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Departments of Surgery and Cell Biology and Neuroanatomy (W.C.E.), University of Minnesota, Minneapolis, Minnesota 55455
Address all correspondence and requests for reprints to: William C. Engeland, Ph.D., Department of Surgery, Box 120 UMHC, University of Minnesota, Minneapolis, Minnesota 55455. E-mail: engel002{at}tc.umn.edu
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Abstract |
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4-isomerase (3ßHSD) and cytochrome P450
11ß-hydroxylase (P45011ß) messenger RNA (mRNA) in adult rat
adrenals after stimulation in vivo. In Exp 1, adrenals
were collected from rats injected with saline or ACTH for 1, 2, 3, or 4
days. Adrenal sections from saline-treated rats showed uniform
expression of 3ßHSD mRNA that extended from the adrenal capsule to
the medullary border. In contrast, P45011ß mRNA showed high levels in
the outer fasciculata and low levels in the inner
fasciculata/reticularis. In response to ACTH, the integrated density of
3ßHSD hybridization did not increase until 4 days. The integrated
density of P45011ß hybridization increased in ACTH-treated rats
between 1–4 days due to increased hybridization in the inner
fasciculata/reticularis. In Exp 2, rats were treated with ACTH or
saline, and adrenals were harvested at 4, 8, or 24 h. The
hybridization density of 3ßHSD did not change after ACTH or saline
injection. Increased expression of P45011ß mRNA was observed at 4 and
8 h, but not 24 h post-ACTH. In Exp 3, to determine the
response to acute stress, adrenals were collected from rats 24 h
after surgical laparotomy. The integrated density of 3ßHSD labeling
did not change, whereas both hybridization area and mean density of
P45011ß increased. Increased expression of P45011ß mRNA was
observed in the inner fasciculata similar to that observed after ACTH
injection. In addition, adrenal cells were more responsive to ACTH
in vitro after surgical stress. These results suggest
that the rat adrenal cortex can respond to acute stress by
up-regulation of the expression of steroidogenic enzyme genes and that
this occurs in part by increasing the number of cells actively
expressing P45011ß mRNA. The adrenal response after stress most
likely results at least in part from stimulation by ACTH. These
findings suggest that changes in adrenal steroidogenesis in response to
ACTH may result from recruitment of steroidogenic cells to synthesize
and secrete corticosteroids. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (P45021) (2). The late steps in
the steroidogenic pathway are zone specific. In the zona fasciculata
and reticularis, corticosterone synthesis is dependent on the
expression of cytochrome P450 11ß-hydroxylase (P45011ß), which
converts 11-deoxycorticosterone to corticosterone. In the zona
glomerulosa, aldosterone synthesis is dependent on the expression of
cytochrome P450 aldosterone synthase (P450aldo), an enzyme with
11ß-hydroxylase, 18-hydroxylase, and 18-oxidase activities, which
converts deoxycorticosterone to aldosterone. In a recent study, in situ hybridization histochemistry was used to monitor the restoration of adrenal zonation during the course of adrenal regeneration (3). Oligonucleotide probes specific for P450aldo and P45011ß messenger RNA (mRNA) were able to distinguish between glomerulosa and fasciculata/reticularis cells, respectively. As expected, P450aldo mRNA was restricted to the zona glomerulosa, whereas P45011ß mRNA was detected only in the inner cortical zones. However, a nonuniform expression of P45011ß mRNA was observed in normal rat adrenals, in that a large proportion of inner zona fasciculata showed minimal gene expression. In these studies, adrenals were collected from rats under nonstress conditions; thus, it was possible that in response to adrenal activation, the proportion of fasciculata cells that express P45011ß mRNA might increase. As P45011ß enzyme activity is required for the production of corticosterone, increases in the number of fasciculata cells actively expressing P45011ß mRNA and enzyme could represent a mechanism that operates to augment adrenal steroidogenesis in response to stress.
In an exhaustive study performed using hamster adrenals, in vivo ACTH treatment induced differential changes in specific steroidogenic enzymes at the transcriptional level (4). Although transcripts for enzymes in the early part of the steroidogenic pathway were increased by ACTH, P45011ß mRNA was not affected. This work prompted the present set of experiments to assess whether the rat adrenal has the capacity to increase the expression of P45011ß in response to adrenal activation. Experiments were performed to monitor the pattern and intensity of gene expression for P45011ß within the zona fasciculata/reticularis under unstimulated conditions and after stimulation by ACTH or acute stress.
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Materials and Methods |
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Experimental protocols
Exp 1.
To determine the effect of chronic treatment with
ACTH on adrenal P45011ß mRNA, rats were injected daily with ACTH (1 U
ACTHar gel, sc; 1 U = 
10 µg ACTH) or saline in the morning
for 1–4 days and were killed by decapitation at 24-h intervals.
Adrenals were removed, cleaned, immersed in cold isopentane, and frozen
at -70 C.
Exp 2.
To determine the acute response to ACTH, rats were
injected with ACTH (1 U ACTHar gel, sc) or saline in the morning and
killed by decapitation at 4, 8, and 24 h. Trunk blood was
collected for assay of plasma corticosterone and ACTH, and adrenals
were collected and stored as outlined above.
Exp 3.
To determine whether surgical stress increases
adrenal P45011ß mRNA and changes the adrenal sensitivity to ACTH,
rats were anesthetized with sodium pentabarbital (65 mg/kg) and
underwent surgical laparotomy or no surgery. The laporatomy consisted
of subcostal skin and muscle incisions followed by gut and liver
retraction; after 15 min, incisions were closed with suture. At 30 min,
tail vein samples were collected for assay of plasma ACTH and
corticosterone. Rats received antibiotics (Ancef; 10 mg/kg, im), were
kept warm until sternally recumbent, and then were returned to their
cages. After 24 h, rats were killed by decapitation, and trunk
blood was collected for plasma hormone assay. Left adrenals were
frozen. Right adrenals were decapsulated, and the
fasciculata/reticularis cells were dispersed with collagenase as
described previously (5). The zona fasciculata/reticularis cells from
each adrenal were minced separately, placed in medium containing
collagenase (DMEM with 0.2% type 1A collagenase; Life Technologies,
Grand Island, NY), 0.1% deoxyribonuclease (Sigma Chemical Co., St.
Louis, MO), and 4.0% BSA (Sigma)) and incubated at 37 C in 10%
CO2 for 90 min with 15-min intervals of gentle trituration.
Cells were filtered through a wire mesh (100 µm) into incubation
medium (DMEM with 0.4% BSA and 12.0 mM HEPES) and
centrifuged at 200 x g for 5 min at 4 C. After two
washes, viable fasciculata cells, defined by the presence of large
lipid droplets, were plated in 96-well microtiter plates (2.0 x
105 cells/well). After a 2-h preincubation, rat
ACTH-(1–39) (Peninsula Laboratories, Belmont, CA) was added, and cell
suspensions were incubated overnight (16 h) at 37 C in 7%
CO2. Medium was removed and stored at -20 C.
The experiments reported here were performed in complete accordance with the NIH Guidelines for Humane Use of Experimental Animals, and all protocols were approved by the University of Minnesota animal care committee.
In situ hybridization histochemistry
Adrenals were frozen-sectioned (14 µm) and thaw-mounted onto
ProbeOn slides (Fisher Scientific, Fairlawn, NJ). The protocol was
identical to the method of Dagerlind et al. (6). Slides were
incubated at 42 C overnight with 5–10 ng 35S-labeled
probe/ml hybridization solution. Sections were washed five times for 15
min each time in 1 x SCC (saline-sodium citrate) at 54 C, rinsed,
dehydrated, air-dried, and exposed to Biomax film (Eastman Kodak,
Rochester, NY). Some sections were dipped in NTB-2 emulsion (Kodak) and
exposed for 5–7 days. Dipped slides were developed, counterstained
with bisbenzimide (7), and dehydrated. Oligonucleotide probes,
purchased from Keystone Labs (Menlo Park, CA) or from Life
Technologies, were designed to detect 3ßHSD (nucleotides 1294–1329
of type I) (8), P45011ß (nucleotides 814–849) (9), and P450aldo
(nucleotides 918–953) (10) mRNA specifically. A generic P45011ß
probe was designed to detect both P450aldo and P45011ß mRNA
(nucleotides 154–189 of P450aldo and 50–85 of P45011ß) (3). The
specific P45011ß probe was used for quantitation of gene expression.
The specificity of the probes used to detect steroidogenic enzyme mRNAs
was confirmed by three methods. First, it was demonstrated that the
hybridization signal could be blocked by the addition of excess
(100-fold) unlabeled probe to the hybridization solution; second, for
each antisense probe, hybridization was not affected by the addition of
unlabeled probe antisense to another steroidogenic enzyme mRNA; and
third, the use of labeled sense probes for each of the steroidogenic
enzyme mRNA resulted in no hybridization signal (data not shown).
Measurements of integrated hybridization density were used as an index of mRNA levels in tissue sections. Sections from each group of adrenals were hybridized together; autoradiograms were scanned using a UMAX scanner calibrated to a density scale (Stouffer, South Bend, IN) 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]. Areas with positive hybridization were measured after thresholding. Thresholds were chosen to include all areas of positive hybridization within the cortex and were held constant for all measurements for each specific transcript. Background measurements taken of nonhybridized tissue were equal to zero, as were measurements of tissue hybridized for specificity controls described above. For adrenals collected from nonstressed or stimulated rats, the area of hybridization and the mean density for three or four tissue sections per adrenal were measured, and the means were calculated for each adrenal collected from four rats. The integrated density for each section was calculated as the product of hybridization area and mean density.
Photomicroscopy
Optical images of emulsion-dipped sections were collected using
a monochrome CCD camera (Cohu, San Diego, CA), captured with a Scion
LG-3 frame grabber, and processed on a Macintosh IIcx computer using
NIH Image 1.6 and Adobe Photoshop 3.0 software.
RIA
Plasma corticosterone was measured by RIA using a kit (ICN
Biomedical, Costa Mesa, CA). The intraassay coefficient of variation
(CV) was 7.6%, and the interassay CV was 13.3% for a pool value of 78
ng/ml. Plasma ACTH was measured by RIA as described previously (11).
The intra- and interassay CVs were both 12% for a plasma pool with a
concentration of 45 pg/ml.
Data analysis
Differences between groups in mRNA expression and plasma or
medium hormone concentration were determined by ANOVA, and individual
means were compared using Fisher’s least significant difference test.
For all analyses, P < 0.05 was required for
statistical significance. Data are expressed as the mean ±
SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The integrated
density of 3ßHSD hybridization did not change until 4 days of ACTH
treatment, whereas the integrated density for P45011ß increased at 1
day and remained elevated for 4 days relative to that in
saline-injected controls (Fig. 1). The change in
integrated density for each message resulted primarily from an increase
in the total area of hybridization; the mean density of hybridization
for P45011ß increased only at 3 days (Table 1).
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). The generic P45011ß probe also demarcated
the zona intermedia as an area devoid of silver grains between the zona
glomerulosa and zona fasciculata (Fig. 2A). In contrast to the
zone-specific expression, 3ßHSD mRNA was characterized by uniform
expression that extended from the adrenal capsule to the adrenal
medullary border (Fig. 2B). Comparison of the labeling using the
P45011ß and 3ßHSD probes showed that the expression of P45011ß
mRNA was nonuniform, in that large areas of the inner zona
fasciculata/reticularis showed minimal gene expression. This pattern
was observed using either the specific or the generic P45011ß probe
(data not shown). In addition, clusters of cells within the adrenal
medulla expressed P45011ß and 3ßHSD mRNA, confirming the existence
of fasciculata/reticularis cells within the medulla (12). Adrenals
collected from rats injected with ACTH showed an expression pattern of
P45011ß mRNA different from that observed in saline-treated rats.
Using either the specific or the generic P45011ß probe, increased
expression of P45011ß mRNA was observed in the inner zona
fasciculata/reticularis (Fig. 2C). The increase in P45011ß labeling
in the inner cortex after exposure to ACTH generated a pattern of
expression similar to that of 3ßHSD mRNA (Fig. 2D). A high power
photomicrograph showed increased density of silver grains representing
P45011ß mRNA overlying cells in the inner cortex after ACTH treatment
compared to that after saline treatment (Fig. 3). The
enhanced expression of P45011ß mRNA in the inner cortex induced by
ACTH was consistent with the measured increases in hybridization
area.
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In response to ACTH the integrated density of hybridization for
P45011ß increased at 4 and 8 h, but was similar to the control
value by 24 h (Fig. 4). The response reflected
changes in hybridization area; the mean hybridization density did not
change (data not shown). In addition, the integrated density of 3ßHSD
hybridization did not change in response to ACTH (Fig. 4). These
results show a differential increase in P45011ß mRNA by 4 h
after ACTH stimulation. However, unlike Exp 1, no increase was observed
at 24 h relative to the value in saline-injected controls. Film
autoradiograms show clear expansion of the area of P45011ß labeling
within the inner zona fasciculata/reticularis at 4 and 8 h, but
not at 24 h, after ACTH (Fig. 5).
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).
The stress-induced increase in P45011ß hybridization was paralleled
by increased labeling for P45011ß mRNA in the inner cortex (Fig. 7). To determine whether the change in P45011ß
hybridization was associated with a change in adrenal responsiveness to
ACTH, the paired adrenal from the rats exposed to surgery or anesthesia
only was dispersed and tested with ACTH in vitro. The
corticosterone response of adrenal cells from rats exposed to surgery
was increased compared to that of control adrenal cells (Fig. 8).
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Discussion |
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In situ hybridization histochemistry was used to define the
phenotype of steroidogenic cells within the rat adrenal cortex. The
zona glomerulosa and fasciculata/reticularis specifically express
P450aldo and P45011ß mRNA and protein, respectively (13, 14). In
addition, all cortical cells express the enzymes responsible for
processing cholesterol to 11-deoxycorticosterone, including P450scc,
3ßHSD, and P45021 (2). A probe for 3ßHSD mRNA was used to
identify all cortical cells, whereas probes for P45011ß mRNA were
used to identify specifically fasciculata/reticularis cells. The
finding that mismatches occurred was anticipated, as the glomerulosa
cells express P450aldo, not P45011ß, and the intermedia cells express
neither P450aldo nor P45011ß (15). However, the identification of
cells expressing 3ßHSD in the inner zona fasciculata or reticularis
that do not express P45011ß was somewhat unexpected. The observation
required a histological approach that, first, distinguished cortical
from noncortical cells and, second, defined the cortical cell
phenotype. In situ hybridization relied on the use of
adjacent sections to match regions positive for both 3ßHSD and
P45011ß mRNA. The method identified areas in the inner zona
fasciculata and reticularis that were 3ßHSD positive and P45011ß
negative. These areas are referred to as P45011ß negative with the
clear understanding that this designation signifies only that
transcript levels are below the limit of detection of the in
situ hybridization method. In response to adrenal stimulation,
additional 3ßHSD-positive cells in the same areas became P45011ß
positive. The differential change in the pattern of expression of
P45011ß mRNA relative to that of 3ßHSD mRNA was reflected by the
measurement of hybridization area for each transcript. In response to
ACTH or stress, there were increases in the hybridization area for
P45011ß, but not for 3ßHSD. Comparison of the magnitude of
hybridization area for each transcript shows that the size of the area
of P45011ß labeling approached that of 3ßHSD labeling after ACTH
stimulation or surgical stress. These data strengthen the conclusion
that additional cortical cells are expressing P45011ß mRNA after
adrenal stimulation.
Using semiquantitative in situ hybridization histochemistry,
a single injection of ACTH increased P45011ß mRNA at 4 and 8 h;
by 24 h, the expression was reduced in one experiment and remained
elevated in a second. Repeated daily injections maintained P45011ß
transcripts at elevated levels for 4 days. These data suggest that
P45011ß is transcriptionally activated by ACTH in vivo. In
cultured bovine adrenal cells, exposure to ACTH increases P45011ß
mRNA within the initial 6–12 h of stimulation (16). In vivo
studies in rats have failed to show consistent ACTH-induced increases
in adrenal P45011ß mRNA. Using Northern analysis, adrenal P45011ß
mRNA did not increase 24 h after ACTH. However, the results are
difficult to interpret, as in one study P45011ß mRNA was normalized
to housekeeping genes that responded to ACTH (17), and in another study
no statistical confirmation of the data was presented (18). In
contrast, using in situ hybridization, a limited data set
was presented showing that P45011ß mRNA was more abundant in the
outer fasciculata and increased in the zona reticularis at 24 h
after ACTH (19). The variability in the 24-h response to a single ACTH
injection cannot be explained. In each of the studies, pharmacological
preparations and doses of ACTH were used. It is possible that the
metabolism of depot ACTH or the duration of ACTH binding to adrenal
receptors varied between experiments. Taken together, the data suggest
that a single injection of ACTH causes a transient increase in
P45011ß mRNA, which is in the process of returning to baseline by
24 h; subsequent exposure to ACTH increases the response. Despite
the variability in the response at 24 h, the dynamics of the
initial response to ACTH are consistent with the in vivo
responses of steroidogenic enzymes to ACTH in hamster adrenals (4). A
single injection of ACTH increased P450scc and P450 17-hydroxylase
mRNA within 2.5–5 h, with a return toward baseline by 24 h.
Interestingly, no changes were observed in P45011ß and P45021 mRNA
over the same time period. These results show that rapid changes in
steroidogenic enzyme gene expression can occur in vivo in
response to ACTH and that specific enzyme transcripts respond
differentially. The finding that ACTH stimulates P45011ß mRNA in rat,
but not hamster, adrenals may reflect differences in the nature of the
corticosteroids produced in each species. The rat adrenal produces
corticosterone and is dependent on P45011ß activity for
glucocorticoid secretion. Although the hamster adrenal secretes both
corticosterone and cortisol, it is primarily a cortisol producer (4).
It is possible that cortisol is regulated in the hamster adrenal by
ACTH at level of 17-hydroxylase P450, instead of P45011ß.
Therefore, differences in experimental results could be due to species
differences. Nonetheless, the data presented demonstrate the capacity
of the rat adrenal to increase P45011ß gene expression rapidly.
Further characterization of the physiological response to ACTH will
require additional studies using lower doses of ACTH.
To assess whether the changes in the pattern of P45011ß gene expression occurred under more physiological conditions, P45011ß gene expression was monitored after surgical stress. The stress was sufficient to stimulate plasma ACTH and corticosterone acutely, with plasma hormone concentrations returning to baseline by 24 h. Activation of the adrenal by surgical stress increased P45011ß mRNA without changing 3ßHSD mRNA. The change in P45011ß hybridization resulted from an increase in both hybridization area and mean density. These results suggest that surgical stress not only increases the number of cells expressing P45011ß mRNA, but also increases the amount of mRNA per cell. These data clearly show that physiological stimuli produced by stress are able to up-regulate gene expression of P45011ß. The adrenal response could result from activation by ACTH, although ACTH alone increased hybridization area without affecting mean hybridization density. It is possible that factors stimulated by surgical stress in addition to ACTH contributed to the adrenal response. For example, there is ample evidence for innervation of the adrenal cortex by sympathetic nerves (20, 21) and for functional changes in adrenal steroidogenesis in response to alterations in sympathetic nerve activity (22, 23). Adrenergic agonists can induce adrenal P450 gene expression in vitro (24), supporting the possibility that neurotransmitters released from nerve terminals could act directly on cortical cells to increase P45011ß mRNA. In addition, the area of increased P45011ß hybridization in the inner fasciculata lies at the termination of adrenal sinusoids. The innervation of the capsular/zona glomerulosa by neuropeptidergic (25) and adrenergic (26) fibers has been viewed as a site for controlling blood flow to the inner cortex (27). Surgical stress, by activating vasomotor nerves in the outer cortex, might augment P45011ß gene expression by increasing the exposure of inner cortical cells to ACTH through increasing adrenal blood flow (28). Additional experiments are required to assess the possible interaction of ACTH and sympathetic neural activity on stress-induced changes in P45011ß gene expression.
The recruitment of cells in the inner cortex to express P45011ß mRNA after stress represents yet another example of the phenotypic plasticity of the adrenal cortex. Others have focused on the capacity of the zona fasciculata to expand outward at the expense of the zona glomerulosa (14, 19). Treatment with ACTH converts cells from a glomerulosa phenotype to a fasciculata phenotype (29), reflected by a change in the expression of P450aldo to P45011ß. Chronic stress in rats induced by immobilization or repeated injections of hypertonic saline decreases P450aldo mRNA, resulting in hypoaldosteronism (30). The decrease in P450aldo mRNA was associated with an expansion outward of the area of P45011ß mRNA. Interestingly, unlike acute surgical stress, chronic stress in rats did not increase P45011ß mRNA in the inner cortex (30), nor did low intensity stress induced by repeated injections of isotonic saline. It is not clear whether the differential response is dependent on the type of stress applied, its duration, or its intensity. However, these findings suggest that the zona fasciculata has the capacity to expand in response to stress by recruitment of cells to perform the last step in the production of corticosterone, and that the expansion can occur at its inner or outer borders under different physiological circumstances. Although the present study did not assess whether surgical stress resulted in an expansion of P45011ß mRNA outward into the subcapsular region, the hybridization area of P45011ß mRNA remained less than that of 3ßHSD mRNA. Hybridization in the subcapsular area was detectable in adrenals from surgically stressed rats using the generic P45011ß probe or the P450aldo probe (Engeland, W. C., B. K. Levay-Young, and L. M. Rogers, unpublished observations). Presumably, the difference in hybridization area reflects the presence of the zona glomerulosa and zona intermedia 24 h after surgery.
Although the data presented show that the zona fasciculata has the capacity to augment the number of cells expressing P45011ß mRNA, additional studies are required to determine whether the change in gene expression results in a change in steroidogenic function. In response to surgical stress, changes in P45011ß mRNA were associated with increases in adrenal responsiveness to ACTH in vitro. It is possible that augmented responsiveness results from an increased number of cortical cells expressing P45011ß activity. No experiments have been performed to measure changes in enzyme activity in response to surgery. Surgery-induced changes in adrenal responsiveness to ACTH could also occur independently of changes in enzyme expression. Experiments have been initiated to determine whether P45011ß protein increases in parallel with P45011ß mRNA. Preliminary results using immunocytochemistry show that expansion of the number of cells in the inner cortex expressing P45011ß protein increases after ACTH treatment (Engeland, W. C., and C. Wotus, unpublished observations). Ongoing experiments are being performed to compare the time courses of expression of P45011ß mRNA and protein after ACTH administration and stress.
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Footnotes |
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Received November 20, 1996.
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References |
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5-4 isomerase cDNAs and differential
tissue-specific expression of the corresponding mRNAs in steroidogenic
and peripheral tissues. J Biol Chem 266:583–593This article has been cited by other articles:
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H. Raff, B. M. Jankowski, E. D. Bruder, W. C. Engeland, and M. K. Oaks The Effect of Hypoxia from Birth on the Regulation of Aldosterone in the 7-Day-Old Rat: Plasma Hormones, Steroidogenesis in Vitro, and Steroidogenic Enzyme Messenger Ribonucleic Acid Endocrinology, July 1, 1999; 140(7): 3147 - 3153. [Abstract] [Full Text]
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 W. C. Engeland and B. K. Levay-Young Changes in the glomerulosa cell phenotype during adrenal regeneration in rats Am J Physiol Regulatory Integrative Comp Physiol, May 1, 1999; 276(5): R1374 - R1382. [Abstract] [Full Text] [PDF]
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C. Wotus, B. K. Levay-Young, L. M. Rogers, C. E. Gomez-Sanchez, and W. C. Engeland Development of Adrenal Zonation in Fetal Rats Defined by Expression of Aldosterone Synthase and 11{beta}-Hydroxylase Endocrinology, October 1, 1998; 139(10): 4397 - 4403. [Abstract] [Full Text] [PDF]
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J.-G. Lehoux, A. Fleury, and L. Ducharme The Acute and Chronic Effects of Adrenocorticotropin on the Levels of Messenger Ribonucleic Acid and Protein of Steroidogenic Enzymes in Rat Adrenal in Vivo Endocrinology, September 1, 1998; 139(9): 3913 - 3922. [Abstract] [Full Text] [PDF]
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) showed increased responsiveness to ACTH
compared to those from control rats exposed to anesthesia only (•).
Each point represents the mean ± SE of
four adrenals. There was a significant treatment effect (surgery
vs. control), as determined by ANOVA
(P < 0.05).