This dissertation has been
microfilmed exactly as received 69-10,601
JAMES, Gordon Price, 1936-STEROID HORMONES AND SUBCELLULARPROCESSES.
University of Hawaii, Ph.D., 1968Biochemistry
University Microfilms. Inc.• Ann Arbor. Michigan
STEROID HORMONES
AND
SUBCELLULAR PROCESSES
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE
UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN BIOCHEMISTRY
AUGUST 1968
BY
Gordon Price James
Dissertation Committee:
Howard F. Mower, ChairmanSidney J. TownsleyTheodore WinnickKerry YasonobuJohn B. Hall
iii
ABSTRACT
Aldosterone administered intraperitoneally induced an increase
in the rate of renal RNA synthesis in the rat. A maximum response
of 130 percent of control occurred 1.5 hours after injection.
Following the maximum at 1.5 hours, the rate of renal RNA synthesis
oscillated about control. Three cycles in the rate of kidney RNA
synthesis occurred within 4.5 hours after injection with no indication
of a decrease in amplitude.
Renal RNA synthesis is stimulated to a maximum of 210 percent
of control 30 minutes after an intravenous aldosterone injection.
Following the maximum at 30 minutes, the rate of kidney RNA synthesis
oscillated about control but with longer periods and greater amplitude
than when the hr,wone was given intraperitoneally. Aldosterone induced
oscillations in renal RNA synthesis occurred in normal, adrenalec-
tomized and hypophysectomized rats. Aldosterone in doses of 0.07 pg
to 2.5 pg was effective in inducing the oscillations.
Intravenous administration of cortisol or aldosterone diminished
the rate of splenic RNA synthesis in rats. Inhibition to 70 percent of
control occurred within four hours after hormone injection. Following
the initial inhibition, a rapid increase occurred to approximately
160 percent of control. The maximum occurred at five and six hours
for cortisol and aldosterone, respectively. A rapid decrease below
control level followed the stimulation. The aldosterone induced
oscillations in splenic RNA synthesis was observed in normal,
adrenalectomized and hypophysectomized rats.
The hormone induced oscillations in renal and splenic RNA
synthesis appear to be unrelated. The possibility is suggested that
the oscillations are unique functions of the respective tissues and
that they are independent of external control.
iv
v
TABLE OF CONTENTS
AB~AAcr ...
LIST OF TABLES
LIST OF ILLUSTAATIONS
I. INTRODUCTION ...
II. MATERIALS AND METHODS
. v
vii
viii
1
9
A. Materials obtained commercially. . 9B. Materials obtained by preparation . . . . . . . 9C. Methods: Animals, hormones, injections, doses 10
1. Methods: RNA polymerase. . . . 11a. Nuclear isolation . . . . . . . . 12b. RNA polymerase assay. . . . . . . 12c. Measurement of radioactivity. . . 14d. Spironolactone injections. . . . . 14
2. Methods: cell-free protein synthesis . . . . 15a. Isolation of components of cell-free system 15b. Cell-free incubations . . . . . . . . . . . 15c. Protein and RNA determinations. . . . . . . . 17d. Sucrose density gradient analysis of polysomes 18
III. RESULTS AND DISCUSSION. . . .. ... 19
A. Effect of hormone, added in vitro, on RNA synthesis ..... 19B. Effect of aldosterone, given in vivo, on renal RNA synthesis. 24
1. Effect of 2.5 pg aldosterone, administered ..... 24intraperitoneally, on the rate of kidney RNA synthesis
2. The effect of 2.5 pg aldosterone, administered. . .. 32intravenously, on kidney RNA synthesis
3. Effect of aldosterone, 2.5 pg injected intravenously .. 37on the rate of kidney RNA synthesis of adrenalectomizedrats.
4. Effect of 0.07 ~g aldosterone (injected intravenously) . 40on kidney RNA synthesis of adrenalectomized rats.
5. Effect of aldosterone on kidney RNA synthesis in.. 43hypophysectomized rats
6. Effect of aldosterone plus spironolactone on the. . 43rate of renal RNA synthesis
C. The effect of aldosterone on the rate of RNA synthesis inrat brain . . . . .. . 49
vi
TABLE OF CONTENTS (Continued)
D. Steroid hormones and spleen RNA synthesis . . . . . . 521. Effect of aldosterone on spleen RNA synthesis. . 522. Effect of cortisol on spleen RNA synthesis . . .... 563. Steroid structural requirements for lymphatic. . 61
activity4. Oscillations in spleen RNA synthesis resulting from. 73
injection of steroid hormonesE. Effect of aldosterone on protein synthesis in the spleen. 76
1. Effect of aldosterone on rat spleen ribosomal. . 79distributions in sucrose density centrifugationpatterns
F. Effect of spironolactone on spleen RNA synthesis 82
VI. LITERATURE CITED
IV. SUMMARY AND CONCLUSIONS
v. APPENDIX84
90
91
Table
l.
2.
3.
4.
5.
6.
7.
8.
9.
10.
LIST OF TABLES
Contents
Honnones, dosage and vehicle .
Assay for incorporation of labeled nucleo-tide into nuclear RNA... . .....
Isolation of ribosomal and pH 5 enzymecomponents from rat kidney, brain andsp1een. . . . . . . . . . . . . . . . .
Assay for incorporation of labeled aminoacid into protein. . .
Effect of aldosterone on RNA synthesiswhen added in vitro to isolated ratki dney nuclei. .
Effect of steroid hormone on RNA synthesiswhen added in vitro to isolated rat livernuclei ...-. . . . . . . . . .. .
Schedule for injection of sesame oil,aldactone, saline or aldosterone...
Effect of aldosterone and/or aldactonegiven alone or in combination on the rateof renal RNA synthesis .
The effect of aldosterone on cell-free proteinsynthesis in rat spleen. . ...
The effect of spironolactone on rat spleenRNA synthes is. . . . . . . .. ....
Page
11
13
16
17
21
22
47
48
77
82
vii
vi i i
LIST OF ILLUSTRATIONS
111 ustrati on . Contents Page
Fig. 1
Fig. 2.
Fi g. 3.
Fi g. 4.•
Fi g. 5.
Fi g. 6.
Fi g. 7.
Fi g. 8.
Effect of 2.5 ~g aldosterone, injected . . . . . . 26intraperitoneall{, on the rate of incor-poration of ATP- 4C into nuclear RNA byisolated rat kidney nuclei.
Extended time course of response of RNA ..... 30synthesis in rat kidney nuclei followingan intraperitoneal injection of 2.5 ~galdosterone.
The effect of aldosterone (2.5 ~g admin- .... 34istered intravenously) on the rate of RNAsynthesis in kidney nuclei from normalunoperated rats.
Effect of 2.5)1g aldosterone (injected ..... 39intravenously) on the rate of renal RNAsynthesis in adrenalectomized rats.
Effect of 0.07 )1g aldosterone (injected ..... 42intravenously) on the rate of renal RNAsynthesis in adrenalectomized rats.
Effect of O.l)1g aldosterone (administered .... 45intravenously) on the rate of renal RNAsynthesis in hypophysectomized rats.
The effect of aldosterone (2.5}.lg admin- ..... 51istered intravenously to normal unoperatedrats) on the rate of RNA synthesis inthe brain.
The effect of aldosterone (2.5 JAg admin- ..... 54istered intravenously) on the rate ofincorporation of ATP_14C into spleenRNA in vitro.
ix
LIST OF ILLUSTRATIONS (Continued)
Illustration
Fi g. 9.
Contents
The effect of cortisol (122 pg injected ..intravenously) on the rate of incorporationof ATp_ 14Cinto RNA by isolated rat spleennucl ei in vitro.
Page
. 59
Fi g. 10.
Fi g. 11.
Fi g. 12.
Fi g. 13.
Fi g. 14
Fi g. 15.
Fi g. 16.
The effect of deoxycorticosterone-acetate 64(125 ug injected intravenously) on the rateof incorporation of ATP_14C into RNA byrat spleen nuclei ~ vitro.
The effect of l-dehydrocortisone (152 pg 67injected intravenously) on the rate ofincorporation of ATP_14C into RNA by ratspleen nuclei ~ vitro.
The effect of progesterone (113 pg injected ..... 70intravenously) on the rate of incorporationof ATP_1 4C into RNA by rat spleen nucleiin vitro.
The effect of testosterone (132 pg injected ..... 72intravenously) on the rate of incorporationat ATP_ 14C into RNA by rat spleen nucleiin vitro.
The effect of aldosterone (2.5 pg injected ..... 75intravenously) on the rate of incorporationof ATP_14C into RNA by rat spleen nucleiin vitro. Extended time course is shownfor normal, hypophysectomized and adrenalec-tomized rats.
The effect of aldosterone (2.5 ~g injected ..... 81intravenously) on rat spleen ribosomaldistributions in sucrose densitycentrifugation patterns.
Comparison of effect of 2.5 ~g aldosterone ..... 87on RNA synthesis in kidney and spleen ofnormal rats.
I. INTRODUCTION
Hormones are chemical substances secreted by various tissues
and transported in the blood stream to the organs on which their
effect is produced. Some hormones elicit specific effects from
specific target cells, other hormones are more general in both action
and target. A question that has received considerable attention for
many years is how does the target tissue read the message carried
by the hormone. Several schools of thought have developed concerning
this problem.
One hypothesis, which has never been substantiated, suggests
that hormones act by activating enzymes (58). Hormone action then
would be analogous to that of the vitamins (58). According to this
theory hormones act as completing factors. By interacting with the
corresponding apoprotein, they would produce enzymes of specific
action (59).
A second theory holds that hormones function as allosteric
effectors. Bound to enzymes, not at the active site, they change
the catalytic properties (135). This theory has few proponents
(135, 146).
The theory with the most supporters and experimental foundation
maintains that hormones act by regulating the expression of certain
genes (146). Some hormones function independent of gene activity
(50, 110, 151) but a wide variety of hormones have been shown to
effect gene expression. The link between hormone and gena was first
2
demonstrated using ecdysone, a steroid hormone which functions in the
molting of insects. After injection of ecdysone the following events
are reported to occur: 15-30 minutes, puffing of certain regions
of the chromosome; 1-5 hours, synthesis of RNA; 7-10 hours, protein
synthesis; 20-24 hours, molting(66).
The enzyme dopa decarboxylase functions in the molting process
and an increased rate of synthesis of this enzyme results from
ecdysone injection. Every step in the process from chromosome
pUffing to molting has been experimentally demonstrated (66, 67).
A wide variety of mammalian hormones have also been shown to
function via gene regulation. Representatives from every class of
hormone, including protein, amino acid derivatives and steroids,
have been shown to reproducibly alter DNA melting profiles (52).
This was interpreted as a destabilization of interstrand linkages
in specific areas of the DNA macromolecule. It is believed that
separation of strands resulting from destabilization would make them
available for transcription.
Differing in approach and interpretation was the work of Sekeris
and Lang (121). They report that tritiated cortisone accumulates
in rat liver nuclei when administered in vivo. Fractionation of the---isolated nuclei showed DNA and RNA contained very little radioactivity,
but the histone fraction contained a considerable amount of activity.
Tritiated testosterone was bound to liver histone to a much lower
extent suggesting steroid-target cell specificity.
Histones have been found only in the nucleus associated with
3
DNA. They are believed to function as DNA insulators or gene
regulators (2, 11). The binding of hormone to histone is thought to
modify the histone so that it no longer serves as an insulator. The
exposed part of the DNA would then function as a primer for DNA-
directed RNA polymerase (67).
This hypothosis would predict an increase in RNA synthesis
following hormone administration. Such an increase has been demon-
strated in several laboratories studying a variety of hormones and
tissues: cortisol-liver (71), cortisone-liver (36), testosterone-
seminal vesicles (144), growth hormone-liver (76), ACTH-adrenal (12),
thyroxine-liver (145), and estrogen-uterus (53).
This stimulation in RNA synthesis appears to be reflected in
all RNA fractions (71, 120). Of particular interest are reports of
increased mRNA synthesis (79, 80). Segal et~. (119) observed a
clear estrogenic response from ovarieatomized rats receiving an
intrauterine injection of mRNA isolated from the uterus of estrogen
injected castrated rats. This is consistent with the gene activation
theory in which mRNA is a mediator of the hormone message.
According to the hormone-gene activation hypothesis a stimulation
of protein synthesis would follow the hormone-induced increase in RNA
synthesis. Increased protein synthesis is an early response to
estradiol (101). Within thirty minutes following an intraperitoneal
injection of estrogen there is an increase in the synthesis of a single
uterine protein (102). This occurs prior to a general stimulation in
protein synthesis in the rat uterus. Estrogen has also been shown to
4
increase RNA and protein synthesis in chick oviduct (103).
A single injection of cortisol has beer. shown to stimulate protein
synthesis in rat liver following an increase in RNA synthesis (106).
Tata(131) reports an increase in the rate of in vivo incorporation of
labeled amino acids following a single administration of growth
hormone, thyroid hormone or testosterone.
Hormonal induction of enzyme synthesis has been reported from
many laboratories. Gibberellic acid increases the de novo synthesis
of a-amylase in barley endosperm (139). Glucocorticoids have been
shown to enhance many enzyme activities in the liver including
glycogen synthetase (128), glucose-6-phospatase (141), fructose -1,
6-diphosphatase (96, 142), phosphohexoisomerase (142), aldolase (142),
phosphoenolpyruvate carboxykinase (124), alanine transaminase (115),
and the arginine synthetase system (92). An increased rate of
synthesis of dopa decarboxylase due to ecdysone was mentioned earlier.
Not all workers agree that hormone influence on protein synthesis
occurs via the gene. The differing opinions may result from different
experimental design or from studies on different hormones. For
example, muscle protein synthesis was stimulated by insulin but there
was no prior increase in RNA synthesis (151). This work and other
reports (108) suggest that hormones may function at sites other than
the gene. Insulin (95) and antidiuretic hormone (111) are believed to
influence cell metabolism by regulating cell permeability. Changes
in cell permeability could alter anabolic rates completely independent
of any gene activity. It is quite conceivable that a variety of
mechanisms mediate the commands of the vari~s hormones (132).
5
The hormone of prime interest in this dissertation is aldosterone.
This adrenal corticoid has been shown to influence kidney, salivary
and sweat glands, striated muscle, bone, the gastrointestinal tract,
and blood pressure (98). Its primary function is the fine regulation
of sodium and potassium excretion. When relatively large doses are
given it also has some glucocorticoid activity but this is probably
of little physiological significance.
There is routinely a time lag from thirty minutes to two hours
before aldosterone shows an effect on sodium reabsorption in the
kidney and the effect can last as long as eight hours (6, 48, 116, 126,
134). Aldosterone has been shown to have its effect on sodium
reabsorption in the distal portion of the nephron (137, 138).
In 1961 Crabbe (20) demonstrated an aldosterone effect on sodium
transport using isolated toad bladder. Using the isolated toad bladder
Edelman et El. (31, 32), Porter et El. (109) and Fanestel et El. (34)
were able to demonstrate an increase in RNA and protein synthesis
following the accumulation of tritiated aldosterone in the nuclei.
Edelman et El. (31) reported that the effect of aldosterone on sodium
transport was considerably reduced when the toad bladder was pretreated
with actinomycin 0 or puromycin. This was interpreted to mean that
RNA and protein synthesis were necessary events preceding an aldosterone
effect on sodium transport. Following a latent period of 60 to 90
minutes, sodium transport was stimulated for as long as six hours.
Sharp and Leaf (122) using the isolated toad bladder, reported an
.a1dosterone effect on sodium transport. AftEr a lag of approximately
6
45 minutes sodium transport increased to a peak activity at two to
three hours. The aldosterone antagonists, SC 9420, SC 14266 and
progesterone were able to block the effect of aldosterone.
All these results made it appear that aldosterone functioned
via the gene activation hypothosis. The mechanism of action of
aldosterone as understood in 1966 is reviewed by Sharp and Leaf (123).
The adrenal corticoids, being very similar in structure, show
some overlap in activity. For example, cortisol, corticosterone,
deoxycorticosterone and aldosterone all have the ability to influence
Na/K excretion ratio (117). Liver glycogen deposition is influenced by
cortisol, cortisone, corticosterone and aldosterone (117). Abundant
evidence obtained from both clinical and laboratory experience has
established the role of a variety of steroid hormones in infection
(68, 69, 133).
Thymic involution is also a well known response to steroid
hormones. Adrenal corticoid induced involution of lymphoid tissue was
first described approximately twenty four years ago (27). Subsequent
studies have confirmed this early report. Santisteban and Dougherty
(118) observed lymphatic involution resulting from several adrenal
steroids. Thymic involution in the rat follows the administration
of cortisone, estrogen, testosterone, ACTH and thyroid extract (140).
Angervall and Lundin (4) injected cortisone twice daily into pregnant
rats. Injections continued throughout the pregnancy. Immediately
following parturition mothers and young were killed and thymus and
spleen weighed. Atrophy in both tissues were observed in mothers and
7
and young. Mendelson and Finland (93) report the average weight of. . .
spleen from cortisol treated mice was approximately half that of
control animals. All lymphoid tissues increase in weight following
adrenalectomy (73).
Corticosteroid induced lymphatic involution can be brought about
in three known ways. Steroids cause lymphocytokaryorrhexis, they
inhibit DNA synthesis and they prevent mitosis by destroying the
metaphase stage.
Significant decreases were observed in the incorporation of
tritiated thymidine into DNA of thymus and spleen of rats following
cortisone treatment (73). Prolonged treatment of mice with cortisol
and ACTH decreased the incorporation of labeled nucleotides into
nucleic acids of lymph nodes, spleen and thymus (13). Cortisol
exerts a suppressive effect on DNA synthesis and mitosis in lymphatic
organs for as long as six to eight hours following a single injection.
This is of particular interest because the steroid has been largely
metabolized and removed from the tissue shortly after injection (28).
Stevens et~. (129) reported a decrease in DNA synthesis in rat spleen,
thymus and lymph nodes following cortisol injection.
RNA synthesis in lymphatic tissue is also sensitive to steroid
hormones. Brinck-Johnson and Dougherty (13) observed a decrease in
both DNA and RNA synthesis in spleen, lymph nodes and thymus of mice
receiving cortisol or ACTH. Using isolated rabbit lymph node cells
Kidson (72) was able to detect very rapid alterations in RNA synthesis
following cortisol administration. RNA and DNA synthesis are inhibited
8
37 and 35 percent, respectively, in thymocytes isolated from rats one
hour after cortisol injection (89).
Although aldosterone has been studied extensively as a mineralo-
corticoid, its action on the l~phatic system has received verY-little
attention (51). Mach and co-workers obseeved evidence of anti-
inflammatory action in an Addisonian patient treated-with aldosterone
(88). However, Desaulles (25) reports that in laboratory studies
aldosterone, unlike cortisone, when applied locally, would not inhibit
formation of granuloma tissue around a subcutaneous cotton pellet.
When the work reported in this dissertation was started it
remained to be demonstrated that the mechanism of action of aldosterone
in mammalian kidney was analogous to the mechanism in the toad bladder.
Studies on the effect of aldosterone on rat kidney RNA synthesis
were initiated based on extrapolations from the toad bladder work.
During the course of studies on aldosterone in the kidney it was
convenient and of interest to study the hormone's effect on the rate
of RNA synthesis in lymphatic tissue. The spleen was chosen for these
studies.
9
II. MATERIALS AND METHODS
A. Materials obtained commercially
Aldosterone, cortisol, progesterone, estradiol, UTP, GTP, CTP,
ATP, phosphoenolpyruvate, pyruvate kinase, dithiothreito1, soluble
RNA, DNA and 2-mercaptoethanol were obtained from California Corporation
for Biochemical Research. ATP_14C and L-phenylalanine- 3H were purchased
from Nuclear-Chicago and New England Nuclear Corp. respectively.
Deoxycorticosterone acetate, corticosterone, testosterone and l-dehydro-
cortisone from Mann Research Laboratories. Packard Instrument Co.
supplied 2,5-dipheny1oxazole (PPO) and 1,4-bis-2-(4-methyl-5-phenyloxa-
zoly1)-benzene (dimethyl POPOP). Mi11ipore filters (HAWP 025) were
purchased from Mil1ipore Corporation. Sigma Chemical Co. supplied
deoxycholate (sodium salt) and L-amino acid kits. Tetramethyl
ammonium hydroxide, trichloroacetic acid and diphenylamine were purchased
from Eastman Organic and bentonite U.S.P., from Robinson Laboratory,
Inc. (San Franciso). Crystalline pancreatic RNAse was obtained from
Worthington Biochemical Corp. Spironolactone (aldactone) was obtained
from Searle Chemicals, Inc. All other chemicals were reagent grade.
B. Materials obtained by preparation
1. Bentonite was prepared as described by Petermann (107).
2. Amino acid mixture for cell-free protein synthesis contained
10 pmoles/m1 of each of the 20 common L-amino acids minus
phenylalanine.
10
3. Solutions:
a. Solution A: 0.32M sucrose, 3mM MgC12, O.lmM dithiothreitol.
b. Solution B: 2.4M sucrose, lmM MgC12, O.lmM dithiothreitol.
c. Solution C: 0.25 m sucrose, lmM MgC1 2, O.lmM dithio-
threitol.
d. Solution D: 5 gil PPO, 0.3 gil dimethyl POPOP in
toluene (50).
e. Solution E: 0.02M Tris buffer (pH 7.6), O.lM KC1,
0.04M NaCl, O.OlM M9Ac2, 0.006M mercaptoethanol.
f. Solution F: Solution E plus sucrose to 0.25M.
g. Solution G: Solution F plus 4 mg bentonite per ml.
h. Solution H: Solution E minus mercaptoethanol.
j. Solution J:60 gil naphthaline, 4 gil PPO, 0.2 gil
POPOP, 100 ml/l methanol, 20 ml/l ethylene glycol, in
dioxane (56).
C. Methods: Animals, hormones, injections and doses
Male rats ranging in weight from 120 to 170 grams were used in
all experiments. Food was taken from the animals the night before the
experiment but they were allowed free access to drinking water. Rats
were of the Wistar strain (obtained from a University of Hawaii colony),
or of the Sprague-Dawley strain (obtained from Berkeley Pacific
Laboratories). Adrenalectomies and hypophysectomies were performed
at Berkeley Pacific Laboratories.
All animals obtained from Berkeley Pacific were kept in our
laboratories from five to seven days prior to use. Adrenalectomized
11
rats were given free access to tap water, 1% saline and 10% glucose.
The hormones, dosage and vehicle of each of the steroids used in
this work are shown in Table 1.
Table 1. HORMONES, DOSAGE AND VEHICLES
HORMONE DOSE/ANIMAL VEHICLE
Aldosterone 0.07 or 2.5 }Jg 1. 0% saline-0.5% ethanolCortisol 122 }Jg 1.0% saline-5.0% ethanolProgesterone 113 ).Ig II II
Estradi 01 117 l1g II II
Deoxycorti co-sterone acetate l25}Jg II II
Testosterone l32}lg II II
l-Dehydrocortisone 152 }Jg II II
The hormone was dissolved or suspended in 0.5 ml of the vehicle and
injected intraperitoneal or intravenous as indicated under results and
discussion. Intravenous injections were via the tail vein. Aldosterone,
hydrocortisone, l-dehydrocortisone and corticosterone were dissolved
in the vehicle. Progesterone, estradiol, deoxycorticosterone acetate
and testosterone were injected as suspensions.
1. Methods: RNA polymerase
In each experiment one group of rats received the hormone and a
control group received only the vehicle. Three to five rats comprised
each group. At the specified times following injection the animals
were killed by neck fracture, tissues excised and placed immediately
in ice cold Solution A. Tissues from the hormone treated animals were
12
pooled and nuclei isolated by the method of Widnell and Tata (147).
Tissues from control animals were treated in the same manner.
a. Nuclear isolation
All procedures in the isolation of neclei were carried out at
0-4oC. The pooled tissues were weighed, minced with scissors and
rinsed once with Solution A. A 25% homogenate (1 part tissue, 3 parts
Solution A) was prepared in a Potter-Elvehjem homogenizer. Homogen-
ization was complete after 12 slow up and down movements with a
mechanically driven pestle turning at a moderate speed. The homogenate
was filtered through a double layer of cheese cloth to remove connective
tissue and clumps of unbroken cells. Samples of homogenate, 12.5 ml,
were diluted to 20 ml with Solution A, and then to a final sucrose con-
centration of 0.25M with water. Solution A, 15 ml, was layered under-
neath, and a crude nuclear pellet isolated by centrifugation at 700 g
for 10 minutes in a refrigerated Servall RC-2 centrifuge. The pellet
was resuspended in 12 ml of Solution B. A nuclear pellet was isolated
by centrifuging the suspension for one hour at 50,000 g in the number 50
rotor of the Spinco model L preparative ultracentrifuge. Whole cells,
erythrocytes, mitochondria and other cell particles formed a firm plug
at the top of the tube and were easily removed with a spatula. The
nuclear pellet at the bottom of the tube was resuspended in 1.0 to 2.0 ml
of Solution C.
b. RNA polymerase assay
RNA polymerase activity of isolated nuclei was determined by a
slight modification of the procedure described by Widnell and Tata(53, 1471
Table 2. Assay for incorporation of labeled nucleotide
into nuclear RNA (53, 147)
13
Component
Tris buffer (pH 8.5)MgClDi thi othrei to1NaFGTPCTPUTPATP_14CPEPPyruvate ki naseNuclear suspension (0.1 ml)
Quantity/O.S mlfinal volume
50 j,lmoles2.5 j.lmoles0.44 j,lmoles3 Jimoles0.3 lJITIoles0.3 }lmoles0.3 .umoles0.01 j,lmoles5.0 j,lmoles10.0 pmoles0.2-1. 0 mg DNA*
* The rate of incorporation of ATP_14C into RNA is higherin isolated brain nuclei than it is in isolated kidneyor spleen nuclei. In a typical experiment isolated brainnuclei incorporated 2550 CPM/mg DNA, kidney incorporated250 CPM/mg DNA and spleen incorporated 360 CPM/mg DNA.Because of this difference in incorporation rates, brainnuclei was used at the lower value shown while kidney andspleen were used at approximately the higher value shown.
14
Nuclei were incubated in the assay system shown in Table 2.
After incubation in a Dubnoff Metabolic shaking incubator for 15
minutes at 37oC, the reaction was terminated by the addition of 4.0 ml
of ice cold 0.5N perchloric acid. The precipitate, collected by centri-
fugation in a clinical centrifuge, was washed once with 0.2N perchloric
acid and once with a mixture of ethanol:ether (3:1 v/v), all operations
were carried out at 0-4oC. RNA was extracted from the precipitate by
two successive extractions in 4.0 ml of 10% NaCl containing 0.,25 mg
carrier RNA. Extractions were carried out at 1000C for 30 minutes.
RNA was precipitated from the combined extracts by the addition of
5.0 ml of ice cold 20% trichloroacetic acid. The precipitated RNA
was collected on a millipore filter (HAWP 025) and washed with 2.0 ml
of ice cold 5% trichloroacetic acid.
c. Measurement of radioactivity
Filters containing ATP_14C labeled RNA were air dried for 60
minutes and placed in counting vials. Ten millileters of Solution D
was added and vials were counted in a Packard Tri-Carb liquid-scintilla-
tion spectrometer (model 3003). RNA polymerase activity was determined
in quadruplicate. DNA was determined in triplicate on 0.1 ml aliquots
of the nuclear preparation by the method of Dische(26). Calf thymus
DNA was used as a standard.
RNA polymerase activity was calculated as counts/minute/mg DNA.
RNA polymerase activity in the control group was defined as 100% and
the hormone treated group was calculated as percent of control.
d. Spironolactone injections
A dose of 0.1 mg of spironolactone (SC-9420), dissolved in 0.1 ml
15
of ses arne oil was injected subcutaneously. Aldosterone (0. lpg in 0.5
ml saline) was administered intravenously fifteen minutes later. Ani-
mals serving as hormone controls were injected intravenously with 0.5 ml
saline. Four groups of rats were injected using the following combina-
tions: oil-saline, lactone-saline, oil-aldosterone, and lactone-
aldosterone. Each group contained three animals. Thirty minutes after
aldosterone, or aldosterone vehicle, injection animals were killed,
nuclei from kidney isolated and nuclear RNA polymerase assayed in vitro.
2. Methods: Cell-free protein synthesis
In each experiment one group of rats received aldosterone and a
control group received the vehicle. Each group contained ten to
twelve rats. At the specified times following injection the animals
were killed by neck fracture, tissue excised and immediately placed in
ice cold Solution E. Tissue from hormone treated and control animals
were pooled separately.
a. Isolation of components of cell-free system
After mincing tissue with scissors and homogenizing in a Potter-
Elvehjem hom.ogenizer, subcellular components were isolated by the method
of Adiga et~. (1) as described in Table 3. Nine to ten hours were
required from the time the animals were killed until cell-free com-
ponents were isolated. Protein synthesis was determined immediately
after isolation of cell-free system, polysome profiles were obtained
on preparations stored frozen over night.
b. Cell-free incubations
The components of the cell-free incubation mixture are shown in
Table 4. All the components of the incubation mixture, except ribosomes
16
Table 3. Isolation of ribosomal and pH 5 enzyme components
from rat kidney, brain or spleen.
tPpt: discard2 hr
J105,000 g microsomal pellet:suspended in 1 ml of 105,000 9supt plus 4.5 ml of Solution G:added 10% DOC* to 1% final conc.,layered onto 5.5 ml of Solution Econtaining 1M sucrose: centrifuged4 hr at 105,000g
Supt: centrifuge 30min. a1 30,000 9
Supt: centrlfugeat 105,000 9 .
I
Fresh minced tissue from 10 to 12 rats homo-genized in Solution G (1:1 w/v), centrifuged
. 10 min. at 10,000.9
t tPpt: discard
fSupt: adjusted topH 5.2 with 1M HAc,centrifuge 10 minat 10'rOO 9
Supt: discard Ppt; pH 5 enzymedissolve in Solution Eand adjust to pH 7.6 withcpnc. tris to give a finalprotein conc. of 10 mg/ml
Ribosomal pellet: rinsed Supt: discardwith 5 ml of Solution G,suspended in 4 ml of SolutionE to a concentration ofapproximately 4.5 mg rRNA/ml
*DOC - Deoxycholic acid
17
andpH 5 enzymes, were added in a total volume of 0.1 mle - After adding
0.1 ml of ribosomes and 0.1 ml of pH 5 enzymes, the incubation mixture
was diluted to 0.5 ml by the addition of 0.2 ml of Solution E.
labeled amino acid was incorporated into protein during a one-hour
incubation at 370 C. The incubation was stopped by the addition of 5 ml
of solution containing 5% TCA and cold phenylalanine. The TCA precipi~
tate was washed successively with hot TCA (900 C), cold TCA, alcohol-
ether (2:1) and finally allowed to air dry at room temperature. The
dried precipitate was dissolved in 0.2 ml of tetramethylammonium
hydroxide, 1.8 ml of ethanol was added and after mixing a 0.2 ml
aliquot was counted in 10 ml of Solution J.
Table 4. Assay for incorporation of labeled amino acidinto protein (Adiga et al., 1)
Composent Quantity/0.5 mlfinal volume
Tris buffer (pH 7.6)Amino acid mixture2-MercaptoethanolMagnesium acetateATPGTPPEPPEP kinasel- 3H PhenylalanineRibosomal suspensionpH 5 enzyme
10 pmoles0.025 ml6 pmoles5 )..111101 es2.5 pmoles0.5 )lmoles1.25 }lmo1es6 ",g10 pcuri es0.1 ml (0.45 mg0.1 ml (1 mg ~f protein)
c. Protein and RNA determinations
Protein was determined by the method of lowry et~. (86).
Messenger RNA was determined by the procedure described by Fleck et al.(4l)
18
with crystalline bovine serum albumin added as carrier.
d. Sucrose density gradient analysis of polysomes
Polysome preparations were resolved into components of discrete
particle size using sucrose density gradient centrifugation. Five
milliliters of 50% sucrose in Solution H was placed in the bottom of
1 X 3 inch cellulose nitrate tubes. Layered on top of the 50% sucrose
was a linear gradient (15-35% w/v in Solution H) prepared with a mixing
device supplied by Buchler Instrument Company. Polysome preparations
in 1.0 to 2.0 of Solution F were gently layered on top of the gradient
and centrifuged for four hours at 25,000 rpm in an SW 25.1 swinging
bucket rotor in a Spinco Model L ultracentrifuge.
After centrifugation, 50% sucrose in Solution H was injected
near the bottom of the tube at a rate of 3.0 ml/minute using an Isco
Model 180 Density Gradient Fractionator (Instrument Specialties Co.).
UV absorption at 254 mp was continuously monitored with an Isco Model
UA-2 UV analyzer.
19
RESULTS AND DISCUSSION
A. Effect of steroid· hormone on RNA synthesis when added in vitro
to isolated rat kidney and· liver nuclei.
Studies on isolated toad bladder have shown that aldosterone
accumulates in the cell nucleus (31). Several laboratories have
shown that an early response to aldosterone is an increase in the
rate of RNA synthesis (20, 31, 109, 146). Using actinomycin D
it has been shown that RNA synthesis is a necessary event preceding
an aldosterone effect on sodium transport. Blocking RNA synthesis
prevents the expression of aldosterone at the sodium transport
level (31).
It remained to be demonstrated that aldosterone functioned in a
similar manner in mammals. Interest, therefore, was directed toward
the mechanism of action of aldosterone in the mammal. Using the rat,
experiments were performed in which steroid hormone effect on nuclear
RNA- synthesis was assayed.
Experiments were designed that would indicate if aldosterone,
when added directly to isolated kidney nuclei, could influence the
rate of RNA synthesis. This design was attractive, because if
aldosterone could function physiologically when given in vitro, it
would eliminate the necessity of intermediates, i.e., factors in the
plasma or cytoplasm.
Precedent for this approach has appeared in the literature.
Ecdysone, a steroid hormone that induces molting in insects, causes
an increase in RNA synthesis when added directly to isolated blOWfly
20
larval epidermis nuclei (30). At the mammalian level the in vitro
approach has produced confl i cting resul ts. Dukes'et'al. (30) report--that RNA synthesis is stimulated when isolated rat liver nuclei are
incubated in the presence of cortisone. Cortisol also increases RNA
synthesis when incubated ~ vitro with rat liver nuclei (87). The
capacity of uterine chromatin from ovariectomized rats to serve as
template for DNA-dependent RNA polymerase is markedly enhanced by the
presence of 17 - estradiol (7).
Drews and Bondy (29) were unable to confirm the above work. They
report that when cortisol is added to the incubation medium it is
taken up by rat liver nuclei in small amounts, but does not stimulate
nuclear RNA synthesis. In agreement with Drews and Bondy is the work
of Dahmus and Bonner (22). They report that the administration of
hydrocortisone in vivo causes increased template activity to be
developed in vivo but that the addition of hydrocortisone directly
to isolated, noninduced chromatin has no such stimulatory effect.
In the work reported in this dissertation, aldosterone, at several
different doses, was added directly to freshly isolated rat kidney
nuclei from both normal and adrenalectomized rats. The effect of
aldosterone on RNA synthesis was determined by comparing against
nuclei not receiving the hormone. The results are shown in Table 5.
Experiments 1 and 2 of Table 1 show a significant stimulation
due to aldosterone. Experiment 3, using the same dose range, indicates
an inhibition that is not statistically significant. In experiment 4
no difference was seen between hormone treated and control. A number
21
of repetitions of this type experiment gave the same unpredictable
results. Adrenalectomy did not influence the results.
Table 5. Effect of aldosterone on RNA synthesis when added in vitroto isolated rat kidney nuclei.
Exp. llg Aldosterone/ RNA synthesis: Significance*mg DNA percent of control
1. 239 116.8 0.052. 64 115.2 0.053. 94 95.4 n.s.
224 91.0 n.s.4. 1 100
*Student IS "til test
The experimental design was expanded to include several other
steroid hormones and rat liver nuclei. Table 6 shows the effect of
several steroid hormones when added in vitro to rat liver nuclei.
In experiment 1 of Table 6. aldosterone. estradiol and
deoxycoricosterone show no significant effect on RNA synthesis while
cortisone acetate shows a mild but significant stimulation. Experiment
2 of Table 6 indicates that the cortisone acetate effect is not
reprodu ci b1e.
The inconsistent results shown in Tables 5 and 6 remain unexplained
and allow no conclusions concerning the mechanism of steroid hormone
action. Several parameters. acting alone or in combination. may have
22
Table 6. Effect of steroid hormone on RNA synthesis when added in vitroto isolated rat liver nuclei.
Exp. Hormone mg Honnonej RNA synthesis: Significancemg DNA percent of
control
1. Aldosterone 0.095 108 n.s.Cortisone- 1. 017 128.8 0.03acetateEstradiol 0.4 112 n.s.Deoxycortico- 0.4 102 n.s.sterone
2. Cortisone- 1.017 108 n.sacetate
influenced these experiments. In the isolation of nuclei it is quite
likely that some change was effected in the nuclear membrane. Because
this experimental design required that the hormone get into the nuclei,
it is possible that random changes in the nuclear membrane affected the
entry of the steroid. The amount of hormone added to the isolated
nuclei is also of concern. Although a range of concentrations was used
in the case of aldosterone-kidney nuclei, it is not known if any of the
doses used were physiological.
The possibility that factors outside the nucleus were required for
aldosterone to have an effect on nuclear RNA synthesis was also considered.
Several laboratories have demonstrated that aldosterone is bound to
serum albumin, and perhaps other serum proteins (19, 23, 24, 94) ..
Litwack et~. (83) have shown an intracellular binding of cortisol
23
prior to enzymatic induction. Corticosteroid-binding globulin from
human plasma has been isolated and partially characterized (97).
Hollander and Chiu (60) reported ~ vitro binding of cortisol by a
substance in the supernatant fraction of mouse lymphosarcoma. Fiala
and Litwack (39) have demonstrated the binding of a cortisol
metabolite in liver supernatant.
Aliquots of kidney homogenate or blood was added to the nuclear
RNA incubation mixture to determine if they might contain some factor
required by aldosterone for its effect on kidney RNA synthesis. No
affect was observed when kidney homogenate or blood was used in the
incubations.
The i nabil i ty of the ~ vitro sys tern to work is in agreement
with Fimognari et~. (40) who concluded from similar studies that
aldosterone has no effect under these conditions. The negative results
from the in vitro studies allow no conclusions concerning the mechanism
of action of aldosterone.
24
B. The effectof,aldosterone, given~'~"on:the'rateofrenal
RNA synthesis assayed· in vitro.
1. Theeffectof2~5~gald6sterone administeredintraperitoneally
on the rate of kidney RNA synthesis.
Aldosterone (2.5 ~g) was injected intraperitoneally into normal
un ope rated male rats while a control group received the hormone vehicle
(ethanolic-saline). Three to four rats were in each group and they
were treated identically and concurrently. At a series of times
following administration of hormone or vehicle the animals were killed,
kidney nuclei isolated and the rate of incorporation of ATP-14C into
nuclear RNA determined.
A stimulation in the rate of RNA synthesis beginning as early as
30 minutes following injection is shown in Figure 1. The earliest
significant stimulation is observed at one hour while the maximum
effect occurs between 1.25 and 1.5 hours. The maximum rate of between
130 and 135 percent of control is short lived, for a rapid return
toward control level is observed by 1.75 hours. Figure 1 clearly
demonstrates that one early response of rat kidney to aldosterone is
an increase in RNA synthesis.
Shortly after obtaining these results several reports appeared in
the literature concerning similar studies. Castles and Williamson
(16, 17) reported the effect of aldosterone on in vivo incorporation of
orotic acid-6-14C into rat renal microsomal RNA. Their experimental
design differed in several respects from the one described in this
dissertation. A much lower dose (approximately 0.01 times ours) was
25
Figure 1. Effect of 2.5 ~g aldosterone, injectedintraperitoneally, on the rate of incorporation ofATP-14C into nuclear RNA by isolated rat kidney nuclei.Vertical bars show the confidence limits with aconfidence coefficient of 0.95. See Appendix forconfidence limits calculations.
120
140
..Joa: 130I-zoU
LLoI-ZL&JUa:lIJ 110Q.
0.5 1.0 1.5 2.0
HOURS AFT ER INJECTION
26
27
injected subcutaneously to adrenalectomized rats and the effect of
the honnone was followed using an in vivo incorporation of orotic
aCid-6-14C into RNA. Our design was an intraperitoneal injection into
nonnal rats and following the effect of the honnone using an in vitro
incorporation of ATP_14C.
Keeping these differences in mind, it is of interest to compare
the results of Castle and Williamson with those in Figure 1. At one
hour they obtained no significant effect, at 1.5 hours they observed
a significant stimulation to 135 percent of control and at two hours
they report 118 percent of control. Their lag time was longer than
ours but we both see maximum stimulation at about 1.5 hours followed
by a decrease in activity.
Fimognari et~. (40) reported similar results. Following the
subcutaneous injection of 2.0 ~g aldosterone into adrenalectomized male
rats the rate of incorporation of orotate-3H into various kidney
fractions was detennined in vitro. One hour following injection they
reported an increase in activity in the nuclear fraction to approximately
127 percent of control (not significant) and at 1.5 hours 123 percent of
control (significant at 0.05 level).
Forte and Landon (42) injected 22~g aldosterone intravenously
into adrenalectomized rats and assayed for an effect of the honnone on
the in vivo incorporation of (14C)orotic acid into renal RNA at several
times after injection. Stimulations to approximately 150 percent of
control at 30 minutes and approximately 215 percent of control at 60
minutes were reported. By two to three hours after injection the
28
incorporation of (14C) orotic acid into renal RNA was again approaching
the control level.
An early, but short lived, effect of aldosterone on rat kidney
RNA synthesis has now been reported by four laboratories. To the
author's knowledge, Figure 1 is the first definition of a time course
of the effect.
Referring to Figure 1, it is apparent that after a brief stimulation,
activity returns rapidly toward control. We were interested in the
complete time profile of the response so the activity at two hours post
hormone injection was determined. The observation that the rate of RNA
synthesis had fallen below control level at two hours post hormone
injection led to extended studies with the results shown in Figure 2.
Duplicate determinations at 2.0, 2.5, and 3.0 hours were all made on
di fferent days.
Recovering from the initial stimulation, the rate of RNA synthesis
in the kidney goes into a series of oscillations. Figure 2 shows three
maxima and three minima. All maximum values are above control while
all minimum values are below control. The period of the oscillations
varies between 1.0 and 1.5 hours. No sign of decay is seen by 4.5
hours even though three cycles have been described by that time.
Several laboratories have demonstrated an increase in kidney RNA
synthesis resulting from aldosterone administration. This has been
interpreted to be a step in the mechanism of action of aldosterone.
However, it seems unlikely that aldosterone would alternately stimulate
and inhibit the same parameter as a function of time. The author feels
Figure 2. Extended time course of response of RNAsynthesis in rat kidney nuclei following anintraperitoneal injection of 2.5 ~g aldosterone.Vertical bars show confidence limits with aconfidence coefficient of 0.95.
N1.0
\
\
q~ z
0....uw
Iq -:>ZC\I
IX:W....lL.q «
C\I
enIX::::>0
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o
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ov
000~ C\I
000 0o en (I) r--
10~J.NO:> .::JO J.N3:>~3d
31
that, under the experimental conditions described, the entire time
course of aldosterone affect on renal RNA synthesis is shown in
Figure 1. The oscillations shown in Figure 2 would then be the affect
of displacing a steady state system. That is, the initial affect,
stimulated RNA synthesis, becomes the effector. Figure 1 would then
be a study of aldosterone mechanism while Figure 2 becomes a study of
renal homeostasis or servo controls.
The experimental design was altered slightly to see if the
oscillations would persist and if there would be any change in their
features. Aldosterone (2.5 ~g in 0.5 ml) was injected intravenously
instead of intraperitoneally. All other conditions remained unchanged.
Changing the route of injection changes two parameters. First, any time
involved in getting the hormone from the peritoneal cavity into the
blood stream is eliminated by an intravenous injection. Second, and more
important, when given intravenously in the tail vein, the hormone
bypasses the liver on the way to the heart before distribution. When
given intraperitoneally the hormone is absorbed into the small veins of
the visera and from there it goes directly to the liver before going to
the heart for distribution.
Aldosterone is efficiently removed from the circulation by the
liver, conjugated and excreted in the feces and urine. Ayers et al. (5)--
studied the disappearance of aldosterone-3H from the plasma of dogs.
In normal dogs the ~ of disappearance of aldosterone was 27 minutes.
In dogs with chronic hepatic congestion secondary to throacic caval
constriction the t~ of disappearance was 85 minutes. In hepatectomized
32
dogs t~ of disappearance was 200 minutes. These studies indicate that
the liver is the major organ responsible for the metabolism of
aldosterone.
In a similar study, Hollander et~. (61) show a t~ of disappearance
of aldosterone from plasma to be 38 minutes in normal dogs. The
disappearance of aldosterone from various tissues paralleled the plasma:
kidney t~ = 34 minutes, heart t~ = 35 minutes and liver t~ = 34 minutes.
The metabolism of aldosterone in rat liver and the excretion of
the metabolites have been reported by Kohler et~. (75) and McCaa and
$ulya (91).
Changing from the intraperitoneal to the intravenous route of
injection gives a much larger effective dose and eliminates time lag
due to absorption of steroid from the peritoneal cavity. This should
be reflected in an earlier response of greater magnitude.
2. The effect of aldosterone administered intravenously on kidney
RNA synthesis.
The effect of 2.5 ~g aldosterone, administered intravenously, on
the rate of kidney RNA synthesis as a function of time post injection
is shown in Figure 3. An initial maximum stimulation is seen at 30
minutes. The stimulation is very brief for activity returns to control
within one hour of the peak stimulation. The strong initial stllmulation
is followed by strong oscillations. Two maxima and two minima are seen
within five hours. Both maxima are above control and both minima are
below control as was the case in Figure 2.
Comparing Figure 3 with Figure 2, one sees that injection of
Figure 3. The effect of aldosterone (2.5 ~gadministered intravenously) on the rate ofRNA synthesis in kidney nuclei from normalunoperated rats.
ww
220
2101 ~200
190
180
-I 1700~ 160z0u 150u.. 1400
...Zl&J 120u0::l&J 110ll.
100
90,
'~~/t80-170-1 \f601 i ii
0.5 1.0 1.5 2.0 2S 3.0 3.5 4.0 4.5 5.0
HOURS AFTER INJECTION
w.j::>
35
hormone by the intravenous route gave an earlier and larger response
as anticipated. The stronger initial displacement from control
apparently leads to oscillations of greater magnitude and longer
periods. The case for renal homeostasis and servo controls suggested
by Figure 2 is supported by Figure 3.
Figures 1, 2 and 3 were obtained using normal unoperated rats.
An obvious question presents itself: if the oscillations observed in
normal rats are reflection of renal homeostasis, what elements are
functioning in this control system?
Kidney function is autonomous to some extent but fine control
'of renal function is exercised by the endocrine system. Endocrine
influence is effected by antidiuretic hormone, parathyroid hormone,
renotrophic hormones (somatotropin, corticosterone, and cortisol)
and aldosterone (74). Antidiuretic hormone and aldosterone have
opposing roles in renal homeostasis. When aldosterone and antidiuretic
hormone are given in combination to the rat, they act in opposition and
sodium is lost or gained depending on which hormone dose is dominant (45).
It has also been shown that the pituitary gland is essential for the
stimulation of aldosterone secretion by sodium depletion (104).
Despite wide variations in water and electrolyte intake, the
extracellular fluid volume and osmolarity are rigidly controlled. A
decrease in volume is sensed as a decrease in renal perfusion pressure
and leads to the release of the proteolytic enzyme renin from the
juxtaglomerular cells on the kidney. This enzyme acts on the plasma
substrate angiotensin I which in turn is acted on by a converting enzyme
- -
36
yielding an octapeptide angiotensin II.
This potent octapeptide causes the release of aldosterone from
the zona glomerulosa of the adrenal cortex and also acts as a pressor
agent directly on the vascular system. Aldosterone stimulates sodium
reabsorption in the renal tubules leading to an increase in extra-
cellular volume. The expansion in extracellular fluid volume is then
sensed as an increase in renal perfusion pressure and the release of
renin is suppressed.
The osmolarity of extracellular fluid is monitored by osmoreceptors
in the hypothalamus. Hypertonicity is believed to cause the osmoreceptors
to shrink which stimulates neural tracts leading from the osmoreceptors
to the neurohypophysis. Stimulation of the neurohypophysis leads to the
release of antidiuretic hormone. This hormone increases the permiability
of the distal tubules to water resulting in a hypertonic urine and a
decrease in extracellular fluid osmolarity. Good reviews on endocrine
control of renal function are plentiful (74, 143, 150).
The possibility that the oscillations shown in Figures 2 and 3
might be an expression of the complex interaction of the pituitary,
adrenal and kidney in extracellular fluid homeostasis was considered.
Thinking along these lines was encouraged by the report of Friedman
et~. (43) that an intracarotid injection of hypertonic saline caused
oscillations in urine volume. In another report, Friedman et al.--(44)
discussed a direct functional linkage of the pituitary and adrenal in
which these two organs oppose each other in a "see-saw" relationship.
Healy and co-workers (57) demonstrated an oscillation in urinary
37
sodium excretion following angiotensin infusion. This oscillation was
later confirmed by Barracloughet~. (8). Intravenous infusion of
hypertonic saline has been shown to cause persistent oscillations in
renal blood flow (100).
The hypothesis that the oscillations shown in Figures 2 and 3
might result from endocrine influence on kidney function is easily
tested. If the adrenal and pituitary are functioning as opponents in
this system, then adrenalectomy or hypophysectomy should abolish the
oscillations.
3. Effect of aldosterone on the rate of RNA synthesis in kidneys of
adrenalectomized rats.
The time course of response of renal RNA synthesis in adrenalectomized
rats following intravenous injection of 2.5 ~g aldosterone is shown in
Figure 4. The time course from normal rats is shown for comparison.
Except for magnitude, the two curves are identical. The suspicions that
the adrenal gland might be functioning in the oscillations was clearly
in error.
Figure 4 is also of interest for it demonstrates the relationship
between the magnitude of the initial displacement from control and the
magnitude of subsequent oscillations. A greater initial stimulation
appears to lead to oscillations of greater magnitude. It is also
observed in Figure 4 that aldosterone had a greater effect on normal
than on adrenalectomized rats. This is opposite to what would be
expected. According to Koch (74) an excessive dose of aldosterone
results in excessive sodium retention. It was thought that the 2.5 ~g
38
Figure 4. Effect of 2.5 pg aldosterone, injectedintravenously, on the rate of renal RNA synthesisin adrenalectomized rats. Effect in normalanimals is shown for comparison .
.,. --=-
39
210 c::>
"200 ' I
, I
19 , -- - NORMAL,ADRENALECTOMIZED
I
I
170 ,I
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u.. I \0 130I ,
~z 120 I :\ I \I.LI0 \
Ia: 110 \I.LI \ c::>Q. ,
100 \,
\
90 "-e:--f , t'\ .'\ \80 '\ \
" " / \70 c::>
2 3 4 5
HOURS AFTER INJECTION
40
of aldosterone would represent a larger effective dose in adrenalec-
tomized rats and a larger response from the operated animals was
expected.
Friedman et~. (45. 46) injected aldosterone in doses of 5 ~g/lOO 9
to 10~g/10D 9 body weight. This was considered to be a reasonable
maintenance dose for adrenalectomized rats (33). Fimognari et~. (40)
injected 2.0 ~g aldosterone subcutaneously into adrenalectomized
rats to study the effect of the hormone on renal RNA and protein
synthesis. Forte and Landon (42) injected 22 ~g aldosterone intravenously
to study the effect on renal RNA synthesis in adrenalectomized rats. The
2.5 ~g dose of aldosterone used in the studies discussed in this
dissertation is within the dose range reported in the literature and
it is felt by the author that 2.5 ~g of aldosterone is a physiological
dose.
4. Effect of 0.07 ~g aldosterone on kidney RNA synthesis.
A time study was made with a much smaller dose to see if
oscillations would result from a mild initial stimulation. The effect
of 0.07 ~g aldosterone administered intravenously on the rate of renal
RNA synthesis in adrenalectomized rats is presented in Figure 5. The
effect of 2.5 ~g aldosterone is shown for comparison. Even at this
small dose the oscillation persists for a significant stimulation is
observed followed by a siynificant inhibition. It is also of interest
to note the dose dependence of the magnitude of stimulation and the
fact that maximum stimulation occurs at about the same time in spite of
a 36 fold difference in dose.
41
Figure 5. Effect of 0.07 ~g aldosterone, injectedintravenously, on the rate of renal RNA synthesisin adrenalectomized rats. Effect of 2.5 ~galdosterone is shown for comparison. Verticalbars show the confidence limits with a confidencecoefficient of 0.95.
160
0150 1\
I \ --- 2.5 )Jg ALDOSTERONEI 0.07 ).I g ALDOSTERONE
140 I \I
\,I
....J 1300a:.-z0u
u.. 1200
.-Z1LI
110ua:1LI 0a.
/ \
/ \100
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...... / \\
...... / \90 0 0
80
I 2 3 4 5
HOURS AFTER INJECTION
42
43
5. Effect6f'ald6ster6neonrenalRNA'synthesis'in
hypophysectomized rats.
Because the adrenal was found not to be implicated in the
oscillations, attention turned to the pituitary. The effect of
0.1 ~g aldosterone administered intravenously on the rate of
RNA synthesis in kidney of hypophysectomized rats is shown in
Figure 6. The oscillation was again observed indicating the pituitary
was not involved as a control element in the oscillating response
of kidney RNA synthesis to a single aldosterone injection.
6. Effect of aldosterone plus spironolactone on the rate of renal
RNA synthesis.
Large doses of progesterone can block the renal tubular effect of
aldosterone (63, 77, 78). This discovery was important because of the
role of the mineralocorticoids in the production and maintenance of
edema (see review by Kagawa, 65). With this knowledge a search was
undertaken in many laboratories for aldosterone antagonists as potential
therapeutic agents in the treatment of edematous patients.
The first synthetic compounds found to have anti-mineralocorticoid
activity and comparatively free of nonspecific side effects, were the
steroidal 17-spirolactones (18). SC-9420 or spironolactone eventually
became of interest because of its high potency as a mineralocorticoid
antagonist. Given alone it has no effect but when given with a
mineralocorticoid it prevents anti-natriuresis.
The spirolactones are believed to function by competing with the
mineralocorticoids for receptor sites in the renal tubule (62, 64, 81).
44
Figure 6. Effect of 0.1 ~g aldosterone,administered intravenously, on the rate ofrenal RNA synthesis in hypophysectomized rats.Vertical bars show the confidence limits witha confidence coefficient of 0.95.
120
"'"...J 1100"""
a::I-z0(.)
IJ.. 1000
I-ZLaJ(.)
90a::LaJQ.
80
45
I 2 3 4
HOURS AFTER INJECTION
46
By increasi.ng mineralocorticoid dosage the spirolactone block can be
overcome. Lockett and Roberts (85) present evidence that some
spirolactones do function by competing with aldosterone reversibly,
but several other spirolactones apparently have a different mechanism.
SC-11480 and SC-9420 are effective aldosterone antagonists only if the
pituitary is intact (85). In hypophysectomized rats SC-11480 and
SC-9420 are not effective.
In isolated cat kidneys SC-11480 mimics the action of aldosterone
and when SC-11480 and aldosterone are given together their effects are
additive (84). The steroidal lactone SC-5233 has been shown to lower
urine volume, and sodium, potassium and chloride excretion (35), which
is contrary to most of the aldosterone antagonists that have no effect
by themselves.
Because the time course of response of renal RNA synthesis to a
single administration of aldosterone was established, it was now
possible to investigate the action of aldosterone antagonists at the
RNA level. Aldactone (SC-9420) was of particular interest because of its
favorable potency. It is approximately five times as potent as other
spirolactones in anti-aldosterone activity (64). The report of Kagawa
(64) that aldactone functions as a competitive inhibitor of aldosterone
is opposed by the work of Lockett and Roberts (85) who claim aldactone
does not antagonize aldosterone in adrenalectomized rats.
Respecting the conflict in the literature concerning aldosterone
antagonists~ experiments were designed that would indicate if aldactone
(SC-9420) would prevent aldosterone stimulation of kidney RNA synthesis in
47
adrenalectomized rats. The assay for spirolactone effectiveness has
always been at the urinary sodium level. No information has been
found to indicate that an attempt has been made to demonstrate if the
spirolactones block aldosterone stimulation of renal RNA synthesis.
Aldactone (0.1 mg in 0.1 ml of sesame oil) or 0.1 ml of sesame
oil was injected subcutaneously. Fifteen minutes later 0.1 ~g of
aldosterone or its vehicle was injected intravenously. Kagawa (64)
showed an aldactone/aldosterone ratio of 1 mg/l ~g was effective in
blocking aldosterone expression at the sodium transport level.
Four groups of adrenalectomized rats (three animals/group) were
injected according to the schedule shown in Table 7.
Table 7. Schedule for injection of sesame oil, aldactone, saline oraldosterone.
Treatment
Group oil aldactone saline aldosteronel. + +2. + +3. + +4. + +
Thirty minutes after hormone injection, animals were killed and the
rate of renal RNA synthesis was determined using isolated kidney nuclei.
The results are shown in Table 8.
48
Table 8. Effect of aldosterone and/or-aldactone given alone or incombination on the rate of renal RNA synthesis. -Numbers representpercent of control with confidence limits using a confidence coefficientof 0.95. -
Group
1. O-S
2. L-S
3. O-A
2. L-S
119 + 2.86
3. O-A
148.6 + 2.47
4. L-A
180.1 + 4.69
151.8 + 5.23
121.5 + 4.58
O-sesame oil, S-saline, L-aldactone, A-aldosterone
Group 1 vs. 3 shows a significant aldosterone induced stimulation
of 148.6 + 2.47 (percent of control). Group 1 vs. 2 shows a significant
stimulation of 119 + 2.86 due to aldactone. Both aldosterone and
aldactone stimulate RNA synthesis in this system. Group 1 vs. 4 shows
the effect of aldosterone and aldactone when given together, a stimulation
to 180.1 + 4.69 percent of control. This shows the stimulations due to
aldosterone and aldactone when given alone are additive when given together.
Group 2 vs. 4 shows the effect of aldosterone plus-aldactone against
controls receiving aldactone. The significant stimulation to 151.8 + 5.23
percent of control agrees with the group 1 vs. 3 cross and shows that
aldosterone functions the same whether aldactone is present or not. Group
3 vs. 4 gives the same results as group 1 vs. 2 and shows that aldactone
stimulates the same whether aldosterone is present or not.
This work indicates that aldactone does not block the action of
49
aldosterone at the RNA synthesis level. It further indicates that
aldactone itself stimulates renal RNA synthesis. The fact that the
aldosterone and aldactone stimulations are additive suggests that they
both could be increasing transcription but at different loci on the
genes.
The additive effects of aldosterone and aldactone (SC-9420) on
renal RNA synthesis in adrenalectomized rats appears similar to the
results of Lockett and Roberts (84) using isolated cat kidneys. in-the
cat kidneys, aldosterone and SC-11480 were both shown to affect urinary
sodium and their affects were additive. Also in apparent agreement with
the results shown in Table 8 is the claim of Lockett and Roberts (85)
that aldactone does not antagonize aldosterone in adrenalectomized rats.
C. The effect of aldosterone on the rate of RNA synthesis in rat brain.
Aldosterone is known to effect a wide spectrum of tissues other than
the kidney, i.e., salivary and sweat glands, striated muscle, bone,
gastrointestinal tract and blood. Furthermore, Woodbury and Koch (149)
have reported a decrease in sodium and a small increase in potassium in
muscle and brain cells of mice treated with aldosterone for four days.
Interest focused on whether an effect could be demonstrated at the RNA
synthesis level in rat brain following a single intravenous injection
of aldosterone.
At a series of times following intravenous aldosterone injection
(2.5 ]Jg), rats were killed, brain nuclei isolated and RNA synthesis assayed
in vitro. The results, shown in Figure 7, indicate an early stimulation
in the rate of brain RNA synthesis. The stimulation to between 140 to
50
Figure 7. The effect of aldosterone (2.5 ~gadministered intravenously to normal unoperatedrats) on the rate of RNA synthesis in the brain.
51
150
80
70
130
52
145 percent of control at 30 minutes post injection is brief, and as
was observed repeatedly in the kidney, activity dropped rapidly to
below control level.
Comparison of Figures 2 and 7 shows that aldosterone has a similar
effect on kidney and brain RNA synthesis in the rats. However,
quantitatively, ion transport effects must be greatly different.
D. Steroid hormones and spleen RNA synthesis.
Probably no other group of compounds have been shown to possess
more metabolic effects than the adrenal corticosteroids. They are known
to function in lipid, carbohydrate, protein, nucleic acid, and mineral
metabolism. They also influence infection, inflammation and lymphatic
involution.
The effects of many of the adrenal steroids have been studied in
the lymphatic system but aldosterone has received very little attention
with respect to this problem (25, 51, 88). A clean preparation of
rat spleen nuclei can be isolated using the same techniques that were
used in the isolation of rat kidney nuclei. Therefore, it was
possible to study aldosterone effect on splenic RNA synthesis
concurrently with studies on kidney RNA synthesis.
1. Effect of aldosterone on spleen RNA synthesis.
Aldosterone (2.5 ~g) was injected intravenously into normal
unoperated rats. At a series of times following injection the animals
were sacrificed, spleen nuclei isolated and the rate of incorporation of
ATP-14C into nuclear RNA determined. The results are shown in Figure 8.
An inhibition in the rate of RNA synthesis is seen as early as
Figure 8. The effect of aldosterone (2.5 ~g administered intravenously)on the rate of incorporation of ATP_14C into spleen RNA in vitro.Six rats, half receiving aldosterone and half receiving the hormonevehicle, were used in each determination. The incorporation of ATP-14Cinto RNA was accomplished by incubating freshly isolated spleennuclei in an RNA synthesizing mixture for 15 minutes at 370 C. Theeffect of the hormone is expressed as percent of control. Duplicatedeterminations at anyone time were always performed on different days.
U1W
54
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e~ z0
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55
thirty minutes. Inhibition becomes progressively greater with time up
to approximately three to four hours at which time RNA synthesis is
about 78 percent of control. At about four hours post injection, the
rate of RNA synthesis begins to increase, reaching control values by
five hours. RNA synthesis accelerates rapidly after 4.5 hours leadfng
to a very pronounced stimulation by six hours. The two determinations at
six hours were made on different days and show values of 142 and 175
percent of control. The six hour stimulation, although prominent, is
very brief for one sees a return to control by seven hours and a
significant inhibition to 71 percent of control by 7.5 hours.
A comparison of Figures 3 and 9 indicates that RNA synthesis in rat
kidney and spleen are affected in an opposite manner by aldosterone. In
both studies, 2.5 ~g of aldosterone was injected intravenously into
normal unoperated male rats. The different manner in which the two
tissues responded was particularly interesting for it showed that the
same hormone, when injected into the rat, induced opposite affects in
the same enzyme system from two different tissues.
Pena et~. (106) demonstrated that cortisol increased RNA and
protein synthesis in rat liver while at the same time it decreased both
of these reactions in rat thymus. Nakagawa and White made a similar
observation with cortisol (99). That a steroid hormone can affect RNA
synthesis in different tissues of the same animal in an opposite manner
has now been demonstrated using two different steroids and two different
sets of ti ssue.
This phenomena has also been demonstrated with other enzyme systems.
56
Cortisol enhanced interferon production by peritoneal tissue while it
inhibited the same system in the spleen (93). Purine nucleotide
biosynthesis in liver is increased by cortisol but the same reaction is
inhibited in both thymus and spleen (38). The lymphatic tissues are
clearly distinguished from liver, kidney and peritoneal tissue by their
unique response to adrenal steroid hormones.
2. Effect of cortisol on spleen RNA synthesis
The initial aldosterone induced inhibition in spleen RNA synthesis
was consistent with known effects of steroids on lymphatic RNA synthesis
but the secondary responses were quite unexpected. Because of the lack
of literature concerning the effect of aldosterone on lymphatic RNA
synthesis, it was decided to perform a similar study using a steroid
hormone whose effect is well documented.
Cortisol (122 pg in 0.5 ml) was injected intravenously into normal
un operated rats and the affect on the rate of RNA synthesis was followed
as a function of time following injection. The results of this experiment
are shown in Figure 9.
The initial response to cortisol was a decrease in the rate of RNA
synthesis. A maximum inhibition to about 78 percent of control was
reached between three and four hours following hormone injection. At
four hours a very abrupt stimulation sets in and by five hours the rate
of RNA synthesis is approximately 150 percent of control. The two
experiments run at five hours after injection were performed on separate
days and indicate values of 137 and 161 percent of control. The stimulated
rate of RNA synthesis observed at five hours is short lived however and the
57
system returns to control values by seven hours and shows an inhibition
to 87 percent of control by eight hours.
Comparing Figures 8 and 9 one sees two di fferent steroi d hormones
eliciting very similar responses from the same system.
An early inhibition in lymphatic RNA synthesis as a result of
steroid injection has been shown in several laboratories. Pena et a1.
(106) injected cortisol (5 mgj100g of body weight) into normal rats.
Three hours after injection animals were killed, thymic nuclei isolated
and nuclear RNA synthesis assayed. Rats receiving cortisol had
approximately 17 percent less incorporation than control. In seven
separate experiments an inhibition of 10 and 25 percent was shown.
The results of Pena et~. (106) and those shown in Figure 9 indicate
that at three hours after hormone injection the spleen and thymus respond
to cortisol in a similar manner.
Nakagawa and White (99) demonstrated a significant decline in
thymic nuclear RNA polymerase activity within thirty minutes following
an intraperitoneal injection of cortisol. The degree of inhibition
became more significant at subsequent time intervals up to three hours when
their experiments were terminated. At three hours post injection hormone
treated rats showed about 20 percent less activity than controls.
Interesting results are reported from the laboratory of Feige1son
and Feige1son (38). The effect of cortisone acetate on glycine-2- 14C
incorporation in vivo into splenic acid-soluble adenine and into RNA
was determined. Labeled glycine was injected two hours before animals
were sacrificed. Adenine biosynthesis in cortisone treated rats was
Figure 9. The effect of cortisol (122 ~g injectedintravenously) on the rate of incorporation ofATP-14C into RNA by isolated rat spleen nucleiin vitro. Six rats were used for each determination,half received cortisol and half received thehormone vehicle. Duplicate experiments at 3.25and 5.0 hours were run on different days. Effectof hormone is expressed as percent of control.
0100
59
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60
decreased approximately 55 percent within two hours. By four hours
the rate of adenine synthesis begins to increase (minus 50 percent)
and by seven hours the activity has reached control levels. At eight
hours adenine biosynthesis has increased to about forty percent over
control. The initial inhibition in adenine biosynthesis resulting from
cortisone injection is followed by a significant stimulation.
RNA synthesis (38) was also inhibited following cortis'one
administration. At two and four hours RNA synthesis was decreased
forty percent. At six hours activity was down about twelve percent
showing a strong recovery from the low as four hours. At eight hours
post cortisol injection the rate of RNA synthesis was about 20 percent
below control suggesting a secondary inhibition.
The results shown in Figures 8 and 9 are in agreement with the
earlier results of Feigelson. Both show an initial inhibition in
splenic RNA synthesis resulting from steroid administration. Both show
a recovery from the initial inhibition. The recovery shown in
Feigelson's work is not quite to control, while Figures 8 and 9 go well
past control. Following the recovery in Feigelson's work, activity
again appears to decrease, although only slightly, whereas Figures 8 and
9 indicate a strong secondary inhibition.
The difference in magnitude of response between Feigelson's
work and that shown in this dissertation could well be accounted for
by the difference in experimental design. The assay used in this
laboratory was a fifteen minute in vitro incorporation of label where
Feigelson's was a two hour ~ vivo incorporation. Their two hour
61
incorporation period could mask, or at least dilute, a brief change in
activity. A fifteen minute incubation allows for monitoring, more
closely, such brief changes.
Dougherty et~. (28) reported that the accumulation of label in
mice spleen is maximal approximately five to six minutes following
injection of cortisol-14C. Following this early maximum, label
concentration dropped rapidly with a half life of approximately seven
minutes. Similar results were obtained for thymus and lymph nodes.
This suggests that almost the entire time course shown in Figure 9
occurs after most of the hormone has left the tissue. However, it is
possible that the trace of hormone remaining in the tissue could be
responsible for the observed affects.
The inhibitory affects of cortisol on DNA synthesis and mitosis
in lymphatic organs persist for a period of six to eight hours (28).
Dougherty et~. (28) concluded that the hormone must trigger some
event at the cellular level that persists after hormone exit. This
initial event would then be responsible for subsequent affects.
3. Steroid structural requirements for lymphatic activity.
The structure-activity relationships of the anit-inflammatory
steroids have received considerable attention. Numerous steroids,
natural and synthetic, have been assayed in an attempt to increase
potency and decrease side affects. From these studies it appears that
certain functional groups are necessary for activity.
In 1961 Boland (10) listed the following as essential elements of
the steroid molecule: C-4, C-5 double bond, keto-group at C-3, C-ll and
62
C-20, and a beta hydroxy group at C-17. In 1964 Dougherty et~. (28)
observed certain requirements common to all naturally occurring steroids
with lymphatic involution inducing ability. The essential features
differed from Boland's list only in that the C-17 hydroxy was not
essential.
In 1967 Steelman and Hirschmann (127) concluded that our knowledge
of structure-function relationships in the steroid-anti-inflammatory
field is rudimentary at best. They (127) reported that compounds have
been prepared without one or more of the above named elements and they
were shown to be highly active in animal studies. For example, the
C-3 keto group is missing from some of the most potent synthetic
anti-inflammatory agents.
Aldosterone and cortisol (Figs. 8 and 9) both have what Dougherty
et ~.(28) considered to be the essential elements: C-4, C-5 double
bond, and keto or hydroxy groups at C-3, C-ll and C-20. Both of these
steroids induced a decrease in the rate of spleen RNA synthesis. The
effect of several other steroids on spleen RNA synthesis was also
determined. Some of the steroids used in these studies had one or more
of the "essential elements" missing. This work was performed in an
attempt to determine if those elements necessary for anti-inflammatory
activity were also necessary for inhibition of spleen RNA synthesis.
The effect of deoxycorticosterone-acetate (125flg injected
intravenously) on the rate of spleen RNA synthesis is shown in Figure
10. The effect of this steroid is similar to the effects of aldosterone
and cortisol (Figs. 8 and 9) in that the initial response is an
63
Figure 10. The effect of deoxycorticosterone-acetate(125 ~g injected intravenously) on the rate ofincorporation of ATP_14C into RNA by rat spleennuclei in vitro. Six rats were used for eachdetermination, half received DOCA and half vehicle.Duplicate determinations at 3.25 hours were made ondifferent days. Effect of hormone expressed aspercent of control.
...J 1200D:: 0l- I 10 ~z ,..0 0
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2 3 4
HOURS AFTER INJECTION
64
65
inhibition. A brief stimulation is observed following the initial
decrease in activity. Both the initial inhibition and the
subsequent stimulation are milder and of shorter duration than was
the case with aldosterone or cortisol.
Deoxycorticosterone-acetate is missing the C-17 oxygen group
thought to be essential for anti-inflammatory activity, and an acetyl
group has been added at C-21. Figure 10 indicates that this combina-
tion of changes does not prevent the steroid from decreasing RNA
synthesis in the rat spleen.
The effect of l-dehydrocortisone on spleen RNA synthesis is shown
in Figure 11. The introduction of a C-l, C-2 double bond results in
enhancement of anti-inflammatory potency (10) and Figure 11 indicates
that the initial affect on rat spleen RNA synthesis is an inhibition.
The duplicate determinations at 3.25 hours in both the deoxycorticos-
terone-acetate and l-dehydrocortisone curves were made on different
days and illustrate the reproducibility of the system.
The effect of four different steroids on spleen RNA synthesis is
shown in Figures 8 through 11. These four steroids all have the
"essential elements" for anti-inflammatory activity with the exception
of deoxycorticosterone-acetate which is missing the C-ll oxygen group.
The initial affect on spleen RNA synthesis in each case is an
inhibiti on. Extrapolating from the structure-functi on rel ati onships
at the anti-inflammatory level, the initial inhibitions could have
been expected in each case.
The effect of progesterone on spleen RNA synthesis is shown in
66
Figure 11. The effect of l-dehydrocortisone (152 ~ginj ected intravenous ly) on the rate of incorporationof ATP-14C into RNA by rat spleen nuclei in vitro.Six rats were used for each determination. Effectof l-dehydrocortisone on RNA synthesis is expressedas percent of control. Duplicate determinations at3.25 hours were made on different days.
2 3
HOURS AFTER INJECTION
67
68
Figure 12. The initial response is a pronounced, but brief, stimulation,
followed by a decrease in activity to below the control level. The
structure of progesterone differs from deoxycorticosterone-acetate
only in that the latter is acetylated at C-21. This single difference
in structure, however, appears to be responsible for the opposite effects
of these two steroids on spleen RNA synthesis (compare Figures 10 and
12).
The effect of testosterone on spleen RNA synthesis is shown in
Figure 13. The initial affect is observed to be a stimulation
followed by a return to control level by 3.25 hours. The duplicate
determinations at 3.25 hours in progesterone and testosterone studies
were performed on different days and again illustrate the reproducibility
of the system.
Progesterone and testosterone are both missing two of the
"essential elements" for anti-inflammatory activity. Both of thes.e
steroids are missing the C-ll oxygen group and the C-20, C-21 side
chain characteristic of adrenal corticoids. The deoxycorticosterone-
acetate curve (Fig. 10) indicates that the C-ll oxygen group is not
necessary for a steroid to initially inhibit spleen RNA synthesis.
Figures 12 and 13 show that a C-20, C-21 side chain characteristic of
adrenal corticoids is required for an initial inhibition in spleen RNA
synthesis. Based on the observations of Boland (10) and Dougherty (28)
neither progesterone nor testosterone would be expected to possess
anti-inflammatory activity and it is seen (Figs .. 12 and 13) that
neither of them initially inhibit spleen RNA synthesis.
69
Figure 12. The effect of progesterone (113 ~ginjected intravenously) on the rate of .incorporation of ATP-14C into RNA by rat spleennuclei in vitro. Six rats per determination.Duplicate determinations at 3.25 hours were madeon different days. Effect of hormone is expressed~s percent of control.
2 3
HOURS AFTER INJECTION
70
71
Figure 13. The effect of testosterone (132 ~ginjected intravenously) on the rate of incorporationof ATP_14C into RNA by rat spleen nuclei in vitro.Six rats per determination. Duplicate determinationswere made on different days. Effect of hormone isexpressed as pe