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Neuropeptide tyrosine (NPY)-like immunoreactivity in adrenal chromaffin cells and intraadrenal nerve fibers of rats.

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THE ANATOMICAL RECORD 214:321-328 (1986)
Neuropeptide Tyrosine (NPY)-Like
lmmunoreactivity in Adrenal Chromaffin Cells and
Intraadrenal Nerve Fibers of Rats
Department of Anatomy, School of Medicine, Niigata University, Asahimachi 1,
Niigata 951, Japan
The present peroxidase-antiperoxidase imniunohistochemical study
demonstrated that approximately 50%of the total chromaffin cells of the rat adrenal
medulla exhibited NPY-like immunoreactivity. The immunoreactive material was
localized in the core of the chromaffin granules as well as diffusely in the cytoplasm.
By combination of immunohistochemistry with noradrenaline-fluorescence microscopy, all NPY-immunoreactive chromaffin cells are nonfluorescent, indicating that
all NPY-chromaffin cells co-store adrenaline. A comparison of two consecutive sections, each of which was processed for the immunostaining with anti-NPY and antiMet-Enk-Arg-Gly-Leu antisera, respectively, indicated that NPY and preproenkephalin A and its derivatives coexist in approximately one-fifth of the total NPYimmunoreactive cells.
In addition to the NPY-immunoreactive cells, a plexus of NPY-immunoreactive
nerve fibers with varicosities was found in the subcapsular regions of the adrenal
gland. The nerve fibers were often associated with small blood vessels and extended
into the zona glomerulosa. Single NPY-immunoreactive fibers were sparsely distributed in the deeper regions of the cortex and in the medulla. Ganglion cells in the
adrenal gland were not seen exhibiting intensely positive NPY-like immunoreactivity. The NPY-immunoreactive nerve fibers contained abundant small clear vesicles
mixed with a few small and large granular vesicles. The immunoreactive material
appeared on the granular cores as well as in the axoplasm. The NPY fibers were
closely apposed to smooth muscle cells and pericytes of small blood vessels in the
cortex. They were sometimes seen in close apposition to the fenestrated endothelial
cells with a common basal lamina intervening. The NPY fibers also made synaptic
contacts with both cortical and chromafin cells.
The occurrence of several neuropeptides such as enkephalin (Kobayashi et al., 1983; Kondo et al., 1984;
Livett et al., 1982; Schultzberg et al., 19781, substance P
(Kuramoto et al., 1983, vasoactive intestinal polypeptide (VIP) (Holzwarth, 1984; Kondo et al., 19861, and
neurotensin (Lundberg et al., 1982a) has been demonstrated in the adrenal chromaffin cells of various mammals in which catecholamines had been thought to be
the dominant, if not exclusive, biologically active substances. Possible functional significances of these peptides have been proposed in several ways in relation to
the secretion of catecholamines Civett et al., 1983;
Schultzberg et al., 1978). In addition, a recent study has
revealed the occurrence of neuropeptide tyrosine (NPY)
(Tatemoto, 1982; Tatemoto et al., 1982)-like immunoreactivity in the chromaffin cells and nerve fibers of the
adrenal gland of several mammals (Varndell et al., 1984).
In the peripheral nervous system, NPY has been shown
to be a potent vasoconstrictor and to inhibit pancreatic
secretion (Lundberg and Tatemoto, 1982; Lundberg et
al., 1982b). In order to understand the functional significance of NPY in the fulfillment of the adrenal hormone
0 1986 ALAN R. LISS, INC
secretion, several morphological questions should be answered: Do NPY-like immunoreactive cells co-store noradrenaline or adrenaline? Do NPY cells co-store other
peptides whose presence in the medulla is known? Where
is NPY localized in the chromafin cells? What are the
ultrastructural characteristics of NPY-immunoreactive
nerve fibers, and what types of cells do the NPY fibers
make synaptic contacts with? In the immunocytochemical study of Varndell et al. (1984), NPY was colocalized
with noradrenaline and enkephalin in adrenal chromaffin cells, but the description was brief and no details
concerning the fine structure of NPY nerve fibers were
In a series of recent papers detailing the localization
of peptides in the rat adrenal gland, we have reported
the ultrastructure of enkephalin-, substance P-, and VIPlike immunoreactive cells and nerve fibers (Kondo et al.,
1984; Kondo et al., 1986; Kuramoto et al., 1985). The
present study represents the fourth in this series and
Received July 15, 1985; accepted October 18, 1985.
was undertaken to clarify the morphological characteristics of NPY-like immunoreactive cellular and neuronal elements and to answer those questions listed
Five male adult rats weighing 180-200 gm were used
in the present study. Under Nembutal(35 mgkg) anesthesia, the animals were perfused first with 200 ml of
physiological saline through the heart, followed by 200
ml of 4% paraformaldehyde in 0.1 M phosphate buffer,
pH 7.3. The adrenal glands were rapidly removed and
immersed in the same fixative for a n additional 2 hours.
The tissues, after rinsing in 0.1 M phosphate buffer for
1hour, were immersed overnight in the phosphate buffer
containing 30%sucrose; they were frozen in liquid nitrogen and 15-ym-thick sections were made on a cryostat.
The sections were incubated for 12 hours a t room temperature with rabbit anti-NPY antiserum (Amersham
International PLC, England) at a dilution of 1:1,600.
The sites of antigen-antibody reaction were visualized
by the peroxidase-antiperoxidase (PAP) method according to Sternberger (1979). For electron microscopy, the
sections were further postfixed with 1% osmic acid in
0.1 M cacodylate buffer, pH 7.4, for 20 minutes, after
completion of the PAP procedure. They were embedded
in Epon 812 according to conventional procedure and
ultrathin sections were examined after brief staining
with uranyl acetate.
For examination of the colocalization of NPY with
enkephalin or catecholamine, sections were mounted on
glass slides, covered with glycerin and subsequently
viewed and photographed with a fluorescence microscope (Leitz Orthoplan) equipped with appropriate filter
set. It is well established that fixation with 4% paraformaldehyde is adequate for demonstrating noradrenaline fluorescence in the adrenal medulla (Eranko, 1967;
Falck and Torp, 1961). After the fluorescent cells were
photographed, the sections were incubated with the antiNPY antiserum or with the rabbit anti-methionine-enkephaline-arginine-glycine-leucine(Met-Enk-Arg-GlyLeu) antiserum (N. Yanaihara, Shizuoka, Japan) at a
dilution 1:3,000. It is generally agreed that the occurrence of Met-Enk-Arg-Gly-Leu-like immunoreactivity
strongly suggests the presence of preproenkephalin A as
a precursor, and of its derivatives, including Met- and
Leu-Enk (Kilpatrick et al., 1981;Noda et al., 1982). For
quantitative analyses of the coexistence of NPY and
Met-Enk-Arg-Gly-Leu, ten sets of two consecutive sections with a thickness of 2.5 pm at intervals of 25 pm
were made from the adrenal medulla on a cryostat. One
of the consecutive sections in each set was immunostained with the NPY antiserum, and the other with
Met-Enk-Arg-Gly-Leu antiserum. All NPY-immunoreactive cells containing nuclear rofiles in a square field
with a size of 0.28 x 0.44 mm i?were counted first and
cells in a n adjacent section which corresponded to the
NPY-immunoreactive cells and simultaneously showed
Met-Enk-Arg-Gly-Leu-likeimmunoreactivity were subsequently counted.
The immunoreactive cells appeared in small groups or
singly and were distributed in the medulla without any
topographical relations to sinusoids or to the boundary
region between the cortex and the medulla (Figs. 1, 3).
The cells were polygonal in shape and lacked cytoplasmic processes. The intensity of the immunoreaction
varied from cell to cell and the immunoreactive material
within the cytoplasm appeared granular. All nuclei were
free of immunoreaction. In fluorescence microscopy, approximately one-fifth of the total chromaffin cells emitted blue-white fluorescence specific for noradrenaline.
The fluorescing noradrenaline cells occurred in small
groups or singly and were distributed throughout the
adrenal medulla (Fig. 2). After processing the same sections for immunohistochemistry, none of the fluorescent
noradrenaline cells were immunoreactive for NPY (Figs.
1, 2).
By comparing the two consecutive sections, each of
which was processed for the immunostaining with antiNPY and anti-Met-Enk-Arg-Gly-Leuantiserum, respectively, approximately one-fifth (16.1 & 6.9%, n = 10) of
the total NPY-immunoreactive chromaffin cells exhibited positive immunoreactivity to the Met-Enk-Arg-GlyLeu antiserum simultaneously (Fig. 4).
In immuno-electron microscopy of the NPY-positive
chromaffin cells, many of the cytoplasmic granules had
rounded cores with high electron density due to the
immunoreaction (Fig. 6). The intensity of the cellular
immunoreaction in light microscopy depended on the
number of the highly electron-dense granules in the
cytoplasm. The cytoplasm of the cells showed a coarse
appearance with moderate increase in electron density.
NPY-like lrnrnunoreactivity in lntraadrenal Nerve Fibers
A distinct plexus of NPY-like immunoreactive nerve
fibers with varicosities encircled small blood vessels
which penetrated the capsule and coursed in the subcapsular regions of the adrenal gland (Fig. 5). The immunoreactive nerve fibers extended into the zona
glomerulosa where they surrounded blood vessels and
cortical cells. Single varicose nerve fibers with the NPYlike immunoreactivity were distributed sparsely in the
zona fasciculata and reticularis of the cortex and in the
medulla. Ganglion cells exhibiting intensely positive
NPY-like immunoreactivity were not seen in the medulla or cortex.
With immuno-electron microscopy the NPY-positive
nerve fibers were identified as electron-dense profiles,
0.3-0.8 pm in diameter, and enclosed by thin processes
of Schwann cells (Figs. 7-12). The immunoreactive nerve
fibers contained abundant small clear vesicles, 45-50
nm in diameter, mixed with a few small granular vesicles of similar size and with larger granular vesicles,
90-100 nm in diameter. The immunoreactive material
was localized in the core of both small and large granular vesicles, and also in the axoplasm surrounding vesicles and mitochondria. However, the interior of the small
clear vesicles was free of the immunoreaction.
The NPY-immunoreactive nerve fibers often were
closely apposed to smooth muscle cells and pericytes of
small blood vessels in the cortex: a n interstitial space of
100-200 nm intervened between the components (Fig.
NPY-like Immunoreactivity in the Chromaffin Cells
9). The apposed surface of the nerve fibers was largely
Approximately 50% of all chromaffin cells in the ad- denuded of Schwann cell sheaths. Denuded nerve fibers
renal medulla exhibited NPY-like immunoreactivity. with NPY-like immunoreactivity sometimes were in
Figs. 3 , 4 . Immuno-light micrographs showing NPY-immunoreactive
Figs. 1 , 2. Immuno-light micrograph showing NPY-immunoreactive
chromaffin cells (Fig. 1) and fluorescence micrograph showing fluores- chromaffin cells (Fig. 3) and Met-Enk-Arg-Gly-Leu-immunoreactive
cent noradrenaline cells (Fig. 2) on the same section of the rat adrenal chromaffin cells (Fig. 4) on two consecutive sections of the rat adrenal
medulla. Note all NPY-immunoreactivechromaffin cells (*) are nonflu- medulla. Note some chromaffin cells (*I immunoreactive both for the
two antisera. x750.
orescent. V: sinusoidal blood vessels. ~ 3 1 0 .
Fig. 5. Immuno-light micrograph showing NPY-immunoreactive
nerve fibers with varicosities in the subcapsular region (C), the zona
glomerulosa (G),and fasciculate (F) of the rat adrenal cortex. Note the
close association of the immunoreactive nerve fibers with small blood
vessels (V). ~ 2 8 0 .
close apposition to the fenestrated endothelial cells of
cortical capillaries. The nerve fibers and endothelium
were separated by a 70-nm space, and a common basal
lamina intervened (Fig. 10). Immunoreactive nerve fibers were directly apposed to both cortical and chromaffin cells, but were separated by a n intercellular space
approximately 20 nm in width (Figs. 7, 8). Membrane
specializations were not found at the apposition site. On
several occasions, NPY-immunoreactive and nonreactive nerve fibers, or two NPY-immunoreactive nerve
fibers, were seen to be in direct contact with each other,
and simultaneously with cortical cells. At the contact
sites between two immunoreactive nerve fibers, a membrane density was seen on one of the apposed membranes (Figs. 11,12).
As controls for immunohistochemistry, sections of the
adrenal medulla were incubated with anti-NPY and
anti-Met-Enk-Arg-Gly-Leu antisera preabsorbed with
NPY and Met-Enk-Arg-Gly-Leu, respectively (10 pgiml
diluted at 1:1,600 and 1:3,000, respectively). Immunoreactive cells or fibers were not recognized in any portions of the adrenal gland.
fined to the chromaffin granules of the immunoreactive
cells. In previous studies, Met-Enk-Arg-Gly-Leu-likeimmunoreactivity also has been demonstrated to be localized within the chromaffin granules (Kobayashi et al.,
1983; Kondo et al., 1984). Furthermore, the co-storage of
serotonin in adrenaline cells recently has been demonstrated immunohistochemically (Verhofstad and Jonsson, 1983).These findings suggest that the four bioactive
substances coexist in a substantial number of chromaffin cells, possibly in chromaffin granules. The coexistence of various combinations of bioactive substances in
the adrenal chromaffin cells suggests the presence of
several subpopulations of chromaffin cells in terms of
bioactive substances contained, in addition to the classical adrenaline and noradrenaline cells.
The present finding that NPY coexists with adrenaline in adrenal chromaffin cells of rats is in contradiction to the previous study of the adrenal medulla of
horses by Varndell et al. (1984). They identified the
NPY-immunoreactive cells as noradrenaline-chromaffin
cells based on the fine structural differences in the chromaffin granules. According to the fine structural criteria for the identification of chromaffin granules in
specimens fixed with glutaraldehyde and osmium tetroxide, noradrenaline granules are characterized by
solid cores of high electron density which often are irregular in shape and located eccentrically. Adrenaline
granules, on the other hand, have rounded cores with
moderate electron density (Coupland et al., 1964). However, in the immuno-electron microscopic study by
By means of immunohistochemistry combined with
fluorescent microscopy, the present study has demonstrated that all NPY-immunoreactive chromaffin cells
also are adrenaline-containing cells, and that some of
the NPY-chromaffin cells co-store preproenkephalin A
and its derivatives. NPY -like immunoreactivity is con-
Fig. 6. Immuno-electron micrograph showing a NPY-immunoreactive chromafin cell (*) next to a nonimmunoreactive cell (Cn). Note
the increase in electron density of most chromafin granules and the
cytoplasm of the immunoreactive cell due to the immunoreaction.
Varndell et al. (19841, the specimens were fixed only
with aldehydes and, therefore, the resulting images did
not show any difference in electron density of two types
of granules. Nevertheless, those investigators did report
“noradrenaline granules” with irregularly shaped cores
in the horse adrenal medulla. The identification of the
Figs. 7, 8. Fine structures of the NPY-immunoreactive nerve fibers
synaptic contacts (dots) with nonimmunorective chromafin cells
(Cn). Note small (short arrows) and large (long arrows) granular vesicles intermingled with numerous small clear vesicles in the immunoreactive nerve fibers. ~ 2 3 , 0 0 0 .
(*) in
two cell types in that study, however, seems less reliable
than the noradrenaline fluorescence method employed
in the present study. This is one of the plausible explanations for the discrepancy.
An alternative explanation for the discrepancy is the
species difference between rats and horses. It is well
Figs. 9, 10. Fine structure of the NPY-immunoreactive nerve fibers
apposition t o pericyte (P)and smooth muscle cell (S)(Fig. 9)
(*) in close
and fenestrated endothelial cell (E) (Fig. 10) in the subcapsular region
of the rat adrenal cortex, n: nonimmunoreactive nerve fiber, Arrows
indicate endothelial fenestrae. x 23,000.
Figs. 11, 12. A NPY-imrnunoreactive nerve fiber (*) and nonimrnunoreactive fibers (n) (Fig. 11) or two adjacent NPY imrnunoreactive
fibers (*) (Fig. 12) in direct contact (arrows)with each other and simultaneously with cortica! cells (Co) containing lipid droplets 6).
Note the
membrane densification (double arrows) on one of the apposed plasma
membrane. x23.000.
known that the proportion of noradrenaline versus
adrenaline-chromaffin cells and the occurrence of chromaffin cells containing a given peptide in the adrenal
medulla varies in different species (Coupland, 1965;
Schultzberg et a]., 1978). It is not unlikely that the
combination of catecholamine and peptides co-stored in
chromaffin cells might differ in different species. Further examinations of the adrenal medulla of various
species by combined immunohistochemistry with fluorescence microscopy would seem necessary to understand more clearly the coexistence of NPY and catecholamines.
Since no ganglion cells with intense NPY-like immunoreactivity were found within the adrenal gland, the
intraadrenal NPY-immunoreactive nerve fibers are regarded as extrinsic in origin. A considerable number of
postganglionic neurons in the celiac ganglion and a
small population of neurons in the myenteric and submucous ganglion are known to exhibit intense NPY-like
immunoreactivity (Furness et al., 1983; Lundberg et al.,
1985). The neuronal connection between the adrenal
gland and the celiac ganglion is well established, and
serves as a route for nerve fibers to pass into and innervate the gland (Pick, 1970). In addition, conventional
electron microscopy combined with histochemical techniques has demonstrated the presence of some postganglionic noradrenergic nerve fibers in the adrenal
medulla (Prentice and Wood, 1975). It is, therefore, reasonable to assume that the intraadrenal NPY-immunoreactive nerve fibers are postganglionic, and that their
cell somas are located in the celiac ganglion. In support
of this assumption, the composition of vesicles in the
NPY fibers is quite similar to that of the postganglionic
noradrenergic nerve fibers; that is, the presence of small
and large granular vesicle's mixed with abundant small
clear vesicles. It is well established that noradrenaline
is stored in the small and large granular vesicles of the
postganglionic sympathetic nerve fibers (Fried, 1980;
Smith, 1972). The occurrence of the immunoreactive
material in small as well as large granular vesicles of
the NPY fibers suggests the coexistence of NPY with
noradrenaline in the vesicular compartments of the
autonomic nerve fibers. This is in marked contrast to
the coexistence of NPY with adrenaline in the chromaffin cells, which is homologous to the postganglionic neurons in ontogeny.
The close apposition between NPY fibers and vascular
smooth muscles suggests that NPY may exert its vasomotor effect as a cotransmitter with noradrenaline. Local intraarterial infusion of NPY is known to induce a
dose-dependent vasoconstriction in the cat submandibular gland (Lundberg and Tatemoto, 1982; Lundberg et
al., 198213)and a similar effect might be expected in the
intraadrenal blood vessels.
The close apposition between NPY fibers and cortical
fenestrated endothelial cells, with a common basal lamina, is quite similar to the topographical relation of the
neurohypophyseal axon terminals and fenestrated capillaries (Bloom and Fawcett, 1975).This similarity infers
that NPY fibers in the adrenal cortex may exert their
effect by way of endocrine secretion.
The present study further disclosed the direct apposition of NPY fibers to certain cortical and medullary
chromaffin cells. The direct apposition is functionally
regarded as a synapse, i.e., the site where synaptic re-
lease and regulation take place, although morphologically, it lacks such membrane specializations as seen in
typical synapse in the central nervous system (Pappas
and Waxman, 1972; Unsicker, 1971).
Based upon the present findings, it might be possible
to suggest several ways of action of NPY in the adrenal
gland, i.e., 1) by being secreted from NPY-chromaffin
cells and influencing the secretory activity of adjacent
chromaffin cells via paracrine or hemocrine action, 2 ) by
being released from the NPY nerve fibers and exerting
its effect on postsynaptic chromaffin cells and cortical
cells through synaptic and endocrine action, and 3) by
being released from the NPY fibers and influencing the
vascular smooth muscle cells to regulate the intraadrenal circulation. In addition, from the finding on the
coexistence of more than two bioactive substances such
as NPY and catecholamines in chromaffin cells and
nerve fibers, it is also necessary to consider its own effect
on NPY-secreting cells and nerves themselves by autoregulation. Physiological and pharmacological analyses
of the effect of NPY in the secretory activity of the
adrenal gland are necessary to examine each possible
way of action of NPY proposed above and those analyses
would be crucial to understand more precisely the nature of the adrenal gland.
The authors wish to thank Dr. N. Yanaihara for a kind
gift of anti-Met-Enk-Arg-Gly-Leuantiserum. They also
acknowledge the technical assistance and secretarial
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