Vol. 276, Issue 5, R1374-R1382, May 1999
Changes in the glomerulosa cell phenotype during adrenal
regeneration in rats
W. C.
Engeland1 and
B. K.
Levay-Young2
Departments of 1 Cell Biology
and Neuroanatomy and
2 Surgery, University of
Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
In situ
hybridization was used to examine cellular differentiation during rat
adrenal regeneration, defining zona glomerulosa [cytochrome
P-450 aldosterone synthase
(P-450aldo) mRNA positive], zona
fasciculata [cytochrome P-450
11
-hydroxylase (P-45011) mRNA
positive], or zona intermedia [negative for both but
3-hydroxysteroid dehydrogenase (3-HSD) mRNA positive].
After unilateral adrenal enucleation with contralateral adrenalectomy
(ULE/ULA), the expression of all mRNA was reduced at 2 days. From 5 to
10 days, P-45011 and 3-HSD mRNA
increased while P-450aldo remained
low; at 20 days, all mRNA were increased. From 2 to 10 days, cells
adjacent to the capsule showed intermedia cell differentiation; by 20 days, the subcapsular glomerulosa cells reappeared. This suggests that after enucleation the glomerulosa dedifferentiates to zona intermedia. The experiment was repeated in rats where the postenucleation ACTH rise
was prevented. Rats underwent ULE with sham ULA (ULE/SULA) or ULE/SULA
with ACTH treatment. Adrenals from ULE/SULA rats expressed increased
P-450aldo mRNA at 10 days and reduced
P-45011 mRNA and adrenal weight at
30 days. ACTH treatment reversed the pattern toward that seen in
ULE/ULA. These findings show that the enucleation-induced dedifferentitation of the glomerulosa cell may result in part from
elevated plasma ACTH and that prevention of dedifferentiation may
result in impaired regeneration.
adrenal cortex; cytochrome P-450
aldosterone synthase; adrenocorticotropic hormone; adrenal enucleation
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INTRODUCTION |
IN RESPONSE TO ENUCLEATION, the adrenal cortex
regenerates from capsular and adherent glomerulosa cells to restore the
outer zona glomerulosa and an inner zona fasciculata/reticularis (8). The glomerulosa cell has been implicated as the stem cell for regeneration (8), yet the absence of glomerulosa-type mitochondrial cristae (22, 33) and the reduction of aldosterone secretion compared
with corticosterone secretion (1) suggest that dedifferentiation of
glomerulosa cells may occur immediately after enucleation. The
synthesis of aldosterone by glomerulosa cells is dependent on
aldosterone synthase, which is the product of the
CYP11B2 gene, whereas the synthesis of
corticosterone by fasciculata and reticularis cells is dependent on
11-hydroxylase, which is a product of the CYP11B1 gene (20, 25). The two genes
are distinct and have been cloned (17, 23), and specific probes can
distinguish cytochrome P-450
aldosterone synthase (P-450aldo) and
cytochrome P-450 11-hydroxylase
(P-45011) transcripts, permitting
identification of glomerulosa and fasciculata/reticularis cells,
respectively (16, 35).
In a preliminary study, in situ hybridization histochemistry was used
to monitor steroidogenic cell phenotype during adrenal regeneration
(6). After enucleation, the expression of
P-450aldo and of
P-45011 mRNA decreased as a
consequence of the loss of adrenal tissue and remained low for 1 wk
after enucleation. In this earlier study, using an oligonucleotide
probe designed to detect both
P-450aldo and
P-45011 mRNA, an area between the
zona glomerulosa and fasciculata was identified in intact adrenals that
did not express either P-450aldo or
P-45011 mRNA (6). A recent
immunohistochemical study (19) has shown that these cells do not
express either P-450aldo or
P-45011 protein. This area was
defined previously as a "sudanophobe zone" because of its reduced
lipid content (34) or a "zona intermedia" based on its position
between the zona glomerulosa and fasciculata (3). Because the zona
intermedia may provide progenitor cells for regeneration, in situ
hybridization histochemistry was used to monitor changes in intermedia
cells after adrenal enucleation over a longer time course.
Optimal regeneration is dependent on establishing a hypocorticoid
cellular environment. Enucleation-induced regeneration is suppressed in
rats administered corticosteroids (31) or in rats in which the paired
adrenal remains in situ (13). To extend this study of normal
regeneration, the pattern of steroidogenic gene expression was
determined during impaired regeneration of enucleated adrenals in the
presence of an intact adrenal.
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METHODS |
Male Sprague-Dawley rats (175-225 g body wt; Sasco Labs) were
housed under a 12:12-h light-dark cycle (on 0700-1900 h) with food
and water available ad libitum. Animals were allowed to acclimate to
the housing facility and light cycle for at least 1 wk before experiments. Under pentobarbital sodium anesthesia (6-7 mg/100 g
ip), rats underwent left adrenal enucleation and right unilateral adrenalectomy (ULE/ULA) or right sham ULA (ULE/SULA). Enucleation consisted of slitting the capsule and extruding the inner cortical and
medullary tissue. Antibiotic (Ancef, 10 mg/kg) was administered, and
animals were kept warm until fully ambulatory, when they were returned
to the animal care facility.
Protocols.
Two experiments were done to establish the time course of the response
to enucleation. At 2, 5, 7, and 10 days (experiment 1) or 10, 20, and 30 days
(experiment 2) after ULE/ULA, rats
(n = 4/group) were killed by
decapitation in the morning, and trunk blood and adrenals were
collected. Two additional experiments assessed regenerative responses
in the presence or absence of an intact adrenal. At 2, 5, and 10 days
(experiment 3) or 30 days (experiment 4) after ULE/ULA or
ULE/SULA, rats (n = 4/group) were killed, and trunk blood and adrenals were collected. In
experiment 3, an additional group of
ULE/SULA rats was treated daily with ACTH [1 U repository
ACTH in gelatin per rat (Rhone-Poulenc, Collegeville, PA)] and killed at 10 days after enucleation. In
experiment 4, an additional group of
rats was included that underwent ULA only. Also, at the end of
experiment 4, nonstress blood samples
were collected by tail clip (10), a maximal dose of ACTH (100 ng rat
ACTH; Pennisula Labs) was injected subcutaneously, and trunk blood was
collected at 15 min after stimulation. Adrenals were removed, cleaned
of fat and connective tissue, immersed in OCT compound,
and frozen in cold isopentane.
Hormone analyses.
Plasma ACTH was measured by RIA as described previously (14). The
intra-assay and the interassay coefficients of variation (CV) on a pool
value of 75 pg/ml were 7.6 and 13.3%, respectively. Plasma
corticosterone was measured by RIA using a kit (ICN Biomedical, Costa
Mesa, CA) as described previously (14). The intra-assay and interassay
CV on a pool value of 100 ng/ml were 7 and 13%, respectively. Plasma
aldosterone was measured by RIA using a kit (Diagnostic Products, Los
Angeles, CA); the intra-assay and the interassay CV for aldosterone
were 15.9 and 21.8%, respectively.
In situ hybridization.
Adrenals were frozen-sectioned (14 µm) and mounted onto ProbeOn
slides (Fisher Scientific, Pittsburgh, PA). In situ hybridization histochemistry was done using a method (7) adapted from that of
Dagerlind et al. (4). Slides were incubated at 42°C overnight with
5-10 ng of 35S-labeled probe
per milliliter of hybridization solution. Sections were washed five
times for 15 min each in 1× SCC at 54°C, rinsed, dehydrated,
air-dried, and exposed to Biomax film (Kodak). Some sections were
dipped in NTB-2 emulsion (Kodak) and exposed for 3-7 days. Dipped
slides were developed, counterstained with bisbenzimide (30), and
dehydrated. Oligonucleotide probes, purchased from Keystone Labs (Menlo
Park, CA) or from GIBCO (Grand Island, NY), were designed to detect
cytochrome P-450 side-chain cleavage
(P-450scc; nt 1368-1403 of rat
P-450scc; Ref. 26), 3-hydroxysteroid dehydrogenase (3-HSD; nt 1294-1329 of type I; Ref. 36),
(P-45021OH; nt
608-643 of mouse CYP21 cDNA; Ref. 24),
P-45011 (nt 814-849; Ref. 23), and P-450aldo (nt 918-953; Ref.
17) mRNA specifically. A "generic" P-45011 probe was designed to
detect both P-450aldo and
P-45011 mRNA (nt 154-189 of
the P-450aldo and 50-85 of the
P-45011). For all probes,
specificity was confirmed by demonstrating the absence of hybridization
when using the corresponding sense probe and/or when excess
(100×) unlabeled probe was added to the hybridization solution.
Sections from each group of adrenals and from all treatment groups
within an experiment were hybridized in the same assay. Film exposure
was chosen to ensure that the density of hybridization was below film
saturation. Autorads were scanned using a UMAX scanner calibrated to a
density scale (Stouffer) using a Macintosh IIci computer and the public
domain NIH Image program [by W. Rasband (NIH) and available from
the Internet by anonymous FTP from
zippy.nimh.nih.gov]. Image analysis provided information
concerning both the mean density of hybridization (i.e., mean pixel
value) and hybridization area (i.e., pixel number). Mean density values
were corrected for background hybridization by subtracting mean density
values obtained by scanning areas of adrenal sections that did not
express steroidogenic enzyme mRNA (i.e., adrenal medulla in intact
adrenals and fibrin clot in regenerating adrenals). Hybridization area
was measured for each transcript after autothresholding. For each
adrenal, the mean density and area of hybridization for three to five
randomly chosen tissue sections were measured; means were calculated
for each adrenal collected from a group of four rats. To control for differences in steroidogenic enzyme mRNA between adrenals that could
result from sampling at different levels of the adrenal cortex,
measurements for intact adrenals were made on sections containing the
adrenal medulla. For regenerating adrenals where the adrenal medulla
was removed, values averaged for each adrenal represented levels
throughout the full extent of the gland. In all cases, adjacent
sections were hybridized for
P-45011 and P-450aldo mRNA.
Data analyses.
The relative levels of P-450aldo,
P-45011, and 3-HSD mRNA were
compared between groups using ANOVA; individual means were compared
using the Fisher's least-squares differences test. Plasma hormone
concentrations were subjected to logarithmic transformation before
ANOVA to reduce variance. For all statistical analyses, P < 0.05 was required for
statistical significance.
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RESULTS |
In intact adrenals, P-450scc mRNA was
uniformly expressed throughout the adrenal cortex (Fig.
1A);
the pattern of hybridization was similar for 3-HSD and
P-45021OH mRNA (data not shown). As expected, the hybridization pattern for
P-45011 and
P-450aldo mRNA was zone specific in
that P-45011 mRNA was restricted to the inner cortex (Fig. 1C), whereas
P-450aldo mRNA was observed underlying
the capsule (Fig. 1B). This pattern
was observed using either the specific or generic
P-45011 (Fig.
1D). The generic P-45011 probe, which detects both
P-45011 and
P-450aldo transcripts, demarcated the
zona intermedia as an area devoid of silver grains between the
glomerulosa and fasciculata. Because transcripts representing earlier
steps in the steroidogenic pathway (i.e.,
P-450scc, 3-HSD, and
P-45021OH) were expressed in the zona
intermedia, this area contains cells in which steroidogenesis occurs
but is limited by the absence of
P-45011 and
P-450aldo.
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Fig. 1.
Cytochrome P-450 side-chain cleavage
(P-450scc), cytochrome
P-450 aldosterone synthase
(P-450aldo), specific cytochrome
P-450 11-hydroxylase
(P-45011), and generic (g)
P-45011 mRNA in an intact rat
adrenal. Dark-field images showing distribution of silver grains
overlying bisbenzimide-counterstained sections. Labeling for
P-450scc mRNA
(A) is observed in all cortical
cells, whereas labeling for P-450aldo
(B) and specific
P-45011
(C) is observed in cells underlying
capsule and in inner cortical cells, respectively. Labeling for generic
P-45011 mRNA
(D) represents a composite of that
found with P-450aldo and specific
P-45011 probes; cortical area with
reduced silver grains identifies zona intermedia. Junction between
capsular and cortical cells is delineated by arrowheads. Bar, 50 µm.
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Steroidogenic enzyme gene expression after adrenal enucleation.
Hybridization area and density of
P-450aldo,
P-45011, and 3-HSD mRNA were
measured in regenerating adrenals collected at 2, 5, 7, and 10 days and
at 10, 20, and 30 days after enucleation in two separate experiments.
Hybridization area of P-450aldo and P-45011 mRNA was viewed as an index
of the presence of zona glomerulosa and fasciculata/reticularis cells,
respectively. Hybridization area of 3-HSD mRNA reflected total
steroidogenic tissue present in regenerating adrenals, because 3-HSD
mRNA was expressed in all cortical zones (Fig. 1). As expected, in
intact adrenals (time 0)
hybridization area of P-450aldo was
less than that of P-45011 and
3-HSD (Fig.
2A; note
that P-450aldo values are plotted at 5 times). In regenerating adrenals at 2 days postenucleation, hybridization area was reduced for all transcripts. At 5, 7, and 10 days postenucleation, labeling for
P-45011 and 3-HSD mRNA increased
compared with 2 days but remained less than that of intact adrenals; in
contrast, the labeling for P-450aldo
mRNA did not increase from 2 to 10 days postenucleation (Fig.
2A). The pattern at 10 days was
similar in experiment 2; hybridization area was reduced for all transcripts (Fig.
2B). However, at 20 and 30 days,
regenerating adrenals showed labeling for
P-45011 and 3-HSD mRNA that was
equivalent to that of intact adrenals, whereas the labeling for
P-450aldo mRNA remained below that of intact adrenals (Fig. 2B). These
data suggested that regeneration of the zona fasciculata/reticularis is
complete by 20 days, whereas regeneration of the zona glomerulosa
remains incomplete at 30 days. Hybridization density did not change for
any transcript after enucleation (data not shown).
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Fig. 2.
Time course of adrenal P-450aldo,
P-45011, and
3-hydroxysteroid dehydrogenase (3-HSD) mRNA expression
expressed as area of hybridization over section after adrenal
enucleation. Independent experiments with a relatively short
(A) or long
(B) time course were done. Data
represent means ± SE of 4 adrenals collected after unilateral
adrenal enucleation with contralateral adrenalectomy (ULE/ULA);
comparisons are made to intact adrenals collected at time of surgery
(time 0).
* P < 0.05 vs. intact adrenal
(time 0).
# P < 0.05 vs.
day 2.
 P < 0.05 vs. day
10.
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To determine the fate of zona intermedia cells after adrenal
enucleation, emulsion autorads of adjacent sections hybridized with
P-450scc,
P-450aldo, 3-HSD, and specific
P-45011 probes were compared. Zona
intermedia cells were defined as cells negative for
P-450aldo and
P-45011 but positive for
P-450scc and 3-HSD mRNA. In all
samples examined, P-450scc (Figs.
3A and
4A) and 3-HSD (Figs. 3B and
4B) hybridization was seen extending
out to the capsule; days 10 (Fig. 3)
and 30 (Fig. 4) are shown as examples.
At 2-10 days, cells adjacent to the capsule showed an absence of
P-450aldo (Fig.
3B) and
P-45011 mRNA (Fig.
3D). By 20 and 30 days, cells near
the capsule expressed P-450aldo mRNA (Fig. 4B), but not
P-45011 mRNA (Fig.
4D), reestablishing the preenucleation pattern (compare with Fig. 1). These results suggest that adrenal enucleation results in the loss of the glomerulosa cell
phenotype concomitant with the appearance of the intermedia cell
phenotype. The glomerulosa cell phenotype is restored in the late
stages of regeneration (i.e., by 20 days).
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Fig. 3.
Dark-field images of P-450scc,
3-HSD, P-450aldo, and specific
P-45011 mRNA in a
day 10 regenerating rat adrenal.
Labeling for P-450scc
(A) and 3-HSD
(C) mRNA identifies all cortical
cells. Labeling for P-450aldo
(B) and
P-45011
(D) mRNA is not observed in cells
underlying capsule, indicating presence of zona intermedia cells.
Junction between capsular and cortical cells is delineated by
arrowheads. Bar, 50 µm.
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Fig. 4.
Dark-field images of P-450scc,
3-HSD, P-450aldo, and specific
P-45011 mRNA in a
day 30 regenerating rat adrenal.
Labeling for P-450scc
(A) and 3-HSD
(C) mRNA identifies all cortical
cells. Labeling for P-450aldo mRNA
(B) is observed in cells underlying
capsule, indicating presence of glomerulosa cells; cells in inner
cortex label for P-45011 mRNA
(D), indicating presence of
fasciculata cells. Junction between capsular and cortical cells is
delineated by arrowheads. Bar, 50 µm.
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Plasma hormonal responses to enucleation.
Plasma ACTH was elevated at 2, 5, and 7 days after ULE/ULA compared
with 10 days (experiment 1); plasma
ACTH decreased between 10 and 20 days (experiment
2) but did not change between 20 and 30 days after
enucleation (Table 1). Although small
increases in plasma corticosterone were observed between 2 and 10 days
after enucleation (experiment 1),
plasma corticosterone did not vary from 10 to 30 days
(experiment 2). Plasma aldosterone
was at the limit of assay detection in all animals bearing regenerating
adrenals (6 pg/ml). Animals bearing only an intact adrenal were not
included in this experiment, precluding statistical testing. However,
in parallel studies in which blood was collected from rats
(n = 4) under nonstress conditions at
14 days after ULA, plasma ACTH, corticosterone, and aldosterone values
were 34 ± 5 pg/ml, 18 ± 9 ng/ml, and 17 ± 11 pg/ml, respectively.
Steroidogenic enzyme gene expression after adrenal enucleation in
the presence of an intact adrenal.
Analysis similar to experiment 1 was
repeated for P-45011 and
P-450aldo mRNA in 2-, 5-, and 10-day
enucleated adrenals regenerating in the absence (ULE/ULA) or in the
presence (ULE/SULA) of an intact adrenal. There was no difference
between enucleated adrenals from ULE/ULA and ULE/SULA at
days 2 or
5, and the results were similar to
experiment 1 for both time points
(data not shown). However, enucleated adrenals in the presence of an
intact adrenal (ULE/SULA) showed reduced
P-45011 mRNA at 10 days compared
with ULE/ULA (Fig.
5A).
P-450aldo mRNA, on the other hand, was
elevated at 10 days in ULE/SULA adrenals compared with ULE/ULA
adrenals, and there was no difference between ULE/SULA and intact
adrenals at 10 days (Fig. 5B).
Treatment of ULE/SULA rats with ACTH did not significantly affect
P-45011 mRNA (Fig.
5A) but reversed the elevated
P-450aldo mRNA observed at 10 days
(Fig. 5B). The data suggest that the
presence of an intact adrenal impairs regeneration as reflected by
reduced P-45011 mRNA and that
reduced regeneration is associated with increased
P-450aldo mRNA. In addition, treatment of ULE/SULA rats with ACTH decreases
P-450aldo mRNA, suggesting that
elevated plasma ACTH observed in ULE/ULA rats contributes to decreased
expression of P-450aldo.
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Fig. 5.
Comparison of adrenal P-45011 and
P-450aldo mRNA expression at 10 days
after adrenal enucleation between rats bearing a single regenerating
adrenal (ULE/ULA) or one regenerating and one intact adrenal
(ULE/SULA). Comparisons are made to intact adrenals collected at time
of surgery (time 0). Additional
ULE/SULA rats received ACTH treatment (1 U/day) for 10 days. Data
represent means ± SE of 4 adrenals.
* P < 0.05 vs. intact adrenal
(time 0).
# P < 0.05 vs.
day 10 ULE/ULA. + P < 0.05 vs.
day 10 ULE/SULA.
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To confirm that the presence of an intact adrenal suppresses adrenal
regeneration as reflected by adrenal mass and to extend the comparison
to normal regeneration, treatment groups were compared at 30 days after
enucleation. Adrenal weight and hybridization area for
P-450scc and
P-45011 were decreased in the
regenerating adrenal from ULE/SULA rats compared with ULE/ULA or intact
adrenals (Fig. 6,
A-C).
As in experiment 2 at
day 30 (Fig.
2B), ULE/ULA adrenals were similar
to intact adrenals in all of these parameters, that is, fully
recovered. In contrast, the expression of
P-450aldo mRNA was the same in the
regenerating adrenals from ULE/ULA and ULE/SULA rats. As in
experiment 2 at day
30 (Fig. 2B),
P-450aldo was reduced in the
enucleated adrenals compared with intact adrenals (Fig.
6D). These results show that the
presence of an intact adrenal suppresses adrenal regeneration as
reflected by adrenal weight and by expression of
P-450scc and
P-45011 mRNA. The presence of an
intact adrenal does not impair recovery of
P-450aldo mRNA. However,
P-450aldo mRNA was not elevated at 30 days of impaired regeneration unlike 10 days after enucleation
(experiment 3), suggesting that
P-450aldo mRNA may reach a plateau as
regeneration is suppressed.
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Fig. 6.
Comparison of adrenal P-45011 and
P-450aldo mRNA expression at 30 days
after adrenal enucleation in rats bearing a single intact adrenal (ULA;
solid bar), a single regenerating adrenal (ULA/ULE; open bar), or one
regenerating and one intact adrenal (ULE/SULA). Paired bars denoted as
ULE-SULA represent values for paired regenerating (ULE; hatched bar)
and intact (SULA; stippled bar) adrenals from ULE/SULA rats. Data
represent means ± SE of 4 adrenals. Values with different
superscripts are significantly different
(P < 0.05) from other treatment
groups. A
: adrenal weight; B: P-450scc mRNA; C:
P-45011 mRNA; D: P-450aldo mRNA.
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Plasma hormonal response to enucleation in the presence of an intact
adrenal.
Plasma ACTH was elevated at 2 and 5 days after ULE/ULA or ULE/SULA
compared with the respective 10-day value (Table
2). However, plasma ACTH was decreased in
the ULE/SULA rats compared with the ULE/ULA rats at 2 and 5 days, but
not at 10 days after enucleation. Plasma corticosterone also decreased
in both treatment groups between 2 and 10 days. Differences in plasma
corticosterone between ULE/ULA and ULE/SULA rats were observed only on
day 2. Because day
2 samples were collected in the afternoon, circadian
changes might have contributed to elevated plasma corticosterone in the ULE/SULA rats. Plasma ACTH and corticosterone values in ULE/SULA rats
treated for 10 days with ACTH were 122 ± 11 pg/ml and 13 ± 6 ng/ml,
respectively, at 10 days after enucleation. Plasma aldosterone was not
assayed in this experiment, since the presence of an intact adrenal
precluded use of plasma aldosterone as a meaningful reflection of
P-450aldo expression in the
regenerating adrenal.
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Table 2.
Comparison of nonstress plasma ACTH and corticosterone between rats
bearing a single regenerating adrenal (ULE/ULA) or one regenerating and
one intact adrenal (ULE/SULA)
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To assess the long-term effect of the presence of an intact adrenal on
enucleation-induced responses, both nonstress plasma corticosteroids
and adrenal responses to a maximal dose of ACTH were measured at 30 days after enucleation. There was no difference in nonstress plasma
ACTH or corticosterone between ULA rats (i.e., a single intact
adrenal), ULE/ULA rats, or ULE/SULA rats (Table 3). As expected, plasma ACTH,
corticosterone, and aldosterone values were increased at 15 min after
injection. In response to ACTH injection, plasma corticosterone and
aldosterone, but not plasma ACTH, were decreased in ULE/ULA rats
compared with ULA and with ULE/SULA rats (Table 3).
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Table 3.
Comparison of plasma ACTH and corticosteroids before and after adrenal
stimulation in rats bearing a single intact adrenal (ULA), a single
regenerating adrenal (ULA/ULE), or one regenerating and one intact
adrenal (ULE/SULA) for 30 days
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DISCUSSION |
The capacity of the adrenal cortex to restore its zonation (8) and
secretory function (32) after enucleation has been thoroughly
documented. Regeneration requires both proliferation to replace tissue
mass and phenotypic differentiation to reestablish zonation (33). To
characterize this process, oligonucleotide probes that specifically
identify glomerulosa and fasciculata/reticularis cells were used with
quantitative densitometry and emulsion autoradiography to delineate the
restoration of adrenal zonation after enucleation. Enucleation removes
the adrenal medulla and the inner adrenal cortex, but not the
glomerulosa and intermedia (data not shown). Surprisingly, the
glomerulosa cell phenotype, as defined by the presence of
P-450aldo mRNA, is downregulated after
enucleation, and the glomerulosa cells assume an intermedia cell
phenotype; the loss of P-450aldo
explains reduced secretion of aldosterone in regenerating adrenals as
reported by others (1) and confirmed in the present study. Restoration
of the fasciculata precedes reestablishment of the glomerulosa; indeed,
full recovery of P-450aldo mRNA had
not occurred by 30 days of regeneration, whereas
P-45011 mRNA had been restored by
20 days. In addition, when P-450aldo mRNA was increased in enucleated adrenals by the presence of an intact
adrenal, recovery of P-450scc mRNA,
P-45011 mRNA, and adrenal weight
were suppressed. The suppression could be partially reversed by ACTH
treatment, suggesting that elevated plasma ACTH resulting from the loss
of steroid negative feedback contributes to the suppression of the
glomerulosa phenotype and the restoration of the fasciculata phenotype.
These data support the contention that the reduction or loss of the
glomerulosa cell phenotype contributes to enucleation-induced
regeneration of the adrenal cortex.
Both systemic and local factors could contribute to the downregulation
of P-450aldo expression after
enucleation of the adrenal. As observed in the present study and shown
by others (2, 12), enucleation results in increases in plasma ACTH. The
presence of an intact adrenal has been shown previously to impair
enucleation-induced regeneration (2, 12), presumably by secreting
corticosterone that acts to suppress plasma ACTH. Because the loss of
the glomerulosa cell phenotype occurs concomitantly with elevated
plasma ACTH, steroidogenic enzyme gene expression was monitored in
regenerating adrenals from rats in which steroid secretion was
maintained by a contralateral intact adrenal. Results showed that
regenerating adrenals collected from rats with a paired intact adrenal
expressed increased P-450aldo mRNA and
reduced P-45011 mRNA at 10 days. Because plasma ACTH was reduced in the presence of an intact adrenal, it is probable that the downregulation of
P-450aldo resulted from elevated
plasma ACTH. This possibility was tested by injecting ACTH to offset
the inhibitory effects of corticosterone produced by the intact
adrenal; results showed that ACTH treatment suppressed P-450aldo mRNA expression. These
findings show that the enucleation-induced dedifferentitation of the
glomerulosa cell may result in part from elevated plasma ACTH. These
data are consistent with earlier work showing that stimulation of
intact adrenals with ACTH decreases P-450aldo expression (11, 29). In
addition to ACTH, local adrenal responses, including hemorrhage, edema,
and infiltration by inflammatory cells (8), could affect
P-450aldo mRNA expression. The
vascular changes could deprive the remaining glomerulosa cells of
nutrients required for survival, resulting in cell death; although cell
death most likely occurs after enucleation, the expression of
P-450scc and 3-HSD mRNA in cells
underlying the capsule supports their viability. However, changes in
blood flow could produce local hypoxia. Because systemic hypoxia
decreases P-450aldo expresson (27),
reduction in tissue oxygenation could contribute to enucleation-induced changes in adrenal cell phenotype. In addition, local inflammation may
play a role by providing cytokines, like tumor necrosis factor and
interleukin-1, that have been shown to inhibit aldosterone responses to
ACTH and ANG II in vitro (21).
The physiological relevance of the dedifferentiation of glomerulosa
cells to intermedia cells to the regenerative response is unknown. It
is intriguing that enucleation results in the expansion of the zona
intermedia, because others have suggested that intermedia cells
represent stem cells for regeneration (18). However, in our initial
studies of adrenal regeneration, proliferating cells as defined by
expression of the Ki67 antigen appeared to be excluded from the zona
intermedia (5). Although a more rigorous examination is required to
establish the phenotype of proliferating cells throughout regeneration,
it is clear that during the initial stages of regeneration,
proliferation is not restricted to the zona intermedia. To establish
the possible significance for the reduction of
P-450aldo in the early stages of
regeneration, it will be necessary to prevent the reduction and assess
the effect on regeneration.
It is also possible that the reduction of
P-450aldo per se is not a required
element for regeneration, but instead is an epiphenomenon of decreased
glomerulosa cell stimulation by ANG II, perhaps via downregulation of
ANG II (AT) receptors. Neither changes in adrenal AT receptors nor
responses of the renin-angiotensin system after enucleation have been
reported to date. Adrenalectomy results in elevated ANG II (28), and in
preliminary studies, ULA/ULE results in increases in plasma renin
activity during the initial 3 days after enucleation (C. Wotus, A. I. Fraticelli, and W. C. Engeland, unpublished observations).
Thus it is unlikely that circulating angiotensin contributes to the
reduction of P-450aldo expression in
regenerating adrenals, because ANG II upregulates the AT receptor and
is a positive regulator of P-450aldo
expression (11, 15, 35). More likely, because
P-450aldo expression decreases after
enucleation, factors like ACTH are required to offset the positive
effect of AT receptor activation on
P-450aldo expression.
Perspectives
The adrenal cortex demonstrates the uncommon capacity to regenerate
after injury produced by transplantation or enucleation. This
phenomenon was described early in this century (see Ref. 13) and
subsequently has been characterized as a regenerative response that
requires both cell proliferation and differentiation (22, 33). The
novelty of the present study stems from the ability to define the
phenotype of the cortical cell during restoration of zonation. The
results implicate modulation of glomerulosa cell differentiation as a
critical component of adrenal cortical regeneration. However, there are
a host of unresolved issues concerning the regenerative process,
including the nature of intermedia cell differentiation, the identity
of proliferating cells in the regenerating cortex, the nature of the
factors that regulate the loss of
P-450aldo expression, and the
mechanism by which glomerulosa cell differentiation is reestablished.
Expression of factors linked to differentiation of adrenal
glomerulosa/intermedia cells, such as Pref-1 (9), may play a role in
these processes. It will be of interest to determine the cellular
mechanisms by which systemic factors like ACTH and ANG II and local
inflammatory factors regulate phenotypic expression and regeneration in
the adrenal cortex.
| |
ACKNOWLEDGEMENTS |
We thank Lisa Rogers and Debra Fitzgerald for excellent technical support.
| |
FOOTNOTES |
This work was supported by the Department of Surgery, Center for Wound
Healing and Reparative Medicine, University of Minnesota, and National
Institute of General Medical Sciences Grant GM-50150.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. C. Engeland,
Dept. of Surgery, Box 120 UMHC, University of Minnesota, Minneapolis,
MN 55455 (E-mail: engel002{at}tc.umn.edu).
Received 1 December 1998; accepted in final form 2 February 1999.
| |
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