer surface of chromaffin-granule membranes. Eur. J. Biochem.,
75: 1-12,
Nagasawa, J . 1977 Exocytosis: The common release
mechanism of secretory granules in glandular cells,
neurosecretory cells, neurons and paraneurons. In:
Paraneurons. New Concepts on Neuroendocrine Relatives. S. Kobayashi andT. Chiba, eds. Suppl. Arch. histol.
jap., 40: 31-47.
Normann, T. C. 1976 Neurosecretion by exocytosis. International Review of Cytology, 46: 1-77.
Reed, P. W., and A. Lardy 1972 A23187: A divalent cation
ionophore. J. Biol. Chemistry, 24 7: 6970-6977.
Schober, R., C. Nitsch, U. Rinne and S. J. Morris 1977 Calcium-induced displacement of membrane-associated particles upon aggregation of chromaffin granulae. Science,
195: 495-497.
Smith, A. D. 1971 Summing up: Some implications of
the neuron as a secreting cell. Phil. Trans. Roy. Soc. Lond.
B., 261: 423-437.
Thorn, N. A., J. T. Russell, C. Torp-Pedersen and M. Treiman 1978 Calcium and neurosecretion. Ann. N. Y. Acad.
Sci., 307: 618-639.
Yates, R. D., J. A. Mascorro, J.T. Hansen and I.-L. Chen
1976 Comparison of the structure of carotid and subclavian bodies and abdominal paraganglia. In: SIF Cells.
Structure and Function of the Small Intensely Fluorescent Sympathetic Cells. 0. Eranko, ed. DHEW Publication No. (NIH), 76-946, pp. 54-65.
PLATE 1
EXPLANATION OF FIGURES
1 A glomus cell of a carotid body incubated in a control medium containing 1 mM calcium but no ionophore. Numerous dense-cored vesicles are seen randomly distributed in t h e cytoplasm. N = Nucleus of glomus cell. X 24,000.
2 A glomus cell from a carotid body incubated in a medium containing 16 @M A23187
but no calcium. Note similar distribution and morphology of dense-cored vesicles as
in the control carotid bodies (fig. 1). x 18,600.
CALCIUM-INDUCED EXOCYTOSIS FROM GLOMUS CELLS
M Gronblad, K. E Akerman and 0 Eranko
PLATE 1
391
PLATE 2
EXPLANATION OF FlGURES
3 Glomus cell after incubation in a medium containing both 16 pM A23187 and 1 mM
calcium. A dense-cored vesicle is in contact with the plasma membrane (arrow) and
another is in the process of being extruded (double-arrow). Note the scarcity of
dense-cored vesicles in the cytoplasm of the glomus cell. N = Nucleus of glomus
cell. x 25,000.
4 Higher magnification of the vesicle fusing with t h e plasma membrane marked with
a n arrow in figure 3. Note inward bulging of glomus cell plasma membrane opposite
to the site of fusion of the vesicle with the cell membrane. x 43,000.
5 Higher magnification of the vesicle marked with double-arrow in figure 3. x 42,000.
6 Coated pit a t bottom of invagination of glomus cell plasma membrane in a peripherally located glomus cell after incubation in a medium containing both calcium and
the ionophore A23187. Coated pit marked with arrows. CV, cluster of coated vesicles. x 40,000.
392
CALCIUM-INDUCED EXOCYTOSIS FROM GLOMUS CELLS
M Gronblad, K E Akerman and 0 Eranko
PLATE 2
393
PLATE 3
EXPLANATION OF FIGURES
7
Peripherally located glomus cell in a carotid body incubated with both calcium and
the ionophore A23187. A membrane invagination containing electron-dense material, possibly representing a site of exocytosis, is seen (arrow). N, nucleus of glomus
cell. x 35,000.
8 Higher magnification of membrane area marked with an arrow in figure 7.
x 74.000.
394
CALCIUM-INDUCED EXOCYTOSIS FROM GLOMUS CELLS
M Gronblad, K E Akerman and 0 Eranko
PLATE 3
395
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incubation, exocytosis, a23187, induction, carotid, glomus, body, rat, calcium, ionophore, cells
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