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

Iq ::I:

o

10o

30

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

160 ,..J

I0a: 150 ,~z c::>00 140 I \

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

\/

...... / \\

...... / \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

o....oCDo 0o OlooNo~ovoCD

CD

Q

e --------e--------

e

e~ z0

It)~0w,z

Q v 0::Wl-LL

e ~e e en

e ~ 0:::::>0:I:e

oCD

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

co

/

oCO

oen

oooo

(\Jo",

ov

oIt)

o(D

z0

10 i=0l&J..,~

a: v l&J....

I.L. :::>03:

oI'-

'O~iNO~ ~O iN3~~3d

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

"0100

lL.

~a0

I-90

z ~I.LJ 800D::I.LJa..

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