Vol. 283, Issue 5, H1811-H1818, November 2002
Presence of cellular renin-angiotensin system in chromaffin
cells of bovine adrenal medulla
Jun Ming
Wang1,2,
Dirk
Slembrouck1,
Junhui
Tan2,
Lut
Arckens3,
Frans H. H.
Leenen2,
Pierre J.
Courtoy4, and
Werner P.
De
Potter1
1 Laboratory of Neuropharmacology and Neurobiology,
Department of Medicine, Universitaire Instelling Antwerpen,
University of Antwerp, B-2610 Antwerp,
Belgium; 2 Hypertension Unit,
University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7,
Canada; 3 Laboratory of Neuroendocrinology
and Immunological Biotechnology, Zoological Institute, Katholieke
Universiteit Leuven, B-3000 Louvain, Belgium;
4 Cell Biology Unit, Louvain University Medical
School, B-1200 Brussels, Belgium
 |
ABSTRACT |
The presence of a local renin-angiotensin
system has been established in organs that serve as angiotensin
targets. In this study, the expression of angiotensinogen mRNA and
subcellular localization of renin, angiotensin-converting enzyme, and
angiotensin II were investigated in bovine adrenal medullary cells in
primary culture. By light microscopy, expression of angiotensinogen
mRNA, immunoreactive renin, angiotensin-converting enzyme, and
angiotensin II were readily detectable only in the chromaffin cells.
The density distribution of renin and angiotensin II in sucrose
gradients suggested a concentration in chromaffin granules, a
localization directly confirmed by immunoelectron microscopy. Reverse
transcriptase-polymerase chain reaction and sequencing confirmed
the expression of angiotensinogen in bovine chromaffin cells and the
adrenal medulla. In addition, in vitro autoradiography indicated that
both angiotensin-converting enzyme and angiotensin type 1 receptors
were present in the adrenal medulla. These results provide the first
direct evidence that chromaffin cells in the adrenal medulla are not
only the target for angiotensin but should also be considered as
potential local angiotensin-generating and -storing cells.
in situ hybridization; immunocytochemistry; radioligand
autoradiography; angiotensinogen
| |
INTRODUCTION |
THE
RENIN-ANGIOTENSIN SYSTEM (RAS) exerts a wide variety of actions
on the cardiovascular, renal, endocrine, and nervous systems in both
physiological and pathological conditions (13). These actions are mediated by ANG II and other shorter angiotensin fragments (31, 33). There is now considerable evidence that ANG II
and its active shorter fragments are produced and act locally in a number of target organs including the brain, anterior pituitary, testes, ovaries, adrenal glands, sympathetic ganglia, kidneys, heart,
blood vessel walls, and eyes (2, 11, 15, 34, 38). However,
where and how angiotensins are locally formed is still controversial.
One hypothesis proposes that angiotensinogen (AGT)
or ANG I or ANG II
directlysecreted from one type of cell is taken up from the
extracellular fluid and subsequently converted into the active peptides
by an adjacent, different cell type (8, 11). In contrast,
abundant experimental evidence supports the proposal that a complete
intracellular RAS can be responsible for the formation of angiotensins
in a single cell (2, 6, 7, 12, 17, 22, 25, 26).
ANG II has long been known to stimulate catecholamine release from the
adrenal medulla both in vivo and in vitro (19). Long-term stimulation of isolated bovine adrenal medullary chromaffin cells by
ANG II modulates the gene expression of catecholamine biosynthetic enzymes and proenkephalin (35, 36, 39). These findings
indicate that ANG II can act locally within the adrenal gland by
interaction with ANG II receptors to stimulate adrenal medullary cells.
A number of studies indicate that the adrenal medulla may possess an
intrinsic RAS. Reninlike material has been found in the human
adrenal medulla (23), and angiotensin-converting enzyme (ACE), renin, as well as ANG II are present within the rat adrenal medulla (3, 4, 16). The expression of renin and AGT
proteins and mRNAs has also been reported in pheochromocytoma and PC12 cells (28, 32). The above evidence therefore suggests that adrenal medullary cells are targets for ANG II, not only carried by the
circulation but also generated locally by an intrinsic RAS. However,
comprehensive evidence for the existence and local generation of all
components in normal adrenal medullary cells is still lacking. For
example, in the studies mentioned above, it remained to be clarified
whether the various components of RAS in the adrenal medulla are of
local or systemic origin (11, 32). Our previous studies
(40, 41) demonstrated a selective uptake of ANG II into
chromaffin cells but did not rule out the possibility of local
angiotensin generation inside these cells.
To test for the existence of a local RAS, it is essential to
demonstrate that all components are not derived from the circulation but are of local origin. To this end, in addition to an investigation at the tissue level, this study was performed on cultured adrenal medullary cells, which may also provide in the future a relevant and
powerful system to study the regulation of the expression of its
components. Three levels of resolution were addressed. At the tissue
level, ACE and angiotensin type 1 (AT1) receptors were
demonstrated by radioligand binding and autoradiography. In addition,
AGT expression was measured by RT-PCR. At the cellular level, AGT gene
expression was assessed with both RT-PCR and in situ hybridization with
a biotinylated oligodeoxynucleotide probe. This was compared with
immunofluorescence localization of renin, ACE, and ANG II. At the
subcellular level, renin and ANG II were further demonstrated with
immunoelectron microscopy and their distribution was determined by
analytical subcellular fractionation.
| |
MATERIALS AND METHODS |
Cell culture.
Bovine adrenal glands were obtained from the local slaughterhouse, and
primary cultures of chromaffin cells were prepared as described
previously (40, 41).
Antibodies.
Polyclonal rabbit anti-ANG II antiserum was purchased from Amersham
(Little Chalfont, UK). Goat anti-rat renin antiserum was a generous
gift of Dr. T. Inagami (Vanderbilt University School of Medicine,
Nashville, TN) (24). To verify antibody
specificity, rabbit anti-ANG II and goat anti-renin antiserum were
preabsorbed for 2 h with 100 µg/ml ANG II (synthetic human
peptide, 99% purity; Sigma, Steinheim, Germany) or 2 mg/ml rat renin
granule lysate [prepared as described by Sagnella and Peart
(30)], as appropriate, or 2 mg/ml BSA for control.
Monoclonal mouse anti-rat renin antibodies (IgG2a) were purchased from
Swant (Bellinzona, Switzerland). Monoclonal antibodies against ACE
(clones 4051 and 3502) were purchased from Chemicon (Temecula, CA).
Monoclonal antibodies against bovine chromogranin A (CGA; an IgG1) were
prepared and characterized in our laboratory (9).
Fluorescent secondary reagents as well as all nonspecific sera were
from Jackson ImmunoResearch Laboratories (West Grove, PA).
Immunofluorescence.
This procedure was performed as described previously (40,
43). Samples were incubated overnight at 4°C with primary
antibodies (1:800 dilution for rabbit anti-ANG II antiserum; 1:1,000
for goat anti-renin antiserum; 1:1,000 for mouse anti-ACE antibodies; 1 µg/ml for mouse anti-CGA antibodies). Secondary antibodies [for ANG
II labeling, 1:200 donkey IgG anti-rabbit IgG conjugated with tetramethylrhodamine isothiocyanate (TRITC) or FITC; for ACE and CGA,
1:200 donkey IgG anti-mouse IgG conjugated to TRITC; for renin, 1:400
biotinylated donkey IgG anti-goat IgG] were applied for 1 h at
room temperature in the dark. To visualize biotinylated donkey
anti-goat IgG, specimens were further incubated for 1 h with 1:600
FITC-conjugated streptavidin.
To test for colocalization of ANG II with renin or ACE or CGA, samples
were incubated overnight with a mixture of the ANG II primary antiserum
and either CGA or ACE or renin primary antibodies and then bound
antibodies were demonstrated with a mixture of the appropriate
secondary antibodies. To test for colocalization of renin with CGA, a
sequential labeling was performed. Renin labeling was carried out as
described above (1:1,000 goat anti-renin antiserum overnight at 4°C,
followed by 1:200 biotinylated donkey IgG anti-goat IgG, and
then 1:600 FITC-conjugated streptavidin, both for 1 h at room
temperature). This was followed by CGA labeling (1:1,000 mouse
anti-bovine CGA antibodies overnight at 4°C, and then 1:200
TRITC-conjugated donkey IgG anti-mouse IgG for 1 h at room temperature).
Preparations were analyzed by confocal laser scanning microscopy
(MRC-600; Bio-Rad, Richmond, CA). Immunofluorescence experiments were
performed more than five times in each case and were confirmed independently by immunoperoxidase (data not shown).
Immunoelectron microscopy and quantification of labeling.
After 40-min fixation, cell pellets were embedded in Araldite CY 212. Ultrathin sections were collected on gold grids and etched with 3%
H2O2 in PBS for 10 min. Sections were then
preincubated with 1% BSA in PBS supplemented with 5% normal goat or
rabbit serum for 15 min. Samples were further incubated overnight with rabbit anti-ANG II antiserum (1:200) or monoclonal mouse anti-renin antibodies (1:600) in 1% PBS-BSA with 1% normal serum. Grids were then incubated, respectively, with goat anti-rabbit IgG conjugated with
colloidal gold (5 nm, 1:100; Sigma) or biotinylated goat anti-mouse IgG
(1:100) followed by streptavidin colloidal gold (5 nm, 1:200; Sigma)
for 1 h at room temperature. The samples were postfixed with 0.1%
osmium tetroxide for 10 min and contrasted with 3% lead citrate for
30 s.
Immunogold labeling was performed three times. For quantification of
labeling intensity, at least 10 micrographs were randomly taken and
gold particles were counted on granules and the remaining cell
components (referred to as the "rest of the cytoplasm"). The
surface area of both cell compartments was determined, and the labeling
intensity (gold particles/µm2) was calculated.
Subcellular fractionation and radioimmunoassay.
Subcellular fractionation of postnuclear fractions of chromaffin cells
in linear sucrose density gradients (0.3-1.7 M) was performed as
described previously (40). Reninlike activity was measured
by the production of ANG I with the renin-ANG I RIA kit from Du Pont
(Boston, MA). RIA reagents and protocol for ANG II were supplied by
Amersham. Samples contained 20 U/ml aprotinin and 10 mM EDTA to prevent
proteolysis. In our hands, the detection limits were 7 fmol/assay for
ANG I and 5 fmol/assay for ANG II. Intra-assay variations were
1-5%, and interassay variations were 2-7%.
In situ hybridization.
Nonradioactive in situ hybridization cytochemistry was performed as
described by Darby and Sernia (7) with the minor
modifications previously described (43). Biotinylated
antisense oligonucleotide probe complementary to bases 122-169 of
the human AGT mRNA sequence (14) was used.
As negative controls, cells were 1) pretreated with 0.005%
RNase A (Boehringer Mannheim, Mannheim, Germany) in PBS for 1 h at
37°C before hybridization with the biotinylated oligonucleotide probe, 2) incubated for 1 h at 37°C with a
prehybridized mixture containing the biotinylated antisense
oligonucleotide and 10-fold excess unlabeled complementary sense
oligonucleotide, or 3) incubated with the hybridization
buffer alone. In situ hybridization of human and bovine liver biopsies
was used as positive control. In situ hybridization experiments were
repeated four times.
RNA extraction and RT-PCR.
Total RNA was extracted from cells with TRIZol reagent (Life
Technologies, Grand Island, NY) according to the manufacturer's instructions. Three micrograms of total RNA of each sample were reverse
transcribed by Superscript II reverse transcriptase (Life Technologies)
using 0.5 µg of the oligo(dT) as primer at 42°C for 50 min. Bovine
AGT cDNA was amplified from the total cDNA by PCR. Oligonucleotides
corresponding to human AGT 148-167 and 886-905 nucleotide
positions were used. The sequences are as follows: 5'-TAYATACAYCCNTTYCAYCT and TTCATYTTNCCYTGRAARTG. These correspond to
highly conserved amino acid sequences in human, sheep, and rat AGT. PCR
was performed with Taq DNA polymerase (Fisher Scientific Canada) with a Fisher buffer containing 3 mM Mg2+ under the
following conditions: 94°C for 1 min, 50°C for 35 s, 72°C
for 45 s, and 40 cycles in a DNA thermal cycler (PCT-2000; MJ
Research, Watertown, MA). RT-PCR products were analyzed by 0.8%
agarose gel electrophoresis and visualized by ethidium bromide staining
under UV light. Bands were quantified with Bio-Rad Quantity One
software and are presented as relative intensity by reference to
-actin. Bovine liver was used as a positive control. Lymphocytes, isolated as previously described (42), were used as a
negative control. PCR products were directly sequenced by an automated system using fluorescence-labeled dideoxynucleotides (Applied Biosystems, Foster City, CA).
Autoradiography.
The tissue distribution of ACE and AT1 receptors was
assessed in the bovine adrenal medulla by in vitro autoradiography with an iodotyrosyl derivative of lisinopril 125I-labeled 351A
(125I-351A) (5) or 125I-labeled
[Sar1,Ile8]ANG II
(125I-[Sar1,Ile8]ANG II) as
ligands, respectively (Washington State University Peptide
Radioiodination Service Center, Pullman, WA). Adrenal gland cryostat
sections (20 µm thickness) were dehydrated and incubated with the
appropriate radioligand under the conditions as described by Chai et
al. (5) and Zhuo et al. (44), as appropriate.
After sections were washed and exposed to Kodak scientific research
film, the image was analyzed by densitometry and converted to
femtomoles per milligram of wet weight of tissue by reference to the
calibrated relative optical density of 125I standards with
an AIS/C computer-assisted image analysis system (Imaging Research, St.
Catherine, ON, Canada). For ACE, specific binding density was
calibrated as total minus nonspecific values. The latter was measured
in the presence of 10 µM EDTA in the binding buffer (5)
because divalent cation depletion reversibly inactivates the enzyme and
completely blocks specific binding of its 125I-351A
substrate. For AT1 receptor, nonspecific binding, measured in the presence of 1 µM unlabeled ANG II, was similarly subtracted to
yield specific binding density.
| |
RESULTS |
Immunoreactivity of ANG II, ACE, and renin in bovine adrenal
medullary chromaffin cells.
By immunofluorescence of primary bovine adrenal medullary cell
cultures, there was a clear punctate labeling for ANG II, ACE, and
renin in all chromaffin cells (based on overall appearance and specific
immunolabeling), at the exclusion of nuclei. Preabsorption with 99%
pure ANG II or renin granule lysate, as appropriate, abolished these
signals (Fig. 1, B and
D). Both ANG II (Fig. 1A) and renin (Fig.
1C) were clearly restricted to those cells that contained
the chromaffin marker acidic CGA (Fig. 2,
B and D), the major soluble protein in
catecholamine storage vesicles (37), and were not detected
in nonchromaffin cells, which do not contain this protein.
Colocalization in the same cell of ANG II with renin or ACE
immunoreactivity is illustrated by comparison of Fig.
2E with Fig. 2F and of Fig. 2G with
Fig. 2H, respectively.
View larger version (85K):
[in this window]
[in a new window]
|
Fig. 1.
Antibodies against either ANG II or renin specifically label
chromaffin cells. Cells were incubated with rabbit anti-ANG II
(A, B) or goat anti-renin antiserum
(C, D) that had been preincubated with BSA
(A, C) or pure ANG II (B) or
renin-enriched kidney granule lysate (D). Whereas
sham-adsorbed serum produces a clear cytoplasmic labeling of chromaffin
cells, signal becomes negligible after preabsorption on the appropriate
antigen. Asterisk indicates a nonchromaffin cell. Scale bar, 10 µm.
|
|
View larger version (82K):
[in this window]
[in a new window]
|
Fig. 2.
ANG II, angiotensin-converting enzyme (ACE), renin, and
chromogranin A (CGA) immunoreactivities colocalize in chromaffin cells.
ANG II (A) and renin (C) immunoreactivities are
limited to bovine adrenal chromaffin cells that also show
immunostaining for CGA (B, D). Renin
(E) and ACE (G) immunoreactivities similarly
colocalize with ANG II (F, H) in the same
chromaffin cell. Asterisks indicate cells that do not contain CGA.
Scale bars, 10 µm.
|
|
Subcellular distribution of ANG II and renin by isopycnic sucrose
gradient centrifugation.
For analytical subcellular fractionation studies, postnuclear fractions
of chromaffin cells were equilibrated in linear sucrose gradients,
after which protein, norepinephrine, and ANG II contents as well as
NADPH-cytochrome c reductase and renin activities were measured in each fraction (Fig. 3). The
distribution of ANG II was similar to that of norepinephrine. Most of
these constituents appeared in the loading zone, which contains all
proteins and components derived from the cytosol or released by damaged
organelles (fractions 1-3; top of the gradient).
The particulate material was very dense (fractions
13-14; bottom of the gradient), suggesting a granular
localization in norepinephrine-containing particles. As to renin
distribution, in addition to a minor component in the loading zone,
there were two major peaks of particulate material, one at
fractions 8-10, corresponding to the peak of the
endoplasmic reticulum marker enzyme NADPH-cytochrome c
reductase, and a very dense peak at fractions 13 and
14, i.e., at the position of the chromaffin granule marker
norepinephrine. Thus a major part of renin can be attributed to
subcellular particles, including very dense granules containing
sedimentable norepinephrine and ANG II. In preliminary experiments, it
was found that most CGA codistributed with norepinephrine (data not
shown).
View larger version (48K):
[in this window]
[in a new window]
|
Fig. 3.
Subcellular distribution of ANG II and renin in linear
sucrose density gradients. Fractions are numbered from top to bottom.
Data are presented as means ± SD from 3 different experiments.
The first 3 fractions correspond to the loading zone, at
left of the dotted line. Note that particulate ANG II
distribution coincides with that of the particulate norepinephrine in
bottom fractions, whereas particulate renin bimodal distribution
overlaps partially with those of NADPH-cytochrome c
reductase and protein and partially with the dense peak of
norepinephrine.
|
|
Ultrastructural localization of ANG II and renin by immunoelectron
microscopy.
At the ultrastructural level, immunogold labeling of chromaffin cells
confirmed the granular localization of ANG II (Fig. 4A) and renin (Fig.
4B). Immunogold labeling was highly enriched over chromaffin
granules, which contained ~70% of all gold label for both ANG II and
renin. Labeling intensity for ANG II reached 108 gold
particles/µm2 over chromaffin granules (as compared with
6 particles/µm2 over the rest of the cytoplasm; i.e., a
18-fold enrichment), and labeling intensity for renin reached 265 gold
particles/µm2 over chromaffin granules (compared with 19 particles/µm2 over the rest of the cytoplasm; i.e., a
14-fold enrichment). About 65% (63/96) of the chromaffin granules were
labeled with gold particles for ANG II and about 83% (90/109)
for renin. Because the majority of granules were labeled by
either renin or ANG II, one must conclude that at least a substantial
fraction of chromaffin granules indeed contained both antigens.
View larger version (113K):
[in this window]
[in a new window]
|
Fig. 4.
Immunoelectron microscopic localization of ANG II and
renin in chromaffin granules. ANG II (A) and renin
(B) are visualized by immunolabeling with 5-nm colloidal
gold. Gold particles are concentrated over chromaffin granules
(arrows). Scale bars, 100 nm.
|
|
AGT mRNA expression is restricted to chromaffin cells.
To search for the presence of the ANG II precursor AGT in chromaffin
cells, we resorted to in situ hybridization with a biotinylated oligonucleotide probe to AGT mRNA (Figs. 5 and
6).
Specificity of hybridization is shown in Fig. 5A. AGT mRNA
appeared to be localized in chromaffin cells, as judged from their
typical round appearance, but not in nonchromaffin cells. Samples in
which mRNA was first digested with RNAse A before hybridization or
incubated either with a prehybridized mixture of antisense and
complementary sense oligonucleotides or with hybridization buffer alone
showed low background (Fig. 5, B-D). Signal for AGT
mRNA was clearly localized in cells also showing CGA (compare Fig. 6,
A and B) and renin (Fig. 6, C and
D) immunoreactivity.
View larger version (96K):
[in this window]
[in a new window]
|
Fig. 5.
Specific detection of angiotensinogen (AGT) mRNA
expression in bovine chromaffin cells by in situ hybridization. Cells
were incubated with biotinylated antisense probe (A), no
probe (B), or probe prehybridized with excess unlabeled
complementary sense oligonucleotides (C) or were pretreated
with RNase A before hybridization with the probe (D).
Staining is considerably reduced in controls (B-D).
Scale bar, 10 µm.
|
|
View larger version (130K):
[in this window]
[in a new window]
|
Fig. 6.
Colocalization of AGT mRNA and renin in chromaffin cells.
Combination of in situ hybridization using biotinylated AGT antisense
probe (A, C) with immunolabeling for CGA
(B) and renin (D) shows that AGT mRNA colocalizes
with renin in CGA-containing cells. Scale bars, 10 µm.
|
|
For more direct evidence of the expression of AGT in chromaffin
cells, we used RT-PCR followed by sequencing. As shown in Fig.
7A, a ~700-bp RT-PCR product
was observed in bovine chromaffin cells and the adrenal medulla as well
as the bovine liver but not in bovine lymphocytes. Quantification is
presented in Fig. 7B. To exclude contribution by
contaminating genomic DNA, a control PCR with total RNA as template was
performed: no band was observed (data not shown). The 688-bp fragment
was further sequenced and found to correspond to a sequence of a
224-amino acid AGT fragment in an open reading frame. This fragment
showed 93% identity with sheep, 69% with rat, 64% with mouse, and
62% with human AGT.
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 7.
Expression of AGT mRNA in chromaffin cells in
culture. RT-PCR was performed with total RNA extracted from bovine
liver (positive control; lane 1), adrenal medulla
(lane 2), chromaffin cells (lane 3), and
lymphocytes (negative control; lane 4). A: RT-PCR
products were analyzed by 0.8% agarose gel, with -actin used as a
loading control. B: relative mRNA intensity
(AGT-to--actin ratio). ND, not detectable.
|
|
Demonstration of ACE and AT1 receptors in bovine
adrenal medulla by radioligand autoradiography.
Specific binding of the ACE radioligand lisinopril
125I-351A was readily detected in the bovine adrenal gland,
as shown in Fig. 8A, and
showed an approximately fourfold (689 ± 46 fmol/mg wet wt tissue;
mean ± SD, n = 6) higher intensity in the medulla
than in the cortex (183 ± 28 fmol/mg). For control, binding was
completely abolished by EDTA (Fig. 8B). Similarly, the
specific binding of the AT1 receptor radioligand
125I-[Sar1,Ile8]ANG II in the
bovine adrenal gland was also demonstrated by autoradiography (Fig.
8C) but was approximately threefold lower in the medulla (417 ± 45 fmol/mg) than the cortex (1,195 ± 98 fmol/mg).
For control, this signal was completely abolished by excess cold ligand
(Fig. 8D).
View larger version (125K):
[in this window]
[in a new window]
|
Fig. 8.
In
vitro radioligand binding distribution of ACE (A) and
AT1 receptor (C) in bovine adrenal gland. In the
presence of EDTA (B) or excess unlabeled ANG II
(D), binding was completely abolished. Binding density
(fmol/mg wet tissue) can be derived from computer-generated pseudocolor
gradient presented at right.
|
|
| |
DISCUSSION |
In contrast to the well-understood circulatory RAS, a satisfactory
definition of a local RAS is still elusive. The existence of all
components of the system within tissues that are targets for
angiotensins is now well accepted (2, 11, 15, 17, 29, 34,
38). However, the presence of all components of the RAS
in the same cells would provide an attractive model to study its
intracellular organization and regulation (26). Such an
intrinsic RAS has been proposed by Marley et al. (18) for the adrenal medulla, although evidence was fragmentary and obtained from different species (4, 16, 18, 20, 23, 41). The present study provides comprehensive evidence for this proposal, based
on the combination of in situ hybridization, RT-PCR,
immunocytochemistry, and subcellular fractionation approaches on the
same material, demonstrating coexpression in chromaffin cells of AGT
mRNA with immunoreactive renin, ACE, as well as ANG II. The presence of ANG II receptors on bovine adrenal medullary cells has already been
demonstrated by two groups, including ourselves (39, 40). At the tissue level, we further demonstrate by in vitro radioligand autoradiography that chromaffin cells also contain ACE and
AT1 receptors.
The concept of angiotensin production by an intracellular mechanism is
supported by a number of studies. Typical examples are the
demonstration of the presence of AGT, renin, ACE, ANG I, and ANG II in
cultured neuroblastoma cells (6, 25, 26) and the
demonstration that AGT, renin, ANG I, and ANG II occur in renal
cortical cells (7, 12). AGT and renin were found in the
same human uterine decidual cell (17). AGT mRNA, renin, and ANG II were also found in the superior cervical ganglion neurons (43). In addition, the RAS in dog ventricular myocytes and
stellate ganglia could be upregulated with ventricular pacing
(2) and by high-frequency preganglionic stimulation
(15), respectively.
However, this attractive concept still meets with some difficulties.
Some early experiments demonstrated that AGT and renin were located in
different cells. In the brain stem, for example, AGT mRNA and protein
are present in astrocytes, whereas renin occurs in neurons
(11). Therefore, it has been suggested that AGT generated
by one cell type is either taken up by adjacent renin-containing cells
and further processed therein to active angiotensins or processed
extracellularly to ANG I or ANG II, which are then taken up (11,
21). However, the localization of AGT and renin in the same
types of cells (neuronal, renal cortical, or uterine decidual cells or
ventricular myocytes) (2, 6, 7, 12, 17, 22, 25, 26) and
even in the same granule has also been reported (12, 22).
By analogy with the latter findings, the subcellular fractionation
analysis combined with granular immunogold pattern reported here
demonstrate that renin and ANG II at least partially colocalize in
dense chromaffin granules. Localization of ANG II in secretory vesicles
was also demonstrated in neurons of the rat subfornical organ, where
immunogold labeling for ANG II was concentrated on large dense-cored
vesicles in axon terminals (27). Codistribution in the
sucrose density gradients of the renin intermediate-density peak with
that of the endoplasmic reticulum marker NADPH-cytochrome c
reductase is also compatible with the suggestion that at least part of
the renin in chromaffin cells is synthesized intracellularly. Furthermore, the present study provides evidence for the availability of an intracellular substrate for ANG II generation, based on the
combination of in situ hybridization with immunofluorescence showing
colocalization of AGT mRNA and renin in the same cell.
Although the probe used for in situ hybridization was selected
from a highly conserved region in sheep, rat, mouse, and human AGT, one
might question the validity of the signal, because oligonucleotides were derived from an heterologous (human) source. To verify the presence of genuine bovine AGT mRNA, a pair of primers was designed to
perform RT-PCR and to sequence its products. The 5' primer (bases
147-168) was selected within the sequence used for the in situ
probe (bases 122-169). The RT-PCR-generated 688-bp fragment derived from the bovine chromaffin cells was sequenced and found to be
identical with the DNA fragment derived from the bovine liver. The high
identity of this DNA fragment with sheep, rat, mouse, and human AGT
validated a posteriori the probe used for in situ hybridization. The
absence of the RT-PCR product in lymphocytes confirmed the cell type
specificity of AGT expression.
The occurrence of ACE at the plasma membrane of rat adrenal medullary
chromaffin cells has been demonstrated by immunoelectron microscopy
(16). In good agreement, the presence of ACE in bovine adrenal medulla was demonstrated in the present study by in vitro radioligand binding autoradiography, with a fourfold higher density in
the adrenal medulla than in the adrenal cortex. Furthermore, immunocytochemistry on cultured chromaffin cells also disclosed ACE
inside ANG II-containing cells, suggesting intracellular synthesis. The
same in vitro radioligand binding autoradiography approach disclosed
the presence of AT1 receptors in the bovine adrenal medulla
at the tissue level, in agreement with previous studies based on
membrane preparations derived from either tissue (1) or
cultured cells (39, 40).
In conclusion, our results show that all components of the RAS are
present in bovine adrenal medulla chromaffin cells and lend further
support to the concept of intracellular generation of angiotensins in
cells. Therefore, chromaffin cells are not only the target but should
also be regarded as the source and site of storage of angiotensins. In
addition, localization of ANG II in secretory granules strongly
suggests that it might be released in a regulated fashion, so as to act
on chromaffin or adrenal cortical cells via controlled autocrine or
paracrine processes.
| |
ACKNOWLEDGEMENTS |
The authors thank Tatiana Lejen (from Dr. Jose-maria Trifaro's
laboratory) for the providing of bovine adrenal medullary chromaffin cells.
| |
FOOTNOTES |
This work was supported in part by the Queen Elizabeth Medical
Foundation, the Belgian program on Interuniversity Attraction Poles,
European Community project BMH4-CT96-1586 (BIOMED 2), and Heart
and Stroke Foundation of Ontario, Canada (HSFO; no. T-4716). F. H. H. Leenen is a career investigator of the HSFO. J. M. Wang is currently supported by the Canadian Institutes of Health
Research University-Industry Program (Pfizer Canada). P. J. Courtoy is supported by the Fonds National de la Recherche Scientifique
and Actions de Recherche Concertee, Belgium.
Address for reprint requests and other correspondence:
F. H. H. Leenen, Hypertension Unit, Univ. of Ottawa Heart
Institute, 40 Ruskin St., Ottawa, ON, K1Y 4W7, Canada (E-mail:
fleenen{at}ottawaheart.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 11, 2002;10.1152/ajpheart.01092.2001
Received 12 December 2001; accepted in final form 3 July 2002.
| |
REFERENCES |
1.
Balla, T,
Baukal AJ,
Eng S,
and
Catt KJ.
Angiotensin II receptor subtypes and biological responses in the adrenal cortex and medulla.
Mol Pharmacol
40:
401-406,
1991[Abstract].
2.
Barlucchi, L,
Leri A,
Dostal DE,
Fiordaliso F,
Tada H,
Hintze TH,
Kajstura J,
Nadal-Ginard B,
and
Anversa P.
Canine ventricular myocytes possess a renin-angiotensin system that is upregulated with heart failure.
Circ Res
88:
298-304,
2001[Abstract/Free Full Text].
3.
Berka, JL,
Kelly DJ,
Robinson DB,
Alcorn D,
Marley PD,
Fernley RT,
and
Skinner SL.
Adrenaline cells of the rat adrenal cortex and medulla contain renin and prorenin.
Mol Cell Endocrinol
119:
175-184,
1996[ISI][Medline].
4.
Chai, SY,
Allen AM,
Adam WR,
and
Mendelsohn FA.
Local actions of angiotensin II: quantitative in vitro autoradiographic localization of angiotensin II receptor binding and angiotensin converting enzyme in target tissues.
J Cardiovasc Pharmacol
8, Suppl10:
S35-S39,
1986.
5.
Chai, SY,
Mendelsohn FA,
and
Paxinos G.
Angiotensin converting enzyme in rat brain visualized by quantitative in vitro autoradiography.
Neuroscience
20:
615-627,
1987[ISI][Medline].
6.
Clemens, DL,
Clauser E,
Celio MR,
and
Inagami T.
Generation of angiotensinogen by cultured neuroblastoma and glioma cells.
Brain Res
364:
205-211,
1986[ISI][Medline].
7.
Darby, IA,
and
Sernia C.
In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys.
Cell Tissue Res
281:
197-206,
1995[ISI][Medline].
8.
De Mello, WC,
and
Danser AH.
Angiotensin II and the heart: on the intracrine renin-angiotensin system.
Hypertension
35:
1183-1188,
2000[Abstract/Free Full Text].
9.
Dillen, L,
De Block J,
Van Lear L,
and
De Potter W.
Enzyme-linked immunosorbent assay for chromogranin A.
Clin Chem
35:
1934-1938,
1989[Abstract/Free Full Text].
10.
Dostal, DE,
Booz GW,
and
Baker KM.
Regulation of angiotensinogen gene expression and protein in neonatal rat cardiac fibroblasts by glucocorticoid and -adrenergic stimulation.
Basic Res Cardiol
95:
485-490,
2000[ISI][Medline].
11.
Ganong, WF.
What regulates the production and secretion of angiotensinogen in the brain?
Front Neuroendocrinol
15:
78-81,
1994[ISI][Medline].
12.
Hunt, MK,
Ramos SP,
Geary KM,
Norling LL,
Peach MJ,
Gomez RA,
and
Carey RM.
Colocalization and release of angiotensin and renin in renal cortical cells.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F363-F373,
1992[Abstract/Free Full Text].
13.
Jeunemaitre, X,
Gimenez-Roqueplo AP,
Celerier J,
and
Corvol P.
Angiotensinogen variants and human hypertension.
Curr Hypertens Rep
1:
31-41,
1999[Medline].
14.
Kunapuli, SP,
and
Kumar A.
Molecular cloning of human angiotensinogen cDNA and evidence for the presence of its mRNA in rat heart.
Circ Res
60:
786-790,
1987[Abstract/Free Full Text].
15.
Kushiku, K,
Yamada H,
Shibata K,
Tokunaga R,
Katsuragi T,
and
Furukawa T.
Upregulation of immunoreactive angiotensin II release and angiotensinogen mRNA expression by high-frequency preganglionic stimulation at the canine cardiac sympathetic ganglia.
Circ Res
88:
110-116,
2001[Abstract/Free Full Text].
16.
Laliberte, F,
Laliberte MF,
Alhenc-Gelas F,
and
Chevillard C.
Cellular and subcellular immunohistochemical localization of angiotensin-converting enzyme in the rat adrenal gland.
Lab Invest
56:
364-371,
1987[ISI][Medline].
17.
Li, C,
Ansari R,
Yu Z,
and
Shah D.
Definitive molecular evidence of renin-angiotensin system in human uterine decidual cells.
Hypertension
36:
159-164,
2000[Abstract/Free Full Text].
18.
Marley, PD,
Bunn SJ,
Wan DC,
Allen AM,
and
Mendelsohn FA.
Localization of angiotensin II binding sites in the bovine adrenal medulla using a labeled specific antagonist.
Neuroscience
28:
777-787,
1989[ISI][Medline].
19.
Mazur-Ruder, M,
Feuerstein G,
Roll D,
and
Gutman Y.
Selective reduction of adrenal medulla response to angiotensin induced by suppression of renin-angiotensin.
Eur J Pharmacol
59:
261-266,
1979[ISI][Medline].
20.
Mendelsohn, FA.
Angiotensin II is concentrated or locally produced in rat adrenal gland.
Clin Exp Pharmacol Physiol Suppl
7:
3-7,
1982[Medline].
21.
Mercure, C,
Ramla D,
Garcia R,
Thibault G,
Deschepper CF,
and
Reudelhuber TL.
Evidence for intracellular generation of angiotensin II in rat juxtaglomerular cells.
FEBS Lett
422:
395-399,
1998[ISI][Medline].
22.
Mungall, BA,
Shinkel TA,
and
Sernia C.
Immunocytochemical localization of angiotensinogen in the fetal and neonatal rat brain.
Neuroscience
67:
505-24,
1995[ISI][Medline].
23.
Naruse, M,
Sussman CR,
Naruse K,
Jackson RV,
and
Inagami T.
Renin exists in human adrenal tissue.
J Clin Endocrinol Metab
57:
482-487,
1983[Abstract].
24.
Niimura, F,
Labosky PA,
Kakuchi J,
Okubo S,
Yoshida H,
Oikawa T,
Ichiki T,
Naftilan AJ,
Fogo A,
and
Inagami T.
Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.
J Clin Invest
96:
2947-2954,
1995[ISI][Medline].
25.
Okamura, T,
Clemens DL,
and
Inagami T.
Renin, angiotensins, and angiotensin-converting enzyme in neuroblastoma cells: evidence for intracellular formation of angiotensins.
Proc Natl Acad Sci USA
78:
6940-6943,
1981[Abstract/Free Full Text].
26.
Petrossian, G,
and
Oliver JA.
Synthesis of angiotensinogen by renin-containing neuroblastomas.
Am J Physiol Cell Physiol
257:
C185-C189,
1989[Abstract/Free Full Text].
27.
Pickel, VM,
and
Chan J.
Co-localization of angiotensin II and 
-aminobutyric acid in axon terminals in the rat subfornical organ.
Neurosci Lett
193:
89-92,
1995[ISI][Medline].
28.
Racz, K,
Pinet F,
Gasc JM,
Guyene TT,
and
Corvol P.
Coexpression of renin, angiotensinogen, and their messenger ribonucleic acids in adrenal tissues.
J Clin Endocrinol Metab
75:
730-737,
1992[Abstract].
29.
Ruzicka, M,
and
Leenen FHH
Relevance of angiotensin II for cardiac hypertrophy and failure induced by cardiac volume overload.
Heart Failure Rev
3:
169-181,
1999.
30.
Sagnella, GA,
and
Peart WS.
Studies on the isolation and properties of renin granules from the rat kidney cortex.
Biochem J
182:
301-309,
1979[ISI][Medline].
31.
Santos, RA,
Campagnole-Santos MJ,
and
Andrade SP.
Angiotensin-(1-7): an update.
Regul Pept
91:
45-62,
2000[ISI][Medline].
32.
Sernia, C.
Location and secretion of brain angiotensinogen.
Regul Pept
57:
1-18,
1995[ISI][Medline].
33.
Sernia, C,
Wyse B,
Tey SK,
and
Leong SL.
Receptors for (3-8) angiotensin in brain cells. AngIV binding in brain cells.
Adv Exp Med Biol
396:
253-263,
1996[Medline].
34.
Sernia, C,
Zeng T,
Kerr D,
and
Wyse B.
Novel perspectives on pituitary and brain angiotensinogen.
Front Neuroendocrinol
18:
174-208,
1997[ISI][Medline].
35.
Stachowiak, MK,
Goc A,
Hong JS,
Poisner A,
Jiang HK,
and
Stachowiak EK.
Regulation of tyrosine hydroxylase gene expression in depolarized non-transformed bovine adrenal medullary cells: second messenger systems and promoter mechanisms.
Brain Res Mol Brain Res
22:
309-319,
1994[Medline].
36.
Stachowiak, MK,
Jiang HK,
Poisner AM,
Tuominen RK,
and
Hong JS.
Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells. Molecular mechanisms and nature of second messenger systems.
J Biol Chem
265:
4694-4702,
1990[Abstract/Free Full Text].
37.
Taylor, CV,
Taupenot L,
Mahata SK,
Mahata M,
Wu H,
Yasothornsrikul S,
Toneff T,
Caporale C,
Jiang Q,
Parmer RJ,
Hook VY,
and
O'Connor DT.
Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules.
J Biol Chem
275:
22905-22915,
2000[Abstract/Free Full Text].
38.
Veerasingham, SJ,
and
Leenen FHH
The brain renin-angiotensin system and salt-sensitive hypertension.
In: Angiotensin II Receptor Blockade: Physiological and Clinical Implications, edited by Dhalla NS,
Zahradka P,
Dixon I,
and Beamish R.. Boston, MA: Kluwer Academic, 1998, p. 15-32.
39.
Wan, DC,
Marley PD,
and
Livett BG.
Angiotensin II stimulates the expression of proenkephalin A mRNA in cultured bovine adrenal chromaffin cells.
Neuropeptides
16:
141-147,
1990[ISI][Medline].
40.
Wang, JM,
Baudhuin P,
Courtoy PJ,
and
De Potter W.
Conversion of angiotensin II into active fragments by an endosomal pathway in bovine adrenal medullary cells in primary culture.
Endocrinology
136:
5274-5282,
1995[Abstract].
41.
Wang, JM,
Llona I,
and
De Potter WP.
Receptor-mediated internalization of angiotensin II in bovine adrenal medullary chromaffin cells in primary culture.
Regul Pept
53:
77-86,
1994[ISI][Medline].
42.
Wang, JM,
Partoens PM,
Callebaut DP,
Coen EP,
Martin JJ,
and
De Potter WP.
Phenotype plasticity and immunocytochemical evidence for ChAT and D beta H co-localization in fetal pig superior cervical ganglion cells.
Brain Res Dev Brain Res
90:
17-23,
1995[Medline].
43.
Wang, JM,
Slembrouck D,
and
Potter WD.
Expression of angiotensinogen mRNA and localization of angiotensin II and renin in peripheral adrenergic neurons in primary culture.
Biochem Biophys Res Commun
229:
876-881,
1996[ISI][Medline].
44.
Zhuo, J,
Song K,
Abdelrahman A,
and
Mendelsohn FA.
Blockade by intravenous losartan of AT1 angiotensin II receptors in rat brain, kidney and adrenals demonstrated by in vitro autoradiography.
Clin Exp Pharmacol Physiol
21:
557-567,
1994[ISI][Medline].
Am J Physiol Heart Circ Physiol 283(5):H1811-H1818
0363-6135/02 $5.00
Copyright © 2002 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
