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Localization of enkephalin-like immunoreactivity in the cat carotid and aortic body chemoreceptors.

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THE ANATOMICAL RECORD 203:405-410 (1982)
Localization of Enkephalin-likelmmunoreactivity in the Cat
Carotid and Aortic Body Chemoreceptors
Department of Anatomy, The University of Texas Health Science Center at
San Antonio, San Antonio, T X 78284
The purpose of this study was to determine if enkephalin-like
immunoreactivity was present in the glomus cells of the carotid and aortic body
peripheral arterial chemoreceptors. Cat carotid and aortic bodies were reacted
with antisera to met- and leu-enkephalin using the indirect peroxidase-antiperoxidase immunocytochemical method of Sternberger (1979). Both the carotid and
aortic bodies demonstrated clusters of immunoreactive cells for both met- and leuenkephalin. Additionally, met-enkephalin-like immunoreactivity was observed in
many of the dense-core vesicles of the glomus cells of the carotid body. The glomus
cells of these chemoreceptors are known to contain catecholamines which may
modulate chemoreceptor activity. The presence of opioid peptide-like substances
co-existing with the glomus cell catecholamines, perhaps in the same vesicles, may
have important implications for a trophic influence of these peptides on glomus
cell chemoreceptor modulation.
The carotid and aortic bodies comprise several groups of specialized epithelioid cells
which are known t o function as arterial chemoreceptors. These chemoreceptors are sensitive
to changes in the PO>,pCO,, and pH of the arterial blood and form an important component
in a reflex arc which initiates appropriate pulmonary and cardiovascular responses (Comroe, 1974).
Both the carotid and aortic bodies contain an
abundance of catecholamines (CAs),especially
dopamine (DA)and norepinephrine (NE)(Hellstrom and Koslow, 1975; Hansen and Yates,
1975; Mills et al., 1978; Hansen and Christie,
1981).These CAs are stored in dense-core vesicles (Chen and Yates, 1969)which are the most
striking characteristic of the major parenchymal cell type, the glomus (Type I) cell. Glomus
cell CAs are thought to be involved in the initiation and/or modulation of the chemoreceptor
response by acting either transsynaptically or
in a paracrine fashion on adjacent glomus cells
and afferent nerve endings (Osborne and Butler, 1975; McDonald and Mitchell, 1975; Krammer, 1978). In addition to CAs, recent immunocytochemical studies have demonstrated that
enkephalin-like peptides are present in the
cells of the carotid body (Lundberg et al., 1979;
Wharton et al., 1980). This study was initiated
1)to confirm these previous results, 2) to determine where in the carotid body enkephalin-like
0003-276X/82/2033-0405$02.00- 1982 ALAN R. LISS. INC.
immunoreactivity was localized, and 3) to determine if enkephalin-like immunoreactivity
was present in the aortic body chemoreceptors
as well.
Light Microscopic Localization
Four young adult female cats weighing 2-3
kg were anesthetized with sodium pentobarbital (Nembutal, 25 mgikg iv) and then sacrificed
by an overdose just prior to removal of the carotid and aortic bodies. Following removal of
the tissues, one half of the carotid body and all
of the aortic body tissues were immersed in formol sublimate for 4 hours at room temperature. The tissue then was washed overnight in
phosphate-buffered saline (PBS)at pH 7.2 and
paraffin embedded. Five micron serial sections
were collected on chrome-alum-coated slides,
deparafinized, and stored in a freezer until processing for immunocytochemistry.
A modification of the indirect immunocytochemical method of Sternberger (1979) was
utilized. Staining of the tissue sections was
done by placing the slides in a humid chamber
with a moistened mat and applying the reagents directly onto the sections. Initially, sections were rinsed for 10 minutes in PBS and
then incubated for 30 minutes in 4% normal
Received October 8 1981 accepted March 15 1982
goat serum (Cappel Laboratories, Cochranville, PA) in PBS containing 0.3% Triton XlOO
(PBS-Tx).Following this incubation, the sections were washed in PBS and then incubated
in the presence of rabbit anti-met- or leu-enkephalin (Immuno Nuclear Corporation, Stillwater, MN) diluted 1:lOOO with PBS-Tx for 18
hours at 4°C. The sections then were washed in
PBS and incubated for 30 minutes with goat
antiserum to rabbit IgG (Cappel Laboratories,
Cochranville, PA) diluted 1:20 with PBS-Tx.
After another PBS wash, the sections were incubated in peroxidase-antiperoxidase diluted
1:40 with PBS-Tx for 30 minutes. Following a
PBS wash, the sections were incubated in a solution of diaminobenzidine (DAB) containing
H,O, (38 mg DAB in 50 ml of 0.05 M Tris buffer at pH 7.6 and 35 p1 of 30% H,O,) for 3
minutes. After a final PBS wash, the slides
were dried, coverslipped, and viewed in the
light microscope. Control incubations were performed by replacing the primary antisera with
normal rabbit serum N YO) or by incubation in
primary antisera which was preabsorbed to synthetic enkephalin prior to tissue incubation.
The primary antisera were produced in a rabbit with glutaraldehyde bovine thyroglobulin
conjugate. Met-enkephalin had a crossreactivity of 1.6% with leu-enkephalin and less than
0.002% with substance P and &endorphin.Leuenkephalin had a crossreactivity of 29% with
met-enkephalin and less than 0.002% with substance P, @-endorphin,and somatostatin.
Ultras tructural Localization
One half of the carotid body tissue was immersed in phosphate-buffered 4% paraformaldehyde, 0.5% glutaraldehyde for 4 hours a t
room temperature. The tissue then was washed
overnight in PBS at pH 7.2 and cut into very
thin slices with a razor blade.
Staining of the tissue sections was performed
in a small vial attached to a tissue-infiltrating
rotor. Initially, sections were rinsed for 10
minutes in PBS and then incubated for 2 hours
in 4% normal goat serum in PBS. Following
this incubation, the tissue slices were washed
in PBS and then incubated in the presence of
rabbit anti-met-enkephalin diluted 1:500 with
PBS for 18 hours at 4°C. The tissue then was
washed in PBS and incubated for 30 minutes
with goat antiserum to rabbit IgG diluted 1 2 0
with PBS. After another PBS wash, the tissue
was incubated in peroxidase-antiperoxidase diluted 1:40 with PBS for 30 minutes. Following
a PBS wash, the tissue was incubated in a solu-
tion of DAB containing H,O, for 5 minutes. After a final PBS wash, the tissue was processed
for electron microscopy by post-fixing the
tissue for 20 minutes in 1.5% osmium tetroxide in PBS. The tissue then was dehydrated in
a graded series of ethanols, passed through
propylene oxide, and embedded in Epon-Araldite. Thin sections were collected on copper
grids, either unstained or lightly stained with
lead citrate, and examined in the electron microscope. Control incubations were performed by
replacing the primary antisera with antisera
which was preabsorbed to synthetic met-enkephalin (SigmaChemical Company, St. Louis,
MO) prior to tissue incubation.
Following incubation in both met- and leuenkephalin antisera, a positive enkephalin-like
immunoreactivity was observed in the glomus
cells of the carotid body (Figs. 12).Immunoreactive cells often were organized in clusters
which were scattered throughout the carotid
body. The peroxidase-antiperoxidase reaction
product was distributed throughout the glomus cell cytoplasm, thereby circumscribing
the centrally located and unstained nucleus
(Fig. 2 ) . A larger number of immunoreactive
cell clusters were observed following metenkephalin incubation compared to leuenkephalin incubation.
Glomus cells of the aortic body chemoreceptors also exhibited both met- and leu-enkephalin-likeimmunoreactivity (Figs. 3,4). Both the
subclavian glomera (aortic body accumulations adjacent to the origins of the subclavian
arteries) (Fig. 3) and aorticopulmonary glomera (accumulations adjacent to the pulmonary arterial trunk and aortic arch) (Fig. 4) possessed immunoreactive cells.
None of the immunoreactive cells observed
in the carotid and aortic bodies following incubation in primary antisera were observed in
sections incubated in normal rabbit serum or
preabsorbed antisera.
Since met-enkephalin-likeimmunoreactivity
was greatest in the carotid body, we attempted
to localize met-enkephalinat the ultrastructural
level. Peroxidase-antiperoxidase reaction product was observed in many of the cytoplasmic
dense-core vesicles of the glomus cells (Figs.
5.6). However, not all of the dense-core vesicles
were stained. Immunostaining was absent in
other glomus cell organelles including the Golgi
apparatus, endoplasmic reticulum, and the
small (40-50 nm) electron-lucent vesicles (Fig.
Fig. 1. Clusters of immunoreactive carotid body glomus
cells following Incubation with met-enkephalin antiserum.
x 210.
Fig. 3. A small cluster of immunoreactive aortic (subclavian) body glomus cells following incubation with metenkephalin antiserum. x 525.
Fig. 2. Several carotid body glomus cells demonstrating
leu-enkephalin-like immunoreactivity. Note the peroxidaseantiperoxidase reaction product in the cytoplasm surrounding the central unstained nucleus. x 840.
Fig. 4. Met-enkephalin-like immunoreactive glomus
cells in the aorticopulmonary glomera. x 210.
Pig. 5. Ultrastructural localization of met-enkephalinlike imrnunoreactivity in some of the densecore vesicles of
the carotid body glomus cells (arrows). Reaction product is
absent in the smaller clear-core vesicles (arrowheads). the
Golgi, adjacent nerve fibers (*), and processes of the sup
,, ,-_.
Fig. 6. Higher power view of the met-enkephalin-like immunoreactivity observed in the dense-core vesicles of the
glomus cells. M, mitochondrion. x 84,000.
5). Likewise, no reaction product was observed
in adjacent nerve endings or in the supporting
(Type 11)cells (Fig. 5).
None of the immunoreactive sites observed
in the dense-core vesicles of the carotid body
glomus cells following incubation in met-enkephalin antisera were observed in tissue incubated in the preabsorbed antisera.
The results of this study confirm the findings of Lundberg et al. (1979) and Wharton et
al. (1980) with regard to the presence of enkephalin-like immunoreactivity in the glomus
cells of the cat carotid body. Additionally, the
results of this study demonstrate for the first
time that met- and leu-enkephalin-like immunoreactivity also exists in the glomus cells of
the cat aortic body chemoreceptors. Finally,
and perhaps most importantly, we have d e m
onstrated met-enkephalin-like immunoreactivity in the dense-core vesicles of the carotid
body glomus cells. These dense-core vesicles
have been shown to contain the CAs which are
present in the glomus cells (Chen and Yates,
1969). Therefore, a t least some of these CAcontaining dense-core vesicles also may contain an opioid peptide-like substance which coexists with the glomus cell CAs.
Functionally, little is known about the role of
opioid peptides in the glomus cells of the peripheral arterial chemoreceptors. However,
opiates do depress respiration (Florez and
Mediavilla, 1977) and both met- and leu-enkephalin decrease the spontaneous chemoreceptor discharge frequency in the carotid sinus
nerve (McQueen and Ribeiro, 1981).The intensity and duration of the depression evoked by
the enkephalins is reduced following naloxone,
suggesting that these peptides act at opiate receptors in the carotid body (McQueen and Ribeiro, 1981). Moreover, opioid peptides affect
the release of classical neurotransmitters (Eidelberg, 1976). In this regard, it is interesting
that we have demonstrated the presence of
met-enkephalin-like immunoreactivity in the
same dense-core vesicles which are thought to
contain CAs. The co-existence of CAs and the
opioid-like peptides have been reported in the
adrenal medulla, with the highest specific activity in the purified adrenomedullary chromaffin granule fraction (Viveros et al., 1979).
Opioid peptides and NE also have been identified biochemically in sympathetic nerves (Wilson et d., 1980) and these investigators suggest that opiate-like peptides and NE probably
co-exist in the large dense-core vesicles of the
nerve. Other paraneurons (Fujita and Kobayashi, 1979), such as the small intensely fluorescent (SIF) cells of autonomic ganglia, also
contain opioid immunoreactivity in addition to
a classical neurotransmitter such as a CA or
acetylcholine (Hokfelt et al., 1980). Therefore,
our demonstration of met-enkephalin-like immunoreactivity in dense-core vesicles which
probably contain CAs strongly supports the
hypothesis that this peptide may function
with the CA as a co-transmitter. Perhaps the
association of CAs and peptides in glomus
cells is involved in the hypothesized modulatory role of these substances during chemoreception. For example, it has been suggested
that peptides may act as co-transmitters while
the amine acts as a primary transmitter (Livett
et al., 1981). A co-transmitter could function as
a trophic factor for the receptor of the primary
transmitter, thereby modulating the sensitivity of the receptor (Costaet al., 1981).Certainly,
further investigation into this intriguing possibility warrants consideration.
The authors wish to thank Ms. Gwynne
Duke for her excellent technical assistance and
Ms. Nora Dimas for typing the manuscript. Dr.
Hansen also wishes to thank Dr. T.H. Williams, The University of Iowa College of Medicine, for graciously permitting him to learn
immunocytochemistry in his laboratory and
Dr. D.C. Herbert for his helpful discussion. Dr.
Karaseks permanent address is the Laboratory of Electron Microscopy, Department of
Pathological Anatomy, Institute of Pathology,
Medical Academy, Lodz, Poland.
This study was supported by NIH grant H L
25508. Dr. Hansen is the recipient of a NIH Research Career Development Award KO4 H L
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like, carotid, chemoreceptor, enkephaline, cat, localization, body, immunoreactivity, aortic
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