PHILLEO, William Wallace, 1939-THE EFFECT OF STEROIDS ON RNASYNTHESIS IN VARIOUS TISSUES OFTHE RAT.

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University of Hawaii, Ph.D., 1969Biochemistry

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THE EFFECT OF STEROIDS ON RNA SYNTHESIS

IN VARIOUS TISSUES OF THE RAT

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

BY

William Wallace Philleo

Dissertation Committee:

Theodore Winnick, ChairmanFrederick C. GreenwoodMorton MandelJohn B. HallTerence O. Moore

ABSTRACT

Single doses of aldosterone were administered to normal rats,

and the effect of RNA synthesis was studied. The maximal stimulation

of the incorporation of l4c_ATP into RNA by nuclei isolated from

rat kidneys was observed 30 minutes after an intravenous injection

of aldosterone. Thereafter, the RNA synthesis decreased to a level

less than that of the control, reaching a minimum at 2.5 hours. The

return to control level was followed by further oscillations in RNA

synthesis. Likewise, in isolated brain nuclei, an oscillation in

RNA synthesis was observed following an intravenous injection of

aldosterone.

The effect of single injections of aldosterone, cortisol, deoxy-

corticosterone acetate, testosterone or progesterone was studied on

RNA synthesis by isolated rat spleen nuclei. It was found that the

l4C_ATP incorporation was initially inhibited following the injection

of aldosterone, cortisol or deoxycorticosterone acetate. The initial

response to an injection of testosterone or progesterone was a

stimulation in the spleen RNA synthesis. The response to the admin-

istration of aldosterone was similar to that of cortisol. Subsequent

to the initial inhibition, a stimulation followed by a return to the

level of the control, was observed and secondary inhibitions were

found. RNA synthesis in the thymus was initially inpibited following

the administration of aldosterone.

RNA synthesis was studied in the nuclei isolated from the kidney

and spleen of adrenalectomized or hypophysectomized rats injected

iv.

with aldosterone. Oscillations were observed in the RNA synthesis of

both tissues of the endocrinectomized rats. It was concluded that

the oscillations in RNA synthesis were not a function of some form

of homeostatic control involving the pituitary and adrenal glands.

Aldactone (SC 9420) was found to modify the RNA synthesis of

nuclei isolated from the kidney, spleen and thymus of intact,

adrenalectomized or hypophysectomized rats. Again, oscillations in

RNA synthesis were observed, however there was the suggestion that

the adrenals or the pituitary might be involved in the response as

observed in the kidney and the thymus.

Experiments were carried out in which both aldosterone and

Aldactone were injected, and their action on RNA synthesis was

observed in the kidney, spleen and thymus. The results indicated that

these two steroids alter RNA synthesis independently from each other.

Analysis of the RNA synthesized in vitro by the isolated nuclei

of the kidney and spleen indicated that only a single species of RNA

was made. This 5 s RNA was identical to that species of RNA presentin nuclei, prior to incubation for the incorporation of l4c-ATP.

v.

TABLE OF CONTENTS

ABSTRACT ••.

LIST OF TABLES .

LIST OF FIGURES

I . INTRODUCTION

iii

viii

x

A.B.C.

D.

E.F.

G.

Statement of the problem . . . . • . . . . .Heterogenity of hormones and their effects •Gene regulation and RNA synthesis, as anearly effect in the mechanism of action ofsome hormones.The proposed mechanism of action of . . • . •aldosterone.Aldosterone antagonism by Aldactone . . . • .Lymphoid tissues and anti-inflammatorysteroids.In vitro synthesis of RNA . . . • . . •

12,

9

1214

16

II. MATERIALS AND METHODS

A.B.C.D.E.F.G.H.IeJ.K.L,.

Materials obtained Commercially .Prepared Materials - solutionsSpecial preparation of reagents .Animals .Experimental treatments .•...Protocol for multiple injectionsIsolation of nuclei .•••...Assay for nuclear RNA synthesis ..••Measurement of radioactivityDNA analysis . . . • . . • •Expression of results ••••RNA extraction and analysis

181819192122222425252626

III. RESULTS AND DISCUSSION

A.B.

In vitro synthesis of RNA by isolated nuclei . •The effect of aldosterone on the synthesisof RNA.

3034

The response of renal RNA synthesis to an . . 34intraperitoneal injection of aldosterone.

The effect of an intravenous injection of • . 37aldosterone on renal RNA synthesis.

The effect of an intravenous injection of . 43aldosterone on renal RNA synthesis inadrenalectomized rats.

vi.

The effect of aldosterone on renal RNA 47in hypophysectomized rats.

The effect of an intravenous injection of 50aldosterone on RNA synthesis in brain ofintact rats.

The effect of an intravenous injection of • . . • • 53aldosterone on spleen RNA synthesis.

The effect of aldosterone on RNA synthesis • • •• 57in the spleen of adrenalectomized orhypophysectomized rats.

The effect of an intravenous injection of 61aldosterone on thYmus RNA synthesis.

The effect of RNA synthesis of hormones . . 61added in vitro to isolated nuclei.

The effect of 2.5 ~g of corticosterone on • . 66renal RNA synthesis.

C. The effect of miscellaneous steroids on . . • • . • 69spleen RNA synthesis.

The effect of an intravenous injection of • • 69cortisol on spleen RNA synthesis.

The effect of an intravenous injection of • . 71deoxycorticosterone acetate on spleenRNA synthes is •

The effect of progesterone or testosterone . . . • . 71on spleen RNA synthesis.

D. The effect of Aldactone on RNA synthesis •..••. 78

The effect of a subcutaneous injection of •Aldactone on RNA synthesis in the kidneyof intact, adrenalectomized or hypo-physectomized rats.

The effect of a subcutaneous injection of .Aldactone on RNA synthesis in the spleenof intact, adrenalectomized or hypo-physectomized rats.

The effect of a subcutaneous injection ofAldactone on RNA synthesis in the thymusof intact, adrenalectomized or hypo-physectomized rats.

78

81

84

E. Effect of multiple .injections of aldosterone • . •and Aldactone on RNA synthesis.

vii.

89

The effect of aldosterone and Aldactone on . • •. 90the synthesis of RNA in the kidney andthymus of intact rats.

The effect of aldosterone and Aldactone on . . .. 92the synthesis of RNA in the spleen andthymus of intact rats.

The effect of Aldactone and aldosterone on • . .. 94the synthesis of RNA in the kidney andthymus of adrenalectomized rats.

The effect of aldosterone and Aldactone on . . •. 94the synthesis of RNA in the spleen andthymus of adrenalectomized rats.

The effect of aldosterone and Aldactone on • • •. 97the synthesis of RNA in the kidney andthymus of hypophysectomized rats.

The effect of aldosterone and Aldactone on • . •. 97the synthesis of RNA in the spleen andthymus of hypophysectomized rats.

The effect of 2.5 ~g and 5.0 ~g of aldosterone 100on the synthesis of RNA in the kidneyand thymus of intact rats.

The effect of 2.5 ~g and 5.0 ~g of aldosterone 100on the synthesis of RNA in the spleenand thymus of intact rats.

F.

G.

H.

Extraction of RNA from isolated nuclei •

RNA from rat kidney nuclei • .RNA from rat spleen nuclei .RNA from rat thymus nuclei

RNA extracted from incubated nuclei .

Kidney nuclei .Spleen nuclei .

Extraction of RNA from incubated nucleifrom control and aldosterone treated rats.

Kidney.Spleen .

105

105107107

. . • • 110

110• • 117

. • 122

122127

IV. SUMMARY AND CONCLUSIONS • . • • • • . • • . . . • • •. 132

V. REFERENCES....................... 136

Table

II.

III.

IV.

V.

VI •

viii.

LIST OF TABLES

Contents Page

Hormones, dosages and vehicles • 21

RNA polymerase assay mixture • 24

Scheme employed for the isolation of RNA from • •• 27resuspended nuclei.

Incorporation of l4C_ATP into nuclear preparations • • 31from brain, kidney, thymus and spleen.

Effect of aldosterone on RNA synthesis when •added in vitro to isolated rat kidney nuclei.

Effect of steroid hormones on RNA synthesis •when added in vitro to isolated rat livernuclei.

VII.

VIII.

IX.

X.

XI.

XII.

The combined effect of Aldactone and aldo-sterone on RNA synthesis in the kidney andthymus of intact rats.

The combined effect of aldosterone andAldactone on RNA synthesis in the spleenand thymus of intact rats.

The combined effect of Aldactone and aldo-sterone on RNA synthesis in the kidney andthymus of adrenalectomized rats.

The combined effect of aldosterone andAldactone on RNA synthesis in the spleen andthymus of adrenalectomized rats.

The combined effect of Aldactone and aldo-sterone on RNA synthesis in the kidney andthymus of hypophysectomized rats.

The combined effect of aldosterone andAldactone on RNA synthesis in the spleen andthymus of hypophysectomized rats.

91

93

95

98

99

Table

XIII.

XIV.

Contents

The "effect of 2.5 and 5.0 ~g of aldosterone .on RNA synthesis in the kidney and thymus ofintact rats.

The effect of 2.5 and 5.0 ~g of aldosterone .on RNA synthesis in the spleen and thymus ofintact rats.

ix.

Page

101.

102

Figure

1.

2.

3.

4.

5·

6.

7.

8.

9·

10.

LIST OF FIGURES

Contents

The incorporation of l4C_ATP into RNA bynuclei isolated from the brain, kidney, thymusand spleen, as a function of the DNA con-centration.

Time course for the effect of a single intra- .peritoneal dose of aldosterone on RNA synthesisin isolated rat kidney nuclei.

Time course for the effect of a single intra~ •venous dose of aldosterone on RNA synthesis inisolated rat kidney nuclei.

Time course for the effect of a single intra- •venous dose of aldosterone on RNA synthesis inkidney nuclei isolated from adrenalectomizedrats.

Time course for the effect of a single intra- •venous injection of 0.1 ~g of aldosterone onrenal RNA synthesis in hypophysectomized rats.

Time course for the effect of a single intra- •venous injection of 2.5 ~g of aldosteroneon RNA synthesis in the rat brain.

Time course for the effect of a single intra-venous injection of aldosterone on RNAsynthesis in isolated rat spleen nuclei.

Time course for the effect of a single intra- •venous injection of 2.5 ~g of aldosterone onRNA synthesis in the spleen of adrenalectomizedor hypophysectomized rats.

Time course for the effect of a single intra-venous dose of 2.5 ~g of aldosterone on RNAsynthesis in the thymus.

Relative rate of RNA synthesis in the nucleiisolated from the kidneys of rats injectedintravenously with 2.5 ~g of corticosterone.

x.

Page

32

40

45

49

52

55

60

68

Figure

11.

12.

Contents

Time course for the effect of a single doseof cortisol on RNA synthesis in isolatedspleen nuclei.

Time course for the effect of a single intra-venous injection of 125 ~g of deoxycortico-sterone acetate on the rate of RNA synthesisin nuclei isolated from rat spleen.

xi.

Page

70

72

13. Time course for the effect of a single injection • . • 74of progesterone or testosterone on spleen RNAsynthesis.

14.

15·

16.

Time course for the effect of a single dose of •Aldactone on the rate of kidney RNA synthesisin normal, adrenalectomized or hypophysectomizedrats.

Time course for the effect of a single dose ofAldactone on spleen RNA synthesis in intact,adrenalectomized or hypophysectomized rats.

Time course for the effect of a single dose of •Aldactone on the RNA synthesis in the thymus ofintact, adrenalectomized or hypophysectomizedrats.

80

83

86

17. Sucrose density gradient profile of RNA extracted •. 106from nuclei isolated from rat kidney.

18. Sucrose density gradient profile of RNA extracted •. 108from nuclei isolated from rat spleen.

19. Sucrose density gradient profile of RNA extracted •• 109from nuclei isolated from rat thymus.

20. Sucrose density gradient profile of RNA extracted •• 112from kidney nuclei incubated for the incorpora-tion of 14c-ATP into RNA.

21. Sucrose density gradient profile of RNA extracted •. 115from kidn~y nuclei incubated for the incorpora-tion of 14C-ATP into RNA.

22. Sucrose density gradient profile of RNA extracted •. 119from sple~n nuclei incubated for the incorpora-tion of 14C-ATP into RNA.

Figure

23.

xii.

Contents Page

Sucrose density gradient profile of RNA extracted.. 121from sple~n nuclei incubated for the incorpora-tion of 1 C-ATP into RNA.

24. Sucrose density gradient profiles of RNA extracted . .124from RNA polymerase assay incubations of kidneynuclei isolated from control and aldosteroneinjected rats.

25. Sucrose density gradient profiles of RNA extracted•• 126from incubations of kidney nuclei isolated fromcontrol and aldosterone injected groups of rats.

26. Sucrose density gradient profiles of RNA extracted. 129from RNA polymerase incubations of spleen nuclei,isolated from control and aldosterone injectedgroups of rats.

27. Sucrose density gradient profiles of RNA extracted. 131from incubations of spleen nuclei isolated fromcontrol and aldosterone injected groups of rats.

INTRODUCTION

A. Statement of the Problem

When this research was initiated, the mechanism of aldosterone

mediated sodium transport in mammals was unclear. The effect of

this hormone on sodium transport in the toad bladder had been well

characterized (Edelman, Bogoroch & Porter-1963 and Sharp & Leaf-

1966). In the toad bladder, it had been shown that actinomycin D,

an inhibitor of RNA synthesis, or puromycin, an inhibitor of

protein synthesis, were capable of inhibiting the physiological

response to aldosterone (Fanestil and Edelman-lg66). In addition,

there was a delay of 60 to 90 minutes after aldosterone administration,

before the onset of sodium resorption (Sharp & Leaf-lg66, Forsham &

Melman-19GB). These facts are consistent with the hypothesis that

the mechanism of action of aldosterone involves an early synthesis

of RNA. In 1966, these findings had not been extended to include mam-

mals. Therefore, it was important to see if aldosterone could

modify the synthesis of RNA in renal tissue.

The initial studies were later expanded to include non-

physiological target tissues, such as the brain, spleen and thymus.

With the finding that aldosterone would elicit a biochemical response

in a non-target tissue, it was of interest to investigate the action

of several different steroids on a single tissue. Among the steroids

studied was Aldactone, a competitive inhibitor of aldosterone. With-

out stressing the point unduly, it should be apparent from the

results, that the concept of a target tissue for a hOl~one may be in

error. It appears that almost any steroid may have a biochemical

effect on all tissues. In themselves, these results should be

considered by others studying the biochemical action of hormones.

Some experiments were devoted to a characterization of the

RNA synthesized in the Mg++ activated RNA polymerase assay system.

This work may be of interest to individuals studying the in vitro

synthesis of RNA. Some very obvious pit falls are pointed out for

researchers, in the field of hormone mechanism, that extend

their work and their conclusions beyond the validity of their

experiments.

B. Heterogenity of Hormones and Their Effects

Hormones have been defined by many researchers (White et al.-

1968, Karlson-1968, Tata-1966, Davidson-1965, Karlson & Sekeris-

1966, Tata-1965, Talwar et al.-1968). From these definitions, it

is possible to conceive of hormones as being specific substances,

secreted by particular organs into the general circulation, which

carries them to sites of action elsewhere in the body; there they

regulate the rates of specific processes, without contributing

significant amounts of energy or matter to the tissues. Certainly

this definition does exactly explain all hormones, but it does

convey an approximate concept.

It is common practice to catagorize hormones according to

their structure, irrespective of the physiological or biochemical

function that they elicit or modify. Epinephrin (Adrenalin) and

thyroxine are examples of hormones derived from the adrenal medulla

and the thyroid gland, respectively. Adrenocorticotrophic hormone

2.

3.

(ACTH) and insulin, representative of the peptide and protein

hormones, are elaborated by the pituitary and the pancreas. The

steroid hormones can be exemplified by the corticosteroids of the

adrenal cortex, and the estrogens and androgens of the ovaries and

testes. The juvenile hormone of insects is the only example of a

hormone that is a homolog of isoprene.

The diverse chemical structures exhibited by hormones is

indeed striking, as is their importance. Virtually every major

process of growth and development, as well as metabolic activities,

is initiated or regulated by hormones (Tata-1966). For the purpose

of this discussion, responses to hormones can be broadly divided

into two major catagories: (1) Those that regulate specific

metabolic activities without affecting the general anabolic, catabolic,

or developmental processes, and (2) Those that control growth

processes and cellular differentiation. This division of hormone-

effects does not infer that all hormones can be nicely segregated

exclusively into one or the other catagory; certainly some hormones

are mutually inclusive.

The mechanism of action of the metabolic regulatory hormones

has often been explained on the basis of some form of direct inter-

action between the hormone an~ the cell membrane, protein or enzyme.

It has been proposed that these sites may involve the rate-limiting

parameter in the metabolic process (Bush-1962, Hechter &Halkerston-

1964, Riggs-1964, Sutherland &Rall-l96o, Tomkins &Maxwell-1963).

For example: (1) The very rapid stimulation of cellular respiration

by Adrenalin is the result of an interaction between the hormone and

4.

adenylcyclase (Sutherland &Rall-1960, Hagen &Hagen-1964, Robinson,

Butcher & Sutherland-1968). (2) Steroids such as estrogen induce

allosteric changes in glutamic dehydrogenase, and this has been

proposed as part of the mechanism by which these steroids produce

their biological actions (Tomkins &Maxwell-1963, Breuer-1965, Tomkins

and Yielding-1964). (3) Vasopressin, which causes vasoconstriction

and anti-diuresis, prevents loss of urinary water, attaches itself

to the outer membrane of the renal cells on which it acts (Li-1968,

Heinz-1967).

The above examples are illustrative of the "direct" action

of hormones. The subject of hormonal control of metabolic activities

by indirect action is more closely related to the subject of this

dissertation.

C. Gene Regulation and RNA Synthesis, as an Early Event in the

Mechanism of Action of Some Hormones.

The hypqthesis that hormones may act by regulating the activity

of certain genes was first set forth by Clever and Karlson (1960)

and was further elaborated by Karlson (1961, 1963). This hypothesis

proposes that some hormones bring about their physiological effect

by derepressing specific parts of the DNA and that there is a

subsequent stimulation of RNA synthesis, followed by an increase

in the synthesis of proteins (enzymes). Thus, it is possible to

explain some of the most interesting aspects of hormone action,

namely: (1) The time lag between administration of the hormone and

the appearance of its physiological effects, and (2) The amazing

variety of these effects (Karlson and Sekeris-1966). Karlson's

..

5.

hypothesis has been further substantiated by the finding that the

physiological effect of some hormones can be blocked by the action

of actinomycin D, an inhibitor of RNA synthesis, or by puromycin, an

inhibitor of protein synthesis (Davidson-1965, Tata-1966, 1967,

Means &Hamilton-1966, Zalokar-1967, Segal., Davidson &Wada-1966,,

Fanestil & Edelman-1966, Sharp & Leaf-1968, De Weer & Crabbe-1968,,

Crabbe-1968, Ludens, Hook & Williamson-1967, Fimognari, Fanestil &

Edelman-1967).

Although numerous examples of hormonal regulation of RNA and

protein synthesis have been described in the literature, it may be

more informative to study a few selected examples in depth, rather

than to gloss lightly over many.

Thyroxin is one of several hormones that is secreted by the

thyroid gland. The principal physiological effects of this hormone

are the stimulation of the basal metabolic rate and growth (Tata-

1965b). Long before these rather gross physiological effects are

observed, there is a sequential series of profound biochemical

events. Within three hours following the administration of tri-

iodothyronine to thyriodectomized rats, there is an increased

synthes.is of the rapidly labeled nuclear RNA in the liver (Tata &

Widnell-1966). By eight hours, there is a specific stimulation in

the synthesis of a type of RNA which has been characterized as

being very much like DNA, and is thought to possibly represent

messenger RNA. Following the early events that leand to the

stimulation of RNA synthesis, there is a considerable time delay.

An increase in the rate of incorporation of amino acids into

6.

mitochondrial and ribosomal protein is not observed until 27 hours

after the hormone injection. Once there is a stimulation in protein

synthesis, there is a marded increase in the synthesis of mito-

chondrial cytochrome oxidase, as well as the microsomal glucose-6-

phosphatase (Tata-l965b) occurs. Subsequent to the stimulated

synthesis of these enzymes, there is an elevated basal metabolic

rate, at 35 hours, that increases to a maximum at about 50 hours

(Tata et al.-l963). It is not until at least 54 hours after the

injection of the hormone, that there is any detectable increase in

the weight of the liver, suggesting synthesis (Tata-l965b). This

sequence of biochemical events is in accord with Karlson's

original hypothesis that hormones elicit their physiological action

by an initial regulation of RNA synthesis.

Cortisone, a steroid hormone produced by the adrenal cortex, is

interesting because of the wide variety of physiological effects

that it elicits. The earliest documented effect of cortiB~ne is an

inhibition of l4C-glycine incorporation into RNA and protein in

lymphocytes and lymphoid tissues (Blecher &White-l957, Feigelson &

Feigelson-l966a). This result was observed as early as 30 minutes

following the injection of the hormone. Cortisone also stimulates

the synthesis of hepatic RNA, an effect observed two hours after

injection (Feigelson, Feigelson & Greengard-l962, Feigelson &

Feigelson-l966a). There is a continued stimulation of RNA and a

subsequent increase in protein synthesis in the liver for three to

four hours (Feigelson, Gross &Feigelson-l962). By five hours,

there is a stimulation in the activity of the enzyme tryptophane

7·

pyrrolase (Thomson & Mikuta -1954). These are the earliest documented

biochemical effects of cortisone. Only after RNA and protein syn-

thesis have been altered, are there any changes in. the dermal concen-

trations of glucosamine, insoluble collagen and schleroprotein.

These changes are noticable at about 24 hours (Houck-1962). The

general catabolic activity of cortisone is reflected by an increase

in the excretion of urinary nitrogen at 48 hours (Clarke-1953). As

an anti-inflammatory steroid, the action of cortisone in reducing

inflammation, is not observable for at least four days (Singer &

Borman-1956). All of these physiological effects of cortisone are

observed long after the earliest alterations in RNA synthesis.

Estradiol is a steroid hormone produced by the ovarial

follicles. Other than the physiological actions of estradiol, which

are many and varied (Davidson-1965, Talwar-1968), the interest in

this hormone relates to the fact that within two minutes after

injection, there is a definite stimulation in nuclear RNA synthesis

in the uterus (Means & Hamilton-1966, Hamilton-1968). Without a

doubt, estradiol stimulates RNA synthesis earlier than any other

hormone reported. The first type of RNA to be stimulated by

estradiol has a base composition similar to that of ribosomal RNA

(Hamilton, Widnell & Tata-1968, Hamilton-1968). Subsequent to this

early action , there is also the stimulation of a second type of

RNA that has a base composition similar to that of the DNA

(Hamilton, Widnell & Tata-1968, Hamilton-1968). These very early

stimulations in RNA synthesis are followed at three hours by the

increased activity of the amino acid activating enzymes in the

unerus (Mcorquodale &Mueller-1958). Actual increase in the

general metabolic activity of the uterus is found from 6 to 16

hours after the injection. This is concomltently followed by an

increased mitotic activity and proliferation of the vaginal

epithelium (Martin-196o, Martin & Claringbold-1963). The action of

estradiol is not limited to the uterus. Within one to four hours

after administration, there is an increased synthesis of liver

phospholipids (Talwar-1963). AlSO, hepatic 5-aminolevulinic acid

synthetase and dehydrase activity are also known to oscillate

following a single injection of estradiol.

The hypothesis that gene-activation is an early event in the

action of some hormones is also based upon more than just the

time relationship between hormone injection, RNA synthesis and

physiological effect. Baker and Warren (1966) have shown that

estradiol stimulates the capacity of uterine chromatin to act as

a template for DNA-dependent RNA polymerase, when the hormone is

injected in vivo. After the in vivo injection of testosterone,

it has been possible to demonstrate that the priming efficency of

chromatin, isolated from rat skeletal muscle is increased (Bruer &

Florini-1966). Liao, Barton, and Lin (1966) have shown a similar

effect upon injecting testosterone propionate and isolating

prostatic chromatin. A number of workers have also shown that

cortisol, administered in vivo, stimulates RNA synthesis in vitro

(Kenney & KUll-1963, Drews & Bondy-1966, Dukes, Sekeris & Schmid-

1966).

8.

9·

Cortisol administered in vitro, increases template activity of rat

liver chromatin by 10-12 percent, however the concentration of

hormone required to elicit this response is quite high (Hahmus &

Bonner-1965, Stackhouse, Chetsanga & Tan-1968).

Taken together, it would seem that this evidence suggests

that certain hormones bring about their physiological responses

by activating specific genes.

D. The Proposed Mechanism of Action of Aldosterone

A number of literature reviews on the biochemistry and

physiology of aldosterone have been published (Laragh-1960, Laragh &

Kelly-1964, Heinz-1967, Sharp & Leaf-1966, 1968). The literature to

be discussed below describes those aspects of the action of aldo-

sterone that are most closely related to the research presented

in this dissertation.

Aldosterone is primarily a mineralocorticoid, that is, it

regulates mineral metabolism. The most profound effect of aldosterone

is on the kidney, however responses have also been observed in such

tissues as the salivary and sweat glands, striated muscle, bone,

the gastrointestinal tract and vascular smooth muscle (Mulorw-

~957). In addition, aldosterone alters electrolyte movement in

huraan erythrocytes (Friedman & Friedman-1958), in rat brain

(Woodbury & Koch-1957) and in human laryngeal carcinoma cells

cultured in vitro (Richards et al.-1966).

In 1961, Crabb~ demonstrated the effect of aldosterone on

sodium transport in isolated toad bladder. The synthesis of RNA

and of protein have also been shown to increase following the

10.

accumulation of tritiated aldosterone in toad bladder nuclei

(Edelman, Bogoroch &Porter-1963, 1964, Porter, Bogoroch & Edelamn-

1964, Fanestil & Edelman-1966). Edelman et ale (1963) have further

shown that the action of aldosterone on sodium transport was

decreased when toad bladders were pretreated with actinomycin D

or puromycin. Ludens, Hook and Williamson (1967) have shown that

actinomycin D inhibits the action of aldosterone only if the tissue

is exposed to the antibiotic prior to the administration of aldosterone.

These data were interpreted to mean that RNA and protein synthesis

must preceed the effect of aldosterone on sodium transport, and are

not secondary to intracellular sodium concentration changes (DeWeer &

Crabb~-1968). These results showed that all types of RNA were

stimulated by aldosterone; however there was the suggestion of a

new species of RNA, not present in the controls. The authors clearly

say that their data was only suggestive.

In the toad bladder, there are definite metabolic requirements

for the active transport of sodium, as stimulated by aldosterone.

The effect is demonstrable only if pyruvate, or compounds which

yield pyruvate in the course of metabolism, are made available to

the bladder (Sharp & Leaf-1965, Kirshberger et al.-1968). Further,

Kirsten et ale (1968) have shown that the activities of the tri-

carboxylic acid cycle enzymes are increased following the addition

of aldosterone. The increase in the magnitude of the enzymatic

activity was correlated with the increase in sodium transport.

Studies with the toad bladder indicate some very interesting

11.

aspects of the mechanism of action of aldosterone, however it is

frequently difficult to extrapolate from the amphibian to the mammal.

A good example of this is seen in a comparison of the differences

between the toad bladder and the rat kidney. In the bladder, there

is good evidence that there are "physiological receptor sites" for

aldosterone (Ausiello & Sharp-1968). Thus, it is possible to carry

out in vitro with the tissue. This is not the case for mammals,

where aldosterone must be injected in vivo and the effect on RNA

synthesis then assayed in vitro. Certainly this is a significant

difference between the two systems. The exact mechanism of how

aldosterone gets into a mammalian cell is not yet understood. It

is known that aldosterone is bound to a specific globular protein

in the blood (Herman, Fimognari & Edelman-1968). Aldosterone may

be transported into cells in this bound form, or by some other

means. The evidence is not yet definitive that aldosterone, itself,

actually interacts with the DNA, to stimulate renal RNA synthesis.

Fanestil & Edelman (1966b) claim that tritiated aldosterone was

found in, or on the nuclei of target tissues in rats. Hollander

et al. (1966) reported that tritiated aldosterone was preferentially

localized along the cell membranes of target tissues in dogs. It is

obvious that there is a conflict on this matter, and until more

definitive research is carried out, the localization of aldosterone

shall remain an uncertainty. Very recently Stumph (1969) has

discussed the evidence that 3H-estradiol actually is localized within

the nucleus. He concluded that the evidence was insufficient to

12.

conclusively say that estradiol actually gets into the nucleus or

the cells of the uterus. The literature is both insufficient and

conflicting.

The action of aldosterone on sodium transport in the kidney

is well established (Barger, Berlin & Tulenko-1958, Gangong &

Mulrow-1958, Ross et al.-1959, Sonnenblick, Cannon & Laragh-196l,

Thorn et al.-196l). In this organ, aldosterone stimulates sodium

resorption at the distal part of the nephron (Vander et al.-1958,

Vander, Wilde &Marvin-1960). There is a time lag of 30 minutes to

two hours after aldosterone injection, before an effect on sodium

resorption occurs, and this response has been shown to last for

six to eight hours (Barger, Berlin & Tulenko-1958, Ganong & Mulrow-

1958, Ross et al.-1959, Sonnenblick et al.-l96l). The physiological

action (sodium transport) is preceeded by a stimulation in the

synthesis of RNA (Castles & Williamson-1965, 1967, Fimognari, Fanestil

and Edelman-1967). Recently Forte and Landon (1968) have shown that

aldosterone stimulates the in vivo synthesis of all species of renal

RNA. These findings are similar to those of Greenman, Wicks and

Kenney (1965) who found that cortisol enhanced not only the synthesis

of a DNA-like RNA, but also stimulated ribosomal and transfer RNA.

It is not clear how these hormones can produce a specific response if

all species of RNA are stimulated uniformly.

E. Aldosterone Antagonism by Aldactone

Aldactone (SC 9420 or spironolactone) was developed as one of

a series of l7-spironolactones that possessed anti-mineralocorticoid

activity (Kagawa, Cella & Van Arman-1957, Kagawa, Sturtevant &

~.

Van Arman-1959). This steroid has the property of reversibly

blocking the mineralocorticoid action of aldosterone, deoxycortico-

sterone and cortisol on the kidneys of rats. Aldactone does not

alter mineral metabolism in the absence of the adrenal glands or

exogenous corticosteroids and does not produce any other classical

endocrine effects of steroids (Kagawa, Sturtevant, & Van Arman-

1959, Liddle-1957). Given alone, Aldactone is a very weak diruetic,

and its action is presumed to be limited by the extent to which

aldosterone is responsible for the condition being treated (Gaunt,

Chart & Renzi-1965). The work of Drill (1962) has shown that

Aldactone also acts on the distal renal tubles.

The action of Aldactone is quite specific; however it has

been shown that there is a compensatory hypersecretion of aldosterone

in rats (Singer-1959) and in man (Davidson et al.-196l), and thus

the use of Aldactone tends to be somewhat self limiting. While

Aldactone can block the renal actions of cortisol in rats (Kagawa,

Sturtevant & Van Arman-1959) and in man (Mills et al.-196l, 1962),

it is interesting that the drug does not possess any anti-inflammatory

activity, nor does it block such action of other steroids such as

cortisol (Drill-1962).

Goodman and Gilman (1965) have reviewed the evidence that

Aldactone is a competitive hinhbitor of the renal actions of

aldosterone. The evidence is based entirely upon in vivo experiments.

These results have shown that the antagonism by Aldactone can be

overcome if one increases the amount of exogenous aldosterone

administered. Using the isolated toad bladder, Porter (1968) has

14.

able to show from a kinetic analysis that Aldactone specifically and

reversibly inhibits the action of aldosterone in active sodium

transport. This research presupposes that there is a direct

competitive inhibition of aldosterone by Aldactone, and the research

was designed to prove this point. No one has looked for any effect

of Aldactone on RNA synthesis, in renal or other tissues.

F. Lymphoid Tissues and Anti-Inflammatory Steroids

The adrenal corticosteroids are generally classified as either

mineralocorticoids or as glucocorticoids, but frequently show some

overlap in the physiological responses that they elicit. The

relative potency of corticosterone, deoxycorticosterone and aldo-

sterone as mineralocorticoids is quite different (Russell-1965).

Cortisol, cortisone, corticosterone and aldosterone all show some

glucocorticoid activity, in that they all increase glycogen

deposition in the liver (Russell-1965). Abundant evidence, obtained

from both clinical and laboratory experience, has established the

role of a number of steroid hormones in combating inflammation

"from infection (Dass & Finland-1953,1957, Thomas-1952, Kruskemper-

1968).

Lymphatic involution is well known response to certain

adrenal hormones. Involution of lymphoid tissues by adrenal corti-

costeroids was described some 25 years ago (Dougherty &White-

1944). Santisteban and Dougherty (1954) observed lymphoid in-

volution following injections of different adrenal steroids.

Weaver (1955) reported that the amninistration of both steroid

hormones (cortisone, estrogen and testosterone) as well as

15.

non-steroidal hormones (ACTH or thyroid extract) caused involution

in rats. Mendelson and Finland (1966) found that the average

weight of the spleen in cortisol treated mice was about half that

of the spleen in control animals.

In addition to the involution of the lymphoid tissues, it

has been shown that certain hormones inhibit both DNA and RNA

synthesis. The incorporation of tritiated thymidine into the DNA

of the thymus and spleen was significantly decreased in rats treated

with cortisol (Knutson & Lundin-1966). The prolonged treatment of

mice with cortisol or ACTH decreased the incorporation of labeled

nucleotides into the nucleic acids of the lymph nodes, thymus and

spleen (Brinck Johnsen & Dougherty-1965). Stevens et ale (1965)

demonstrated that DNA synthesis was inhibited in the same tissues

of rats injected with cortisol. In his work with isolated rabbit

lymph node cells, Kidson (1965) found rapid changes in the RNA

synthesized following the administration of cortisol. The synthesis

of RNA and DNA was inhibited 37 and 35 percent, respectively, in

thymocytes isolated from rats one hour after an injection of cortisol

(Makman 1 Dvorkin & White-1966). Also, it has been reported that DNA

synthesis and mitosis are suppressed for a period of six to eight

hours in the lymph tissues of rats given a.single injection of

cortisol (Dougherty et al.-1964). These findings are of particular

interest because steroids are rapidly metabolized following their

injection (Dougherty et al.-1964).

Although aldosterone has been studied extensively as a mineralo-

corticoid, its action on the lymphoid system has received little

16.

attention (Gaunt, Renzi & Chart-1955). Mach and co-workers (1954)

observed evidence of anti-inflammatory action in an Addisonian

patient treated with aldosterone. However, unlike cortisone, the

local application of aldosterone does not inhibit the formation of

granuloma tissue around a subcutaneous cotton pellet (Desaulles,

Schuler &Meier-1965).

G. In Vitro Synthesis of RNA

With the development of a technique for the isolation of

enzymatically active nuclei from mammalian tissues, it has been

possible to study the DNA-dependent synthesis of RNA (Widnell &

Tata-1964). These authors have described two RNA polymerase reactions,++

one activated by Mg++, while the second is activated by Mn and

ammonium sulfate (Widnell & Tata-1966). The primary difference

between these two reactions appears to be the fact that the RNA

products are different, and depends upon which metal ion is used

to activate the process. When the enzyme is activated by Mg++,

the synthesized RNA was similar in base composition to ribosomal

RNA (Widnell &Tata-1968). These same studies have also shown

that the Mn++-(NH4)2S04 activated RNA polymerase synthesizes an

RNA that has a base composition similar to that of the DNA

(Blackburn & Klemperer-1967, Chambon et al.-1968).++

The confusing aspect of this work is the fact that the Mg

activated RNA polymerase synthesizes only a single product that

has a sedimentation value of 4 to 6 s (Chipchase & Birnstiel-1963, Monjardino &MacGillivary-1968, Wicks, Greenman & Kenney-

1965). In an attempt to clarify just what kind of RNA is synthesized,

17.

Mangan, Neal and Williams (1968) studied the product in detail and

found, just as others have, that the newly synthesized RNA was

comparable in size to transfer RNA, but possessed a base composition

comparable to that of ribosomal RNA. Moriyama (1968) has found that

this type of RNA was closely associated with the nucleolus. Thus,

the suggestion was made that this type of RNA may possibly be the

nuclear precursor of the cytoplasmic ribosomal RNA.

From this brief literature review, it should be apparent that

a number of problems exist. Therefore the research described here

was designed to answer several basic questions: (1) What is the

time course for the action of aldosterone on renal RNA synthesis?

(2) Does aldosterone affect RNA synthesis in such non-target tissues

as the brain, thymus and spleen? (3) What is the effect of adrenal-

ectomy or hypophysectomy on the response to an injection of aldo-

sterone? (5) Is it possible to detect the synthesis of any specific

species of RNA, as the result of an injection of aldosterone?

It is felt that these are all basic questions that are unanswered in

the existing literature.

18.

MATERIALS AND METHODS

A. Materials Obtained Commercially

Chromatographic standard (+) aldosterone, cortisol, progesterone,

estradiol, UTP, GTP, ATP, CTP, phosphoenolpyruvate, pyruvate kinase,

yeast soluble ribonucleic acid and Cleland's reagent (dithiothreitol)

were obtained from CalBiochem Corp. Adenosine-8-14c-triphosPhate

(17.6 mc/mM) was purchased from Nuclear-Chicago Co. Bovine pancreatic

ribonuclease A (EC 2.7.7.16) and electrophoretically purified deoxy-

ribonuclease I (EC 3.1.4.5) were obtained from Worthington Biochemical

Corp. Aldactone (SC 9420) was purchased from Searl Chemicals. Deoxy~

corticosterone acetate, corticosterone and testosterone were obtained

from Mann Research Laboratories. Packard Instrument Co. supplied

2,5-diphenoyloxazole (PPO) and 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-

benzene (dimethyl POPOPO). Millipore filters (HAWP-025) were

purchased from the Millipore Corp. other chemicals were reagent grade

and were used as such, except where noted.

B. Prepared Materials - Solutions

1.

2.

Solution A:

Solution B:

0.32 M sucrose, 3 roM MgC12, 0.1 roM di-

thiothreitol.

2.4 M Sucrose, 1 roM MgC12, 0.1 roM di-

thiothreitol

3. Solution C: 0.25 M sucrose, 1 roM MgC12, 0.1 roM di-

thiothreitol.

4. Solution D:. Scintillation solvent, 5 gil PPO, 0.3 gil

dimethyl POPOP in toluene (Hayes-1956).

19·

C. Special preparation of Reagents

1. Phenol - Loose crystals of phenol were redistilled under

a stream of nitrogen, in an all glass apparatus. To the re-

distilled phenol was added sufficient glass distilled water

to saturate at room temperature. Fresh phenol solutions, not

more than two weeks old, were used for all RNA extractions.

2. Sodium Dodecylsulfate SDS was purchased from Sigma

Chemical Co. and was recrystalized according to the method of

Dingman and Sporn (1962). The SDS was disolved in boiling 96%

ethanol. The hot solution was then filtered without suction.

The volume of ethanol was slightly reduced and allowed to

stand at room temperature until crystals formed. The flakes

of SDS were then collected by suction, on a filter paper? washed

with 25 to 50 ml of diethyl ether and dried in an evacuated

dessicator. The SDS was stored in a brown bottle under

nitrogen.

3. Sucrose - Gradients were prepared exclusively from

reagent grade Merck sucrose. This was the only brand of sucrose

that did not have a strong U.V. absorbance.

D. Animals

Male rats ranging in body weight from 120 to 170 g were used

in all experiments. The animals were obtained from two sources:

(1) Wistar strain, University of Hawaii colony, and (2) Sprague-

Dawley strain, BioScience Animal Laboratories, Oakland, Calif. The

Sprague-Dawley rats were air-freighted to Hawaii and were maintained

in our laboratory for a period of five to seven days preceeding the

20.

experiments. In the studies to be described here, no differences

were observed between the two strains of rats.

The rats were maintained on Purina Laboratory Chow and tap

water, ad libitum, except as noted. As a routine procedure, food,

but not water, was taken from the rats 18 to 20 hours prior to the

experiments, to minimize the variability in food intake among the

rats. The adrenalectomized rats had free access to 1% saline and

tap water, and the hypophysectomized rats were given 1% saline, 10%

glucose and tap water.

In some preliminary experiments, rats were either adrenalectom-

ized or hypophysectomized in this laboratory. With the development

of these techniques, it was possible to observe such facets as the

endocrinectomized rats as fluid intake, weight gain or loss, coat

texture and the appearance of the internal anatomy. The results of

this work are not presented here, but served as a basis for in-

specting animals that were purchased. The results of all endo-

crinectomized rats were based upon animals operated on by BioScience

Laboratories. The adrenalectomies were performed through a single,

dorsal midline incision and the hypophysectomies by means of the

intra-aural route. All operations were done four to seven days

before shipment to Hawaii. During the five to seven days that the

rats were kept in our laboratory, they were observed for changes in

body weight, coat texture, alertness, appetite and fluid intake. At

the time that the rats were killed, all were inspected to determine

if the adrenalectomy or the hypophysectomy was complete. If there

21.

was any question concering the completeness of the operation, the

animal was discarded.

E. Experimental Treatments All experiments were started at

6:30 to 7:00 A.M.

During the course of the work, the animals were injected with

a number of different steroids. A summary of the steroids, dosages,

and vehicles is listed in Table I. Except for Aldactone, the

steroids were first disolved in a small volume of 95% ethanol. The

steroid-ethanol solution was then diluted to the desired concentration

with 1% saline. Because of the insolubility of Aldactone in saline

solutions, sesame oil was used as the vehicle.

Table I. Hormones, Dosages and Vehicles.

Steroid f.J.g/animal Vehicle

Aldosterone 0.07 0.1% ethanol in 1% saline0.1 " " " " "2.5 2.5% " " " "

Corticosterone 2.5 5.0% " " " "Progesterone 113 " " " " "Estradiol 117 " " " " "Cortisol 122 " " " " "Deoxycorticosterone 125 " " " " "

acetateTestosterone 132 " " " " "Aldactone 100 Sesame oil

Steroids, except Aldactone, were injected in a volume of 0.5 ml.

Aldactone was injected in a volume of 0.1 ml. Throughout this

research, injections were made via three different routes: sub-

cutaneous, intraperitoneal andintravenous in the tail vein.

22.

For every time interval, there was a control group and a

hormone treated group of 3 to 5 rats each. Zero time was taken as

the time of the injection, and all rats were thereafter, individually

timed until they were killed. In this manner, all rats were subjected

to a specified time of steroid action, ~O.5 min. The animals were

killed by neck fracture. The desired tissue or tissues were immediate-

ly removed and placed in ice-cold Solution A (homogenizing medium),

as described below.

F. Protocol tor Multiple Injections

In one series of related experiments, Aldactone and aldosterone,

or their vehicles, were injected into the same animals. In this

work, the primary tissues studied were the kidney and the spleen. The

thymus was also studied at every time interval.

As will be described in more detail later, the primary objective

here was to determine if the effects of aldosterone and Aldactone on

RNA synthesis were independent. Thus, the timing of the injections

was such that the response to each steroid was maximal at the time

that the rats were killed. The injection of oil or Aldactone was

subcutaneous, while that of the saline or aldosterone was intravenous.

Four groups of rats were injected per experiment, such that the

following combinations resulted: oil-saline, oil-aldosterone,

Aldactone-saline, and Aldactone-aldosterone. In this way, it was

possible t~ make several comparisons.

G. Isolation of Nuclei

At various stages of the research, nuclei were isolated from

five different tissues: the brain (cerebral hemispheres), liver

23·

(all lobes), kidneys, spleen and thymus. The kidneys or other

tissues from the hormone-treated animals were pooled, and their

nuclei isolated essentially according to the method of Widnell and

Tata (1964). The same procedure was used for control animals.

After the tissues were excised, all steps in the isolation

of the nuclei were carried out at 0 to 40 • The pooled tissues were

weighed, minced with scissors and rinced once with solution A.

One part of tissue was diluted with three volumes of solution A

and homogenized in a glass Potter-Elvehjem tissue grinder.

Homogenization of the tissue was accomplished with 12 slow up-and-

down movements with a mechanically driven Teflon pestle, turning at

a moderate speed. The homogenate was filtered through a double

layer of cheese cloth to remove connective tissue and clumps of un-

broken cells. Aliquots, 12.5 ml, of the homogenate were diluted

to 20 ml with Solution A, and then further diluted to a final

sucrose concentration of 0.25 M, with distilled water. Solution A,

15 ml was layered below, and the crude nuclear pellet was obtained

by centrifugation at 700 g for 10 min. The crude nuclear pellet

was obtained in solution by resuspension in Solution B. A purified

nuclear pellet was obtained by centrifugation of the above suspension

for one hour at 50,000 g. Whole cells, erythrocytes, mitochondria

and general cell debris floated to the top of the centrifuge tube,

where they could be easily removed with a spatula. The purified

nuclear pellet was resuspended in Solution C. Periodically, the

resuspended nuclei were checked for cellular comtamination by means

of an oil-emmersion light microscope.

24.

H. Assay for Nuclear RNA Synthesis

The RNA polymerase activity of the isolated nuclei was

determined by the procedure described by Widnell and Tata (1964),

and Hamilton, Widnell and Tata (1965), with minor modifications.

The resuspended nuclei were incubated in the polymerase assay

medium shown in Table II.

Table II. RNA Polymerase Assay Mixture

Quantity/O.5 mlComponent final volume

Tris-HCl buffer (pH 8.5)MgC12DithiothreitolNaFCTPUTPG'l'Pl4C-ATPPEPPyruvate kinaseResuspended nuclei (0.2-1.0 mg DNA)

50 j.lmoles2.5 "0.44 "3.0 "0.3 "0.3 "0.3 "0.01 "5.0 "

10.0 "0.1 ml

After incubation in a Dubnoff Metabolic Shaking Incubator

for 15 minutes at 370 , the reaction was terminated by the addition

of 4.0 ml of ice-cold 0.5 N HC104. The precipitate from the

incubations was collected by centrifugation in a clinical centrifuge,

washed once with 0.2 N HC104 followed by a mixture of ethanol:ether

(3:1 v/v), and was allowed to dry in the cold.

The l4C-labeled RNA was extracted from the washed and dried

precipitate by two successive treatments with 4.0 ml of 10% NaCl

containing 0.125 mg of carrier RNA/ml. The extractions were

25·

performed at 1000 for 30 min. RNA from the combined extractions was

precipitated by the addition of 5.0 ml of ice-cold 20% trichloro-

acetic acid,. and allowed to stand on ice for at least one hour. The

precipitated RNA was collected on a Millipore filter (0.45 ~ pore

size), washed With 2.0 ml of ice-cold 5% trichloroactic acid and air

dried at room temperature.

I. Measurement of Radioactivity

The air dried filters containing the 14c-labeled RNA precipitate

were placed in counting vials with 10 ml of scintillation solvent,

Solution D. Vials were assayed for radioactivity in a Packard

Tricarb. model 3003 liquid scintillation spectrophotometer.

Correction for non-specific binding of 14C_ATP to the RNA was

determined by stopping the lli~A polymerase reaction immediately after

the addition of the incubation medium to the nuclei. The counts

obtained with this zero time incubation were substracted from the

counts incorporated during a 15 min. incubation. The RNA polymerase

activity was determined in quadruplicate for each experimental point,

for both the control and the hormone treated groups of rats.

J. DNA Analysis

Nucleic acids were extracted from 0.1 ml aliquots of the

resuspended nuclei by two successive treatments at 750 with 0.2 N

HC104' The combined extracts were assayed for DNA by the diphenyl-

amine reaction described by Dische (1955). Calf thymus DNA was

used as the standard. All DNA determinations were performed in

triplicate.

26.

K. Expression of Results

RNA synthes:l.s was expressed as counts per minute of l4C_ATP

incorporated into the acid insoluble precipitate per mg of DNA added

to the reaction mixture. At each time interval following injection,

both a control group and a hormone treated group of rats were

studied. The incorporation of l4C_ATP in the control group was

defined as 100%, that of the hormone treated group was expressed

as percent of the control. The confidence limits were calculated

for the 95% level of significance of the standard error of the mean J

as described by Richmond (1964).

L. RNA Extraction and Analysis

RNA from Isolated Nuclei The extraction of RNA was

accomplished by a combination of the methods described by Sporn

and Dingman (1962), Peacock and Dingman (1967) and Blackburn and

Kemperer (1967). The scheme for the isolation of nuclear RNA is

outlined in Table III. In summary, the RNA extraction involved

breaking of the nuclear membrane with purified SDS in saline,

followed by repeated extractions with redistilled phenol, and finally

precipitation of the RNA with 95% ethanol at -150 • The steps

were carried out entirely in glass centrifuge tubes (Corex). This

minimized the number of solution transfers and reduced the loss of

RNA.

After the final precipitation, the RNA was disolved in 0.1 N

saline, to give a desired volume or concentration of RNA. Analysis

of the biosynthesized product is described below.

27·

Table III. Scheme employed for the isolation of RNA from

resuspended nuclei.

10 min.l

Discardphenol

Aqueous layerI

Make 0.2 M inNaCl, add 1/2vol. phenolI5 min.

ethanol,at -150

~Discardsupernatant

Shake

11 CentrifugeAqueous layer

jAdd 2.5 vol.store 8 hrs.Centrifuge 15 min. ------l

RNA ppt.

1Disolve in0.1 N NaCl

Re-precipitate withethanol, at -150

!Centrifuge 15 min. ---I

RNA ppt.~

Disolve in0.1 N NaCl

~Discardsupernatant.

Resuspended Nuclei

1Add 10 ml of 0.8%

SDS in 1% NaCl

Shake for 30 sec.

1Add 10 ml of phenolr--------- Centrifuge 10 min... 30,000 g

Phenol layer.~

Re-extract with 10 mlof 0.8% SDS in 1% NaCl

'1Centrifuge 10 min.Discard phenol

28.

RNA from Incubated Nuclei - The procedure was that described

for extraction of RNA from isolated nuclei. For this work, the RNA

polymerase reaction mixture was increased from 0.5 to 10 ml, total

incubation volume. The incubation of the nuclei was carried out in

the 30 ml Corex centrifuge tubes. The reaction was terminated by

the addition of 10 ml of 0.8% SDS in 1% NaCl. Thus, the RNA

extractions were accomplished without unnecessary transfers.

Analysis of Extracted RNA Phenol extracted RNA samples

were centrifuged in linear gradients of 5 to 20% (w/w) sucrose in

0.1 N NaCl, 0.01 M sodium acetate, pH 5.0. A volume of 0.5 ml of

50% sucrose was placed in the bottom of each tube. From pre-

liminary work, it was established that centrifugation for eight

hours at 40,000 rpm resolved the major species of nuclear RNA.

Centrifugation for 36 hours at 40,000 rpm was required to adequately

move the low molecular weight RNA. During the deceleration

period, the centrifuge was not braked.

Phenol extraction of RNA was considered to be acceptable when

the 260/280 ~ optical density ratio was 2.0.0.1. An o. D.260/280 ~

within these limits was assumed to represent an RNA concentration

- of 1 mg/ml per 20 optical density units, as described by Sporn and

Dingman (1964). In several experiments, the phenol extracted RNA

was assayed for DNA, as previously described. In all cases, it was

found that contamination of the RNA by DNA was consistently less

than 1%, and usually less than 0.5%.

Determination of the approximate s values for the various

species of RNA was based upon a comparison with catalase (11.3 s)

and yeast soluble RNA (4.1 s). These reference compounds were

centrifuged simultaneously, in separate tubes. The movement of

the phenol-extracted RNA was compared, relative to that of the

!mown compounds.

Sucrose gradients were analyzed on an ISCO analyzer, by

pumping 50% sucrose into the bottom of the tube and forcing thegradient out through the top of the tube. The material was then

passed through a U.V. analyzer that continuously monitered the

optical density at 254 IJlIl. Individual fractions were then collected.

The optical density (260 IJlIl) of the individual fractions was read

on a Beckman D.U. spectrophotometer.

Subsequently, the individual fractions from some of the

gradients were analyzed further by placing them in counting vials

and dehydrating them in a drying oven, at about 800

• Ten milliliters

of scintillation solvent (Solution D) were then added and the

fractions were monitered in the liquid scintillation counter.

RESULTS AND DISCUSSION

A. In Vitro Synthesis of RNA by isolated Nuclei.

With the development of a method to isolate metabolically

active nuclei from mammalian tissues (Widnell & Tata-1964), it was

possible to measure incorporation of radioactively lebeled nucleo-

side triphosphates into an acid insoluble product that has been

characterized as RNA. Table IV shows the results of an experiment

in which nuclei from rat brain, kidney, thymus and spleen were

incubated in the RNA polymerase reaction mixture (see Methods),

and the incorporation of l4C_ATP into an acid insoluble product

was measured.

It may be seen that isolated nuclei synthesize RNA upon in-

cubation. This is evidenced by the requirement that all four

nucleoside triphosphates be present, and by the sensitivity of the

synthesized product to the action of ribonuclease. The omission of

anyone of the individual nucleotides from the reaction mixture

resulted in a considerable reduction of l4C_ATP incorporation in

all of the tissues. The absence of three nucleotides from the

polymerase mixture resulted in a further reduction in the extent of

l4C-ATP incorporation. Following an initial 15 minute incubation

of the nuclei in the mixture, the addition of ribonuclease resulted

in the loss of most of the recoverable radioactivity. These

findings are consistent with reports by others who have assayed

RNA polymerase activity in nuclei isolated from the liver

(Widnell &Tata-1964, 1965, Blackburn &Klemperer-1967), brain

30.

Table 4.

31.

Incorporation of 14C_ATP into nuclear preparations from

from brain, kidney, thymus and spleen.

Acid Insoluble Nucleotide(counts/min./ mg DNA)

RNA PolymeraseReaction Mixture Brain Kidney Thymus Spleen

Completepolymerase mixture 1890 716 565 437

Complete plusRNase (100 J..Lg) 18.0 42.3 34.6 36.6

Minus GTP,22.1 5.6 26.0 2·7UTP, CTP

Minus GTP 79.4 25.6 41.7 28·9

Minus UTP 101.5 27.6 33.5 30.0

Minus CTP 88.0 30·9 55·5 31.8

Nuclei were isolated from homogenized tissues i~cised from 12 ratsand assayed for the in vitro incorporation of C-ATP into RNA,as described in Methods. The incubation conditions were modifiedas shown above. The RNase treatment was carried out for 10 minuteson ice, after the initial 15 minute incubation in the completepolymerase wixture. The RNA synthesis is expressed as counts perminute of 1 C-ATP incorporated into the acid insoluble precipitateper mg of DNA added to the reaction mixture. The values I~recorrected for background and for non-specific binding of C-ATPfound in unincubated controls.

(Dutton & Mahler-1968), and thymus (Nakagawa &White-1967).

The Results shown in Table IV and in Figure 1 clearly in-

dicate that of the four tissues studied, the brain had the most

active RNA synthesis. The RNA synthesis in the brain was approximate-

ly 2.6 times greater than that in the kidney, 3.3 time that in the

thymus and 4.3 times that in the spleen.

32.

A-A 6.\-&-&

Many of the results to be presented in this dissertation are

expressed in terms of counts per minute of l4c_ATP incorporated

into RNA per mg of DNA. Figure 1 demonstrates the'validity of this

method of expressing the results. Varying concentrations of nuclei

(expressed as mg of DNA) were incubated in the reaction mixture, and

it was shown that there was a direct, linear relation between the

l4C_ATP incorporated and the amount of DNA present. Over the range

of 0.05 to 5.0 mg DNA, there was a constant cpm/mg DNA vs mg DNA.

This relationship was true in all four tissues studied. Insufficient

nuclei were isolated from the brain, to assay the highest conc-

entration of nuclei.

34.

B. The Effect of Aldosterone on· the Synthesis of RNA

Aldosterone has been shown to accumulate in cell nuclei of the

isolated toad bladder (Edelman, Bogoroch &Porter-1963). Several

laboratories have demonstrated that an early response to aldosterone

is an increased synthesis of RNA (Crabb~-1961, Edelman Bogoroch &

Porter-1964, Porter, Bogoroch &Edelman-1964, Williams-19G8). Block-

ing RNA synthesis, by the use of actinomycin D, has been found to

prevent the action of aldosterone on the active transport of sodium.

Using the rat as the experimental animal, experiments were

designed to see if aldosterone would have an effect on nuclear RNA

synthesis, and if the kidney was, in fact, the primary target tissue

of this steroid hormone.

The Response of Renal RNA Synthesis to an Intraperitoneal

Injection of Aldosterone Renal RNA synthesis} following an

intraperitoneal injection of 2.5 ~g of. aldosterone into normal rats,

is shown in Figure 2. Although there was an apparent increase in

the RNA synthesis as early as 30 minutes, the first stimulation,

significantly different from the control, occurred at one hours. A

maximum stimulation greater than 130% of the control level, occurred

1.25 to 1.5 hours after the injection. Following the maximum

stimulation, there was a rapid decline, and at 1.75 hours, RNA

synthesis in the hormone-treated rats was 106% that of the control

rats.

Since the RNA synthesis decreased rapidly following the maximum

initial stimulation, it was of interest to see if the action of the

hormone was confined to this initial effect. Figure 2 also shows the

Figure 2. Time course for the effect of a single intraperitonealdose of aldosterone on RNA synthesis in isolated rat kidneynuclei. At time zero, rats were injected intraperitoneally with2.5 ~g of aldosterone. The rats (3 to 5 per group) were killedat the times indicated, and the kidneys were excised ana pooledfor homogenization. The nuclei were isolated and assayed forRNA synthesis as described in Methods. At each time interval,both a control group and a hormone treated group of rats werestudied. The 14C-ATP incorporation into RNA of the controlgroup was defined as 100%, and that of the hormone treatedgroup was calculated as percent of the control. The reproducibilityof the 2.0, 2.5 and 3.0 hour determinations is shown. Theseduplicate determinations were carried out on different days.

W\.II

36.

to

0

~~-I-0IJJ

,.,,-=>Z-0::LLI

NI-L&.

0- «Co

(J)'0 -0::

"-0 :::>0'0

:t:

0 0 0 0 0 0 0V ,." N - 0 0) CD- - - -

10~.LNOO .:10 .LN30~3d

37·

extended time course for the effect of aldosterone. This experiment

demonstrates that following the initial stimulation, the RNA synthesis

was depressed to a level less than that of the control· suggesting

a slight inhibition. Although not significantly different from the

control, the inhibition at two hours was reproducible.

The slight inhibition observed at two hours was followed by

a series of oscillations in RNA synthesis. In the 4.5 hour time

course shown in Figure 2, there occurred three maxima and three

minima. With the exception of the two hour minimum, all were

significantly different from the control. It is of interest to note

that the three maxima were all above the control level of RNA

synthesis, and that the minima were all below the control. The

period of the oscillations varied from 1.0 to 1.5 hours, and there

was no indication of any damping of the oscillations, during the

time period studied.

Using the in vitro assay for the measurement of DNA-dependent

RNA synthesis, this study confirms other reports that the admin-

i~tration of aldosterone resulted in an early stimulation in renal

RNA synthesis (Castles &Williamson-1965, 1967, Fimognari, Fanestil &

Edelman-1967, Forte & Landon-1968).

Oscillations in the renal RNA synthesis following the injection

of aldosterone have not been previously reported. Therefore, it

was of interest to attempt to elucidate some of the factors that

might affect the oscillation.

The Effect of an Intravenous Injection of Aldosterone on

Renal RNA Synthesis It was decided to see if the oscillations

38.

in RNA synthesis were related to the route of administration of the

hormone. Aldosterone (2.5 ~g per rat) was injected into the tail

vein, and the renal RNA synthesis was followed, as a function of time.

Figure 3 shows that an intravenous injection of aldosterone

caused a very rapid stimulation in renal RNA synthesis. Maximum

stimulation, greater than 200% of the control, occurred as early

as 30 minutes following the administration of the hormone, by the

intravenous injection, as compared to a maximal stimulation of 130%

at one hour for the intraperitoneal route. This figure also shows

that after the maximum was reached at 30 minutes, there was a rapid

decrease in the rate of RNA synthesis. At about 1.5 hours, the

synthesis decreased to a level less than that of the control,

indicating an inhibition. Following the initial stimulation, minima

of 72% and 73%, and a maximum of 146% were reached at 2.5, 5.0 and 4.0

hours, respectively. Hence, a single injection of aldosterone

stimulated the renal RNA synthesis, and led to a number of subsequent

oscillations around a control level. The maximum values were above,

and the minimum values were below the control. The period of the

oscillations following the intravenous administration, was about

3.5 hours, for the time period studied.

This demonstrates that nuclear RNA synthesis was stimulated

with the intraperitoneal or intravenous injection of aldosterone.

Therefore, it was possible to show that the route of hormone

administration did alter the observed response in RNA synthesis.

The maximum stimulation resulting from an intravenous injection

occurred earlier, and was of greater magnitude, than that resulting

Figure 3. Time course for the effect of a single intravenousdose of aldosterone on the RNA synthesis in isolated rat kidneynuclei. At time zero, 2.5 ~g of aldosterone was injected into thetail vein. The animals were killed at the times indicated andwere treated as described in Fig. 1.

W\0.

40.

10

/0 z0-0 V I-0LIJ..,Z

0" rt) 0::0 LIJ

} l-LL0 «

/ U)0

0::

0/:::>0

---0' J:0-

000 0 0 0 000N 0 ~ ~ V N 0 ~ Wt\I N - - -

10Y~NOO ~O iN30Y3d

41.

from an intraperitoneal injection. The delayed stimulation following

an intraperitoneal injection may be due to the time required for

absorption of the hormone from the peritoneal cavity. Aldosterone

administered into the peritoneal cavity is absorbed into the blood

were it is carried to the liver and catabolized (Ayeres et al.-1962,

Hollander et al.-1966, Kohler et al.-1964, McCaa & SUlya-1966).

The intravenous injection enabled the hormone to be distributed to

all tissues more rapidly and in greater amounts.

The results of these extended time courses indicated that

after the initial stimulation, there was a rapid decrease followed

by oscillations. It seems unlikely that aldosterone would alternately

stimulate and inhibit the same parameter, as a function of time,

therby causing the observed oscillations in the rate of RNA synthesis.

The half-life of aldosterone in the kidney has been shown to be in

the order of a very vew minutes (Ayeres et al.-1962, Hollander et al.-

1966). In studying the effect of cortisol on lymphatic DNA synthesis,

Dougherty et al. (1964) proposed that the hormone triggers some

cellular event that persisted long after the hormone itself had been

catabolized. Because the effect of aldosterone on RNA synthesis

continued for several hours after the hormone had been metabolized,

it is here proposed that aldosterone, per se, was responsible only

for the initial stimulation in RNA synthesis and that the oscillations

are a consequence of this stimulation. Hamilton et al. (1968) and

Hamilton (1968) found oscillations in the RNA synthesis of the rat

uterus, following the injection of estradiol. These results were

interpreted to mean that estradiol initially stimulated the synthesis

42.

of ribosomal RNA. The subsequent stimulation of RNA synthesis was

possibly due to an increase in the production of messenger RNAs. It

is felt that the oscillations in renal RNA synthesis, described in

this section, are not of the type described by Hamilton (1968). The

oscillations shown here, fluctuate about the control level, their

maxima being above and their minima below the control. In the work

with estradiol, Hamilton et aL (1968) showed that the maxima and

the minima were both obove the level of the control. It is certainly

tempting to suggest that the oscillations described in the present

work may be indicative of the homeostatic changes induced by other

hormones that are required in the regulation of sodium metabolism,

after an injection of aldosterone.

The endocrine system regulates the water and electrolyte

balance through the influence of several hormones. Somatotropin,

corticosterone and cortisol are among those hormones involved, as

are the antidiuretic hormone (ADH), parathyroid hormone, and aldo-

sterone (Koch-1965). Based on studies in neurohypophysectomized-

adrenalectomized rats, ADH and aldosterone tend to have separate

and opposing functions. When ADH and aldosterone are given in

combination, the sodium balance observed is the net effect of these

two opposing stimulii (Friedman et al.-1966). Despite wide variations

in water and electrolyte intake, the extracellular fluid volume and

osmolality are controlled. This control mechanism involves the

complex interaction of the hypothalamus, neurohypophysis, adrenal

cortex, and the kidney (lockett & Roberts-1963, Strong-1966). It

was thought that the oscillations in RNA synthesis, subsequent to the

43.

initial stimulation might be the expression of an interaction of the

pituitary, adrenal and kidney, in the homeostatic regulation of the

electrolyte movement.

The Effect of an Intravenous Injection of Aldosterone on Renal

RNA Synthesis in Adrenalectomized Rats In order to test the

hypothesis that the oscillations in RNA synthesis may be a reflection

of some form of homeostatic control of mineral metabolism, involving

the adrenal and the kidney, aldosterone was administered to adrenal-

ectomized rats, and the RNA synthesis was followed as a function of

time.

The response of the renal RNA synthesis following an intravenous

injection of aldosterone (2.5 ~g) into adrenalectomized rats, is shown

in Figure 4. Time course for the effect of 2.5 ~g of aldosterone on

renal RNA synthesis in intact rats was shown in Fig. 3, and is

reproduced for the purpose of comparison. The administration of

aldosterone into adrenalectomized rats induced an initial stimulation

in the RNA synthesis. As shown in Figure 4, 2.; ~g of aldosterone did

not stimulate RNA synthesis to as great a magnitude as did the same

quantity injected into intact rats. A dosage of 0.07 ~g of aldo-

sterone was also found to initially stimulate RNA synthesis ~n 'che

adrenalectomized rats. The maximum initial stimulation in the intact

and in the adrenalectomized rats occurred at 30 minutes following the

injection.

Others have found that a much smaller dose of aldosterone,

injected into adrenalectomized animals, elicits the same physiological

response as in the normal animals (Barger, Berlin &Tulenko-1958).

44.

Figure 4. Time course for the effect of a single intravenousdose of aldosterone on the RNA synthesis in kidneynuclei isolated from adrenalectomized rats. At time zero,the rats were injected with 0.5 ml of a solution con-taining 2.5 or 0.07 ~g of aldosterone, or the hormonevehicle. The rats were killed at the times indicated andtreated as previously described. The effect of aldo-sterone on renal RNA synthesis in unoperated rats hasbeen previously described, and the time course isincluded here for comparison.

Adrenalectomized, 0.07 ~g

Adrenalectomized, 2.5 ~g

Intact rats, 2.5 ~g

.--eo 0

..J0a:....z0 140 -, \0 I \

I \I \1L

120 I\

0 \, ,I \l- I ,z 100I.LI

(.)-0a:

I.LIQ. 80

60.L--......--.,r---~---.----,r--I 234 5

HOURS AFTER INJECTION

46.

Hence, it is interesting to compare the initial response to aldosterone

in the normal and adrenalectomized rats. In the intact animals, 2.5

~g of aldosterone stimulated renal RNA synthesis to a level greater

than 20Cf{o that of the control. The same dose given to adrenalectomiz-

ed rats produced a response that was only about 150% of the control

level. When the dosage of aldosterone was reduced to 0.07 ~g per

rat, the maximal initial stimulation was subsequently reduced to

about 120% of the control. Therefore, these findings do not support

the in vivo demonstration that aldosterone is more effective in

adrenalectomized, than in intact animals (Barger, Berlin & Tulenko-

1958).

Initially, it was felt that 2.5 ~g of aldosterone might

represent a pharmacological dosage. However, Friedman et al. (1966a,

1966b) injected the same hormone in doses of 5 to 10 ~g/lOOg body

weight, and later it was reported that this was a reasonable maintenance

dosage for adrenalectomized rats (Eilers &Peterson-1964). Fimognari

et ale (1967) injected 2.0 ~g of aldosterone subcutaneously into

adrenalectomized rats and studied the effect of the steroid on renal

RNA and protein synthesis. Forte and Landon (1968) intravenously

injected 22 ~g of aldosterone into adrenalectomized rats and studied

the effect on RNA synthesis. Furthermore, the present findings

indicated that the maximum stimulation in adrenalectomized rats was

concommitantly reduced when the dosage of aldosterone was changed

from 2.5 to 0.07 ~g per rat. Although aldosterone does possess

some weak glucocorticoid activity (Russell-1965), it is unlikely

that the present findings represent such activity. Glucocorticoid

effects are usually measured at large dosages of steroid (Russell-

1965). Also, later in the dissertation it will be shown that an

intravenous injection of 2.5 ~g of corticosterone did not effect

renal RNA synthesis for at least four hours following the injection

(see Fig. 10).

Following the initial stimulation, the pattern of the response

to 2.5 ~g of aldosterone in the adrenalectomized rats was similar

to that in the normal rats. Both responses represent oscillations

in the RNA synthesis. The magnitude of the response in the adrenal-

ectomized rats was less than that in the intact rats, however the

time after injection at which the maxima and minima occurred were

quite similar. The abbreviated time study carried out with the lower

dosage of aldosterone indicated that although the initial maximum

stimulation occurred at 30 minutes following the injection, the

minimum occurred sooner than that for the 2.5 ~g of aldosterone.

The Effect of Aldosterone on Renal RNA Synthesis in

Hypophysectomized Rats Oscillations in renal RNA synthesis were

not greatly altered in the adrenalectomized rats, therefore it was

important to determine if the absence of the pituitary would effect

the oscillations. The effect of 0.1 ~g of aldosterone on the kidney

RNA synthesis in hypophysectomized rats is shown in Figure 5. These

results indicate that there was a maximum stimulation 30 minutes

following the injection. The initial stimulation was followed by

a marked inhibition and a subsequent return to the level of the

control.

When compared to the intact and the adrenalectomized rats, the

48.

Figure 5. Time course for the effect of a single intravenousinjection of 0.1 ~g of aldosterone on renal RNA synthesisin hypophysectomized rats. The animals were killedat the times indicated, and treated as previouslydescribed.

120 0"..J0a:I- 110 0z0u

100l&. 00

I- 90z.IaJua::IaJ 80a..

I 2 3 4HOURS AFTER INJECTION

50.

response to an injection of aldosterone in the hypophysectomized

rats was remarkably similar. There was an initial maximum stimulation

at 30 minutes, followed by a decrease to a level below that of the

control. The meaning of the oscillations induced by aldosterone on

the RNA synthesis is not clear. The oscillations persist in the

intact, adrenalectomized and hypophysectomized rats, and the oscil-

lations appear to be similar in their general profile. This suggests

that the oscillations were not manifestations of the interaction of

the pituitary, adrenals and kidneys. The lack of involvement of the

adrenals or the pituitary suggests that the oscillations in renal

RNA synthesis may not involve electrolyte homeostasis. The results

presented in this section and leater would tend to suggest strongly

that the observed oscillations in RNA synthesis may be a unique

function of the respective tissue studied.

The Effect of an Intravenous Injection of Aldosterone on RNA

Synthesis in the Brain of Intact Rats With the suggestion that

the oscillations in RNA synthesis may not be related to the movement

of sodium, it was decided to see if aldosterone would modify RNA

synthesis in other tissues. It has been shown that aldosterone has

a physiological effect on a variety of tissues other than the kidney.

Woodbury and Koch (1957) have reported that mice treated with

aldosterone for a period of four days showed a decreased sodium and

an increased potassium level in the muscle and brain. For this

reason, it was of interest to see what effect, if any, aldosterone

would have on the rate of RNA synthesis in the rat brain.

The effect of a single intravenous injection of 2.5 ~g of

51.

Figure 6. Time course for the effect of a single intra-venous injection of 2.5 ~g of aldosterone on RNAsynthesis in the rat brain. Animals were killed at thetimes indicated and the cerebral hemispheres weretaken and pooled for homogenization. The isolation ofnuclei and the assay for RNA synthesis was the same asthat described for the kidney. The reproducibilityof the 0.5 and 2.0 hour determinations is shown.

52.

•

./

•

150

..J 140

~ 130J-

~ 120ulL 110

o 100..----+------...Z 90IJJ

~ 80IJJa. 70

60 ~--r-___.-...._.........__.....__.__123

HOURS AFTER INJECTION

53.

aldosterone on RNA synthesis in the brain is shown in Figure 6.

This figure clearly shows that there was an early stimulation of

RNA synthesis. As in the case of the kidney, the initial response

to aldosterone in the brain was maximal 30 minutes after the injec-

tion. In both kidney and brain, the stimulations were then followed

by an inhibition that was suggestive of an oscillation. Although

the maximum deviations from the control were not the' same in the

kidney and in the brain, the general profile of the two responses

were quite similar.

The effect of aldosterone on sodium metabolism appears to be

tissue dependent. Therefore, interpretation of the present results,

in terms of sodium metabolism, may not be clear, since aldosterone

brings about the resorption of sodium in the kidney and the loss"

of sodium from the brain (Woodbury and Koch-1957). In the present

work, it appears that, in terms of RNA synthesis, the response

to aldosterone in the kidney and brain was qualitatively similar.

Because of the apparent confusion between the physiological

and biochemical responses, the research was expanded to include

tissues in which aldosterone was not known to have any effect.

The Effect of an Intravenous Injection of Aldosterone on

Spleen RNA Synthesis Either aldosterone (2.5 ~g) or vehicle

was injected into intact rats. The time course for the effect of a

single intravenous dose of aldosterone on RNA synthesis in the

spleen is shown in Figure 7. These results show that an inhibition

of RNA synthesis occurred as early as 30 minutes following the

injection. The inhibition continued for three to four hours, at

Figure 7. Time course for the effect of a single intra-venous injection of aldosterone on RNA synthesis inisolated rat spleen nuclei. At time zero, the ratswere intravenously injected with 2.5 ~g of aldosterone.The rats were killed at the times indicated, and thespleens were excised and pooled for homogenization.Nuclei were isolated and assayed for RNA synthesis aspreviously described for the kidney. At each timeinterval, both a control and a hormone treated groupof rats were studied.

\J1+".

CD Z

~ 0~o I'- -I-~

U0 CD ILl-':)

~ Z10 -0\ a:

q- ILl0 I-a I.L010 If)

56.

which time the inhibition was maximal at less than 80% of the control

value. Six hours after the injection, RNA synthesis surpassed that

of the control and a maximal stimulation ~'1as observed. Subsequently

there was a rapid decrease and by 7.5 hours there was a marked

inhibition.

The results presented here show that there is a very striking

difference between the effect of aldosterone on the spleen, as

compared to that in the kidney or in the brain. From this and