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Inhibition of ACTH-induced differentiation of cortical cells and their mitochondria by corticosterone in tissue culture of fetal rat adrenals.

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Inhibition of ACTH-induced Differentiation of Cortical
Cells and Their Mitochondria by Corticosterone in
Tissue Culture of Fetal R a t Adrenals'
ARVI I. KAHR12A,4
Department of Anatomy, Harvard Medical School, Boston,
Massachusetts 021 15
ABSTRACT
Tissue cultures of fetal rat adrenals were used to study the
effects of corticosterone on the ACTH-induced ultrastructural differentiation of
cortical cells and their mitochondria. Corticosterone in dosages of 0.2, 2.0, 5.0,
30, and 20 pglml (corresponding to concentrations of 6 X lo-', 6 X
1.5 X
3 X lo+, and 6 X
molar) was added alone or together with
100 mU/ml of ACTH to the culture medium, daily from the sixteenth day of
cultivation up to and including the twenty-first day. Corticosterone alone induced
no ultrastructural changes in cortical cells. Corticosterone in concentrations of
6X
to 3 X
M given with ACTH induced hypertrophy of Golgi appaM inhibited the ACTHratus. Corticosterone in concentrations of 6 X
induced differentiation of cortical cells. However, the nuclear chromatin increased and Golgi apparatus was strikingly hypertrophied. Mitochondria often
aggregated adjacent to the nuclear envelope but their ultrastructure remained
undifferentiated with tubular or tubulovesicular cristae. Ribosomes appeared as
single particles. A marked increase of smooth surfaced endoplasmic reticulum
was noted also in cortical cells treated with 6 X
M of corticosterone.
The present observations suggest that corticosterone acts as an intracellular
inhibitor in cortical cells. It appears to inhibit cytoplasmic protein synthesis at
the ribosomal level and prevents synthesis of cytoplasmic mitochondrial protein
synthesis stimulating factor and the latter, in turn, inhibits the activation of
mitochondrial protein synthesis. A new model is presented to explain the regulation of growth and secretion in the adrenal cortex.
The general role of adrenocorticotrophic
hormone (ACTH) as a regulator of growth
and steroidogenesis in the adrenal cortex is
well-known. Moreover, the pituitary-adrenal axis is controlled by the level of ACTH
in blood which also influences differentiation of cortical cells. Eliminating the
source of ACTH by hypophysectomy results in atrophy of the inner zones of the
adrenal cortex leaving the zona glomerulosa intact. This effect is reversible since
normal physiology and morphology of
adrenal cortex is restored by ACTH stimulation (Sabatini et al., '62; Nishikawa et
al., '63; Idelman, '66). It is generally accepted that the function of these zones is
controlled by ACTH alone and the glucocorticoids produced by the adrenal cortex
in turn regulate the secretion of pituitary
ACTH.
In vitro studies have shown that ACTH
ANAT.REC.,176: 253-272.
is an inductor in the ultrastructural differentiation of cortical cells (Kahri, '66, '68,
'70, '71b; Kahri et al., '70), based on two
different protein synthetic systems, nuclear
and mitochondrial. Recent findings indicate that the nucleus is primary locus of
ACTH stimulation (Kahri, '71b). The
marked mitochondrial differentiation noted
after ACTH treatment presumably results
from stimulation of chloramphenicol-sensitive mitochondrial protein synthesis
(Kahri, '70), which is also mediated by
Received Nov. 10, '71. Accepted Mar. 14, '73.
1 Presented in Tenth Annual Meeting of the American Society for Cell Biology in San Diego, in November 1970. See abstract J. Cell Biol., 47: lola, 1970.
ZSupported in part by research grant GM-06729
from the Institute of General Medical Sciences, National Institutes of Health.
3Supported by a Public Health Service Intemational Postdoctoral Research Fellowship F05 TW
1387-2.
4Author's present address: I1 Department of Pathology, the University of Helsinki, Haartmaninkatu
3, SF-00290 Helsinki 29, Finland.
253
254
ARVI I. KAHRI
cycloheximide-sensitive cytoplasmic ribosomal protein synthesis (Kahri, '71b).
These results suggest that a specific cytoplasmic factor, probably a protein stimulates mitochondrial synthesis and may be
a mediator of ACTH action on the mitochondria. The fmal product of highly differentiated mitochondria of rat adrenal
cortical cells is corticosterone (Sharma et
al., '62; Harding et al., '65; Pkron et al.,
'66; Guerra et al., '66; Kimura and Suzuki,
'67; Kimura, '68). The llp-hydroxylase
enzyme is located in the inner membranes
of the mitochondria (Yago and Ishii, '69).
In vitro studies also indicate that mitochondrial protein synthesis is partially responsible for the production of structural
protein in cristae (Kahri, '70) and the
ACTH-induced structural changes in the
mitochondria correlate very well with the
ACTH-induced increase in enzymatic capacity of mitochondrial llp- and 18-hydroxylase in cultivated cortical cells of
fetal rat adrenal (Kahri et al., '70).
All these findings have stimulated research on the effects of corticosterone, the
endproduct of mitochondrial functional
activity, on the ACTH-induced ultrastructural differentiation of cortical cells and
especially on the ACTH-induced biogenesis
of mitochondria in tissue culture of fetal
rat adrenals. Such research might provide
information on the regulatory mechanism
of growth and protein synthesis in adrenocortical cells.
MATERIAL AND METHODS
Twenty-one day old fetal albino rats of
Charles River strain were sacrificed by decapitation. Their adrenals were explanted
as tissue cultures. The average amount of
tissue per culture was about seven adrenals. The culture method employed was
found in earlier studies (Kahri, '66) to be
suitable for long term cultivation of adrenals. The medium contained 50% Monkey
Kidney Medium A (0.5% lact-albumin hydrolyzate in Hanks' Balanced Salt Solution
-I- 2% calf serum), 25% calf serum and
25% Eagle's Minimum Essential Medium
(Microbiological Association, Bethesda,
Maryland, U.S.A.). Adrenocorticotrophic
hormone (Acthar, Corticotropin Injection,
Armour, Pharmaceutical Company, Chicago, Illinois, U.S.A.), was added to the
culture medium in the amount of 100 mU/
ml every day for six days from the six-
teenth day of cultivation up to and including the twenty-first day. Corticosterone
(A-pregnen-1 lp, 21-diol-3, 20 dione)
(Sigma, Chemical Company, St. Louis,
Missouri, U.S.A.), dissolved in ethanol,
was added in amounts of 0.2, 2.0, 5, 10,
and 20 pg/ml/day (corresponding molar
concentrations of 6 X lo-', 6 X lo-',
1.5 X
3X
and 6 X lo-' M)
separately and together with ACTH every
day for six days from the sixteenth day of
cultivation up to and including the twentyfirst day. The medium was changed every
fifth day of cultivation but not during
stimulation.
Tissue cultures were carefully studied by
phase contrast microscopy during the experiments and before fixation.
Cultures were fixed in situ in 2.5%
glutaraldehyde in Hanks' BSS adjusted to
pH 7.2 at the beginning of fixation and
postfixed in 1% osmium tetroxide with
phosphate buffer (Millonig, '62) or in 1%
osmium tetroxide in Hanks' BSS. After
fixation they were dehydrated in a graded
series of ethyl alcohol and embedded in
situ in a mixture of Epon 812 (Shell
Chemical Corp., New York) and Araldite
6005 (Ciba Products Company, Summit,
N. J., U.S.A. ). Thin sections were cut with
a Sorval MT-1 ultramicrotome using glass
or diamond knives. The sections were
stained with 0.2% lead citrate for 20
seconds (Venable and Coggeshall, '65).
Electron micrographs were taken at original magnifications of 1300-30,000 with a
RCA-3F electron microscope.
RESULTS
The ultrastructure of cortical cells
of fetal TQt adrenals in
tissue culture
Cortical cells of fetal rat adrenals growing in tissue cultures demonstrated undifferentiated ultrastructure of zona glomerulosa cells of rat adrenal cortex. ACTH
stimulation of cortical cells in tissue culture of fetal rat adrenals induces differentiation of these cells into zona fasciculata type cells. The ultrastructural changes
of this transformation include : increase
in size of cells, transformation of small,
INHIBITION OF MITOCHONDRIAL BIOGENESIS BY CORTICOSTERONE
irregular nuclei to spherical nuclei, hypertrophy of the nucleolus and Golgi apparatus, the amount of cytoplasm and smooth
endoplasmic reticulum increases and
desmosome-like thickenings of the plasma
membranes disappear. There is increase in
the number of microvilli and mitochondria
and lipid droplets. Single ribosomes as they
appear in untreated cortical cells, seem to
transform to polysomal clusters. The most
prominent effect of ACTH on the cortical
cells has been seen on the mitochondria.
This involves alteration of the structure of
mitochondrial cristae from tubular or
tubulovesicular to vesicular (600 A vesicles).
Effect of corticosterone on the cortical
cells of fetal rat adrenals in
tissue culture
Using 0.2/pg/ml/6 days (6 X lo-' M )
- 2/&ml/6 days ( 6 X lo-' M ) of corticosterone no ultrastructural changes of
cortical cells in tissue culture of fetal rat
adrenals were observed. The mitochondria
remains undifferentiated and the amount
of smooth surfaced endoplasmic reticulum
remains scant (figs. 1-2). No development
of mic:rovilli or increase in the amount of
lipid droplets were observed. The size and
shape of cells and nuclei remained unchanged and no visible changes in the
amount of nuclear chromatin or hypertrophy of nucleolus were observed. There
was no change in the shape or size of
Golgi apparatus.
Effects of corticosterone on the
ACTH-induced differentiation
of adrenocortical cells
Corticosterone in dosage of 0.2/pg/ml/6
days (6 X lo-? M ) together with 100
mU/n1l/6 days of ACTH induced no inhibition of ACTH-induced differentiation
of cortical cells. There is more hypertrophied Golgi apparatus in these cells than
those treated only with ACTH (fig. 3).
Corticosterone in dosages of 2 pg/ml/6
days ( 6 X
M and 3 X lo-' M) together
with ACTH induced increased hypertrophy
of Golgi apparatus. The Golgi saccules and
vesicles become enormously dilated (fig.
4). The dilated vesicles seem to be empty
and 110 electronopaque material was observed in vicinity of Golgi apparatus. All
255
cortical cells seem to be similarly differentiated and undifferentiated cells were
not observed in significant numbers. Cortical cells enlarged normally and their
cytoplasm was filled with profiles of tubular or vesicular cisternae of smooth surfaced endoplasmic reticulum membranes.
Most cisternae were tubular but vesicular
membranes were prominent in cortical
cells that had the largest, enormously dilated Golgi apparatus. The most mitochondria were well developed and inner
membranes (cristae) were completely
changed to 600 A vesicles. In some cells
the mitochondria were less differentiated
and contained tubular cristae. One typical
feature in corticosterone f ACTH treated
cultures was apparent in living cells using
phase contrast microscopy after the third
day of treatment. Cortical cells became
spherical and the intercellular space enlarged more rapidly than in ACTH-treated
cultures. The number of single ribosomes
and polysomal clusters had decreased. The
amount of lipid droplets decreased in cortical cells exposed to higher dose of corticosterone simultaneously with ACTH.
The most prominent and significant effect
of corticosterone in dosages of 2 pg/ml/6
days- 5 pg/ml/6 days on the ACTHinduced differentiation of cortical cells in
tissue cultures was the enormously hypertrophied Go19 apparatus.
A dosage of 10 pg/m1/6 days of corticosterone together with ACTH induced the
appearance of an increased number of undifferentiated cortical cells. In addition to
its stimulatory effect on the Golgi apparatus there was an increased inhibitory effect
on the development of mitochondrial
cristae and an increase of single ribosomes
and decrease of polysomes. A dosage of
20 ,g/ml/6 days (6 X
M ) corticosterone induced almost complete inhibition
of ACTH-induced differentiation of cortical
cells (fig. 5). The prominent stimulatory
effect that persists in this higher dose of
corticosterone was the highly hypertrophied Golgi apparatus. Also, the amount of
nuclear heterochromatin was greatly induced and was condenced along the nuclear envelope. The cells remained small
as in control cultures and a scant amount
of cytoplasm surrounded their irregular,
oval nuclei. Corticosterone almost com-
256
ARVI I. KAHRI
adrenal homogenates or slices (Birmingham and Kurlents, '58; Lucius et al., '65;
Langecker and Lurie, '57; Fekete and
Gorog, '63; Black et al., '61; Peron et al.,
'60; Vinson, '66; Bohus, '66; Endroczi and
Lissak, '59). It has also been demonstrated
that corticoids inhibit in vitro amino acid
incorporation into adrenal protein (Morrow et al., '67). A 30% and 85% inhibitory
effect on the incorporation of uridine-C14
into total adrenal RNA was observed at
M and
M
concentrations of 3 X
of corticosterone respectively (Ferguson et
al., '67).
As reported earlier (Kahri et al., '70)
cortical cells in tissue culture of fetal rat
adrenals are able to convert progesterone-C'* into deoxycorticosterone (DOC),
corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC), all of which are
main steroids produced by rat adrenals
(see Birmingham et al., '68; Baniukiewicz
et al., '68). ACTH induces significant elevation in the rate of conversion of progesterone-C14into these steroids without inducing any qualitative differences in steroid
production. After three days of stimulation with ACTH there is almost an 80%
increase in the conversion of progesterone-Ci4 to corticosterone and 18-OH-DOC.
Because of a lack of data on the total corticosterone level in the medium during
DISCUSSION
cultivation in the culture system employed,
the approximate amount of corticosterone
Regulation of adrenocortical
in the culture medium is determined ingrowth and function
directly (Kahri, '71a). Results indicate that
The present study has shown that cor- the corticosterone concentration during
ticosterone, a natural steroid produced by cultivation did not exceed the limit of 0.2
rat adrenal cortex, is a regulator of adre- /*.g/ml. This dosage has been shown to innocortical differentiation at the cellular duce differentiation of skeletal muscle
level. It inhibits the differentiation of the myotubes into striated fibres in cultures of
adrenal cortex. In a dosage of 20 /*.g/m.l/6 skeletal striated muscle of the fetal rat
days (corticosterone concentration in diaphragm. If striated muscle of fetal rat
adrenal venous blood is 2-20 pg/ml) corti- diaphragm was cultivated together with
costerone completely inhibits the ACTH- fetal rat adrenals no differentiation
induced differentiation of cortical cells in occured in myotubes. The level of corticosvitro, corticosterone inhibits the mito- terone in the rat blood is 10-7-10-8 M
chondrial biogenesis and cytoplasmic ribo- (Glick et al., '61).
somal protein synthesis. In contrast there
Locus of corticosterone inhibition in the
is n o morphological evidence that it has
ACTH-induced ultrastructural
an inhibitory effect either on nuclear prodifferentiation of cortical
tein synthesis or on ACTH-induced hypercells in vitro
trophy of Golgi apparatus. It has been reported earlier that adrenal glucocorticoids
As is indicated in this study corticostermay diminish the steroid production in one in the presence of ACTH exerts an
pletely inhibited the development of vesicular (600 A ) cristae and mitochondrial
inner membranes remain tubular or tubulovesicular (figs. 6-7). It also inhibited the
ACTH-induced increase of size and number of mitochondria. One very interesting
phenomenon concerned the location of
mitochondria. There was an increased
tendency of mitochondria to touch the nuclear envelope, however, there was no apparent connection between the nuclear
space and mitochondrial matrix (fig. 7).
No abnormalities in the ultrastructure of
mitochondria were observed. Their matrix
showed moderate density and their shape
varied from spherical to long rods. Few
dense matrix granules were present in the
matrix. A few small lipid droplets were
found in cytoplasm. No increase in the
amount of membrane bounded granules
(lysosomes) was detected. Polysomes had
totally disappeared and numerous single
ribosomes were visible in the cytoplasm
(figs. 6-7). The amount of smooth surf aced endoplasmic reticulum was higher
than in untreated cortical cells and appeared as tubular profiles (figs. 5-7). The
intercellular space become dilated and
desmosome-like thickenings of plasma
membrane seemed to disappear. Microvilli did not develop.
INHIBITION OF MITOCHONDRIAL BIOGENESIS BY CORTICOSTERONE
inhibitory effect on the differentiation of
cortical cells, but has no morphological
effect on undifferentiated cortical cell if
added alone. The inhibitory effect of corticosterone seems to be linked to the presence of stimulated protein synthesis induced by ACTH. Corticosterone inhibits
completely the development of mitochondria, microvilli and to a lesser degree the
smooth surfaced endoplasmic reticulum.
It also inhibits the formation of polysomes
and prevents the growth of cells. However, it has no effect on ACTH induced
hypertrophy of the Golgi apparatus and
the increase in nuclear heterochromatin.
Comparing the corticosterone effect on the
inhibitory effects of several protein synthese inhibitors in earlier reports (table 1)
it is possible to localize the corticosterone
inhibitlory site with moderate accuracy.
The corticosterone effects may be compared with the inhibitory effects of actinomycin D, chloramphenicol, puromycin or
cycloheximide. Actinomycin D induces
complete inhibition of nuclear protein synthesis, reducing the nucleolus and nuclear
chromatin, inhibits the ACTH-induced differentiation of cortical cells and the mitochondria in tissue cultures (Kahri, '67, '68)
there ilre no such effects of corticosterone
on the nuclear protein synthesis.
Chloramphenicol, which is generally
considered an inhibitor of mitochondrial
protein synthesis (Kroon, '69) can selectively inhibit the ACTH-induced mitochondrial differentiation without inhibit-
257
ing other cytoplasmic organelles (Kahri,
'70). Comparing the corticosterone effects
on the locus of chloramphenicol inhibition
it very clearly demonstrates that corticosterone has also inhibitory effects on the
cytoplasmic protein synthesis preventing
the development of polysomes, microvilli
and in lesser degree, smooth surfaced endoplasmic reticulum. It does not support
the mitochondrial locus of inhibitory
effects even though corticosterone like
chloramphenicol inhibits the development
of mitochondria.
A third group of protein synthetic inhibitors studied earlier includes puromycin
and cycloheximide although acting at different loci on ribosomes both prevent protein synthesis at the cytoplasmic level.
Puromycin inhibits the ACTH-induced differentiation of cortical cells and mitochondria without inhibiting nuclear protein
synthesis (Kahri, '68) and the effects of
puromycin at the cytoplasmic level are
comparable with the effects of corticosterone. The inhibitory effect of corticosterone
on the formation of polysomes is much
more complete than with puromycin.
Cycloheximide, a potent inhibitor of
ribosomal protein synthesis in eucaryotic
cells has no effect on the mitochondrial
and bacterial protein synthese (Bennet et
al., '65). In our experiments cycloheximide
inhibits the ACTH-induced differentiation
of cortical cells. It also prevents the ACTHinduced differentiation of mitochondria
which showed the existence of cytoplasmic
TABLE I
Comparison of the efTects of different protein synthese inhibitors and corticosterone on the
ACTH-induced differentiation of cortical cells in tissue culture of fetal rat adrenals
Inhibitor
Actinoinycin D
0.002 p g / m l / 6 days
Puromycin
0.1 p g / m l / 6 days
Chloramphenicol
100 p g / m l / 6 days
Cycloheximide
0.15 p g / m l / 6 days
Corticoeterone
20 p g / m l / 6 days
Nucleus Nucleolus
SER
+++ +++
++
Golgi
Polysomes apparatus
Mitochondria
+++ + + +
+
++
+
+++
-
-
++
-
-
-
-
-
+++
-
S S f
-
-
4-
+++
-
-
+++
+A+
+++
+++
Microvillus
Lipids
++
++
++
-
-
+-I-+
+++ +++
+++ ++
References: Actinomycin D: Kahri, '68; Puromycin: Kahri, '68; Chloramphenicol: Kahri, '70; Cycloheximide: Kahri, '71b.
Complete inhibition.
Moderate inhibition.
$-, Weak inhibition.
--, No inhibition.
+++++-:
258
ARVI I. KAHRI
mitochondrial protein synthesis stimulating factor in cortical cells. This factor is
a mediator of the ACTH stimulatory effect
on the mitochondrial protein synthetic
system (Kahri, '71b). As seen in table 1
corticosterone has a similar inhibitory
effect on ACTH-induced differentiation of
mitochondria. Indicating that it may be the
inhibitor of cytoplasmic mitochondrial protein synthesis stimulating factor.
Although it seems that corticosterone
also has other cycloheximide-likeinhibitory
effects on the cytoplasmic ribosomal protein synthesis and is able, at least partially, to prevent the development of
smooth surfaced endoplasmic reticulum
and microvilli and to decrease the number
of lipid droplets in the cytoplasm. However, it has a completely different effect on
the Golgi apparatus and ribosomes. The effect of corticosterone in totally preventing
the development of polysomes is completely
opposite to the effect of cycloheximide.
How selective the corticosterone inhibitory effect is, has not yet been resolved.
However, it is evident that corticosterone
at concentration of 6 X lo-' M inhibits
completely the cytoplasmic mitochondrial
protein synthesis stimulating factor and
prevents the mediation of ACTH-stimulatory action on the mitochondria. Protein
synthesis still occurs in the cytoplasm,
especially during the hypertrophy of Golgi
apparatus which is not inhibited by corticosterone. Further study is also needed to
resolve the corticosterone effects on the
other cytoplasmic structural and functional proteins.
A model for the regulatory mechanism
of growth and protein synthesis in
adrenocortical cells during ACTHinduced differentiation
Corticosterone is synthesized by several
enzymes, from which cholesterol hydroxylating and side chain cleavage enzymes
and 11p-hydroxylase are locating in the
mitochondria and A5-3p-ol steroid dehydrogenase and 21-hydroxylase are cytoplasmic enzymes. Localization of steroid
hydroxylating enzymes in the mitochondria does not itself indicate genes responsible for the formation of these enzymes
are located on the mitochondrial DNA.
However, there is evidence that 1lp-hy-
droxylase is dependent upon mitochondrial
protein synthese (Kahri and Milner, '69).
A correlation between ACTH-induced mitochondrial structural differentiation and increased capacity for llp- and 18-hydroxylation (Kahri et al., '70) has been observed.
Chloramphenicol inhibits ACTH-induced
mitochondrial differentiation (Kahri, '70)
and simultaneously inhibits the ACTHinduced increased 1lp-hydroxylation capacity (Kahri and Milner, '69). These findings support the idea that regulatory
mechanisms are located within the mitochondria, especially the dependence of corticosterone synthesizing enzyme 1lp-hydroxylase for a mitochondrial protein
synthesis. The localization of a regulatory
mechanism in the mitochondria is further
supported by findings that stimulation or
activation of mitochondrial protein synthesis is not induced directly by ACTH. Instead the stimulus is mediated by a cytoplasmic ribosomal protein synthetic factor
which can be inhibited by cycloheximide
(Kahri, '71b). The latter in turn shows that
this mitochondrial regulatory mechanism is
under nuclear control. This factor, a cytoplasmic mitochondrial protein synthesis
stimulating factor, is responsible for the
mitochondrial differentiation and elevated
1lp-hydroxylase capacity in the cortical
cells (Kahri et al., '70) and is inhibited by
the corticosterone inhibitor. It is also clear
that corticosterone and ACTH are not
antagonists. It appears that they are not
acting at the same locus as inhibitor and
as inductor. There is some weak evidence
that despite inhibition of differentiation
and cytoplasmic ribosomal protein synthesis by cycloheximide (Kahri, '71b) or
corticosterone, hypertrophy of nuclear
heterochromatin still occurs, indicating
activation of nuclear protein synthesis by
ACTH. There is also biochemical evidence
that ACTH has stimulatory effect on nuclear RNAs (Bransome and Chargaff,
'64) and induce an increase in RNA polymerase (Farese and Schnure, '67). This
suggests that ACTH has an inductive
action on nuclear protein synthesis. As is
shown in this study corticosterone inhibitor action seems to be located at the level
of cytoplasmic ribosomes. Corticosterone
inhibitor activity in cortical cells may reuresent a highly specific regulatory mech-
INHIBITION OF MITOCHONDRIAL BIOGENESIS BY CORTICOSTERONE
anism that does not function in other cells.
The regulation of adrenocortical cell differentiation and growth may occur in the
following manner as described in the schematic representation in figures 8 and 9.
Corticosterone acts as an intracellular
inhibitor in cortical cells, inhibiting cytoplasmic protein synthesis at the ribosomal
level, thereby inhibiting synthesis of cytoplasmic mitochondrial protein synthesis
stimulating factor and preventing the differentiation of mitochondria. In this way
it prevents the synthesis of inhibitorcorticosterone itself. The purpose of this action of corticosterone is to prevent the differentiation of all cortical cells at the same
time. This mechanism supports the survival of the organism and controls the corticosterone level in the blood. Finally this
increase of the level of corticosterone in
venous blood of adrenal cortex is leading
to the zone reticulum formation in vivo.
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ARVI I. KAHRI
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Cyclic AMP as intracellular mediator of trophic
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PLATE 1
EXPLANATION OF FIGURES
1 Cortical cells in monolayer colony in tissue culture of fetal rat
adrenals, treated with 20 pg/ml of corticosterone for six days from
sixteenth day of cultivation up to and including twenty-first day.
Cortical cells are typical relatively undifferentiated zona glomerulosa
cells. Mitochondria have lamellar cristae. There are no visible changes
in the ultrastructure of cortical cells after corticosterone treatment
X 11,500.
2
Mitochondria in cortical cell in tissue culture of fetal rat adrenals
treated with 20 Fg/ml of corticosterone as described in figure 1.
Typical zona glomerulosa-zona intermedia mitochondria which have
tubular or lamellar cristae and a few 600 A vesicles in the matrix.
There is n o transformation in the ultrastructure in corticosterone
alone treated cortical cells. All ribosomes appear as single free ribosomes. x: 35,000.
INHIBITION OF MITOCHONDRIAL BIOGENESIS BY CORTICOSTERONE
A M I. Kahri
PLATE 1
261
PLATE 2
EXPLANATION OF FIGURE
3
262
Cortical cells in monolayer colony in tissue culture of fetal rat
adrenals, treated with 0.2 p g / m l of corticosterone together with 100
mU/ml of ACTH for six days from sixteenth day of cultivation up to
and including twenty-first day. ACTH has induced transformation of
zona glomerulosa type of cortical cells into zona fasciculata cells.
0.2 pg/rnl of corticosterone has no inhibitory effect on the differentiation of cortical cells. ACTH has induced marked increase in the
amount of smooth surfaced endoplasmic reticulum and transformation of the mitochondria into zona fasciculata which has vesicular
cristae (600 A vesicles) in the matrix. Ribosomes appear mostly as
free polysomes. x 22,000.
INHIBITION OF MITOCHONDRIAL BIOGENESIS BY CORTICOSTERONE
Arvi I. Kahri
PLATE 2
PLATE 3
EXPLANATION OF FIGURE
4
264
Extremely hypertrophied Golgi apparatus in cortical cell in tissue
culture of fetal rat adrenals cultivated 22 days and treated with
10 pg/ml/6 days of corticosterone together with 100 mU/ml/6 days
of ACTH. x 28,500.
INHIBITION OF MITOCHONDRIAL BIOGENESIS BY CORTICOSTERONE
Arvi 1. Kahri
PLATE 3
265
PLATE
4
EXPLANATION OF FIGURE
5
Cortical cells in tissue culture of fetal rat adrenals cultivated 22 days
and treaied with 20 pg/m1/6 days of corticosterone together with
100 mU/ml/6 days of ACTH. Corticosterone has completely inhibited
the differentiation of mitochondria and they are typical zona glomerulosa type with tubular or lamellar inner membranes. ACTH has still
induced in lesser degree development of smooth surf aced endoplasmic
reticulum membranes as tubular profiles. Only Golgi apparatus js
still highly hypertrophied. There is no development of microvilli and
also only a few lipid droplets in the cytoplasm. The nuclei are oval
and nuclear chromatin is highly hypertrophied and nucleoli very
prominent. x 10,500.
INHIBITION OF MITOCHONDRUL BIOGENESIS BY CORTICOSTERONE
Arvi I. Kdiri
PLATE 4
PLATE 5
EXPLANATION OF FIGURES
6
Mitochondria in cortical cell in tissue culture of fetal rat adrenals
cultivated 22 days and treated with 20 pg/m1/6 days of corticosterone
together with 100 mU/ml/6 days of ACTH. Note the typical internal
structure of the zona glomerulosa type mitochondria and the close
contacts of mitochondria with the nuclear envelope. x 40,000.
7 Mitochondria in cortical cell in tissue culture of fetal rat adrenals
cultivated 22 days and treated similarly as described in figure 6.
Corticosterone has inhibited completely the ACTH-induced transformation of mitochondria and they remain typical zona glomerulosa mitochondria. Note the appearance of ribosomes as single particles and
total lack in polysomes. There is also slight ACTH-induced increase
in the amount of smooth surface endoplasmic reticulum as tubular
profiles. x 35,000.
268
INHIBITION OF MITOCHONDRIAL BIOGENESIS BY CORTICOSTERONE
Arvi I. Kahri
PLATE 5
269
PLATE 6
EXPLANATION OF FIGURES
270
8
Schematic representation of the hypothetical role of mitochondrial
protein synthesis in the regulation of differentiation and steroid
secretion of cortical cells of rat adrenals and the role of corticosterone
as an inhibitor of mitochondrial differentiation. cmpssf, cytoplasmic
mitochondrial protein synthesis stimulating factor: 1. Locus of inhibitory effect of actinomycin D, 2. cycloheximide, and 3. chloramphenicol. CAMP (Kahri et al., '72).
9
Schematic representation of the complete inhibition of ACTH-induced
mitochondrial differentiation with corticosterone in concentration of
M in tissue cultures of f e t d rat adrends.
6X
INHIBITION OF MITOCHONDRIAL BIOGENESIS BY CORTICOSTERONE
PLATE 6
Arvi I. Kahri
NUCLEAR
STRUCTURAL GENES
--.------eJ.c
w
J.
corticosterone
r
PROTEIN
I
MITOCHONDRIAL
GENE(S)
mRNA
PROTEIN
IN V I T R O
-
6x ~ o - ~ M
of corticosterone
NUCLEAR
STRUCTURAL GENES
l*
rn
Golgi
X l t e r o n e
- inhibitor
mRNA
’
MI TOCHONDRlAL
GENE(S)
IN V I T W O
6 x lO-’M
of c o r t i c o s t e r o n e
27 1
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induced, inhibition, corticosterone, rat, tissue, acth, cells, differentiation, cortical, culture, adrenal, mitochondria, fetal
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