o‘;'i.—

.— E-

 

REGULATKDN OF FUNCTION AND ACTOMYOSIN
CONTENT OF CARDlAC MUSCLE BY ESTROGEN

“flush for tho Dogma of Ph. D.

MICHEGAN STATE UNIVERSiTY

Theeéoro Mafihew King
1959

This is to certify that the

thesis entitled

Regulation of Function and Actomyosin
Content of Cardiac Muscle by Estrogen

presented by

Theodore Matthew King

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Phxsiology and
Pharmacology

2/? A/ (git/l "/4 U f s2

Major professor é7

 

Date June 30, 1959

 

0.169

   
     

  

LIB R A R Y
Michigan State

University

 

REGULATION OF FUNCTION AND ACTOMYOSIN CONTENT OF CARDIAC
MUSCLE BY ESTROGEN

By
THEODORE MATTHEW KING

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and Applied
Science in partial fulfillment of the requirements

for the degree of ‘ ‘

DOCTOR OF PHILOSOPHY
Department of Physiology and Pharmacology

1959

 

ABSTRACT

Little information is available on the influence of
estrogen on heart muscle. This situation exists in spite
of the known difference in sex incidence of at least some
types of heart disease, the use of gonadal steroids in
treatment of heart disease, and the recognized greater
longevity of females. .This, along with the need for
knowledge concerning the mechanism of action of such
steroids on the cellular level, stimulated this study.

The effects of estrogen on prOperties of the myo-
cardium were noted in albino rats treated as follows:
ovariectomized; sham Operated females; ovariectomized
given 0.1 gamma alpha estradiol daily; orchiectomized;
sham Operated males; and orchiectomized given 1.0 gamma
alpha estradiol daily. On the 30th day of treatment the
animals were weighed and killed. Heart, adrenal, uterus
or seminal vesicles were weighed and dried. Refractory
periods, excitability to square wave stimuli, and active
and passive tension curves were determined in gitrg on
left ventricular columnae carneae.

No significant differences existed in heart weight,
heart-body weight ratios or water content of the myocardi-
um. Expected changes existed in organ-body weight ratios
for adrenal, uterus, and seminal vesicles. No differences
in excitability or refractory periods were noted. Mean-

deve10ped tension at a given length was significantly

ii

greater in intact females than in castrate females, while
develOped tension in estradiol-treated castrates ap-
proached normal values. In the male groups no significant
difference in develOped tension was found. Mean-develOped
tensionscdfintact females and males were not dissimilar.
No differences in passive tension curves existed in any
groups.

In an attempt to investigate the mechanisms involved
in the reduced contractility of isolated surviving left
ventricular columnae carneae of ovariectomized rats, fur-
ther studies were completed. Glycerol-extracted columnae
were prepared, and ventricular actomyosin was quantita-
tively and qualitatively studied. These studies were
completed after 30 days Of treatment in sham Operated
females, ovariectomized females, and the following ovari-
ectomized alpha estradiol—treated groups: 0.1 gamma,

l.O gamma, and 10 gamma. A 100 gamma alpha estradiol
group Of sham Operated animals was also studied.

Maximal A.T.P. induced tension was significantly re-
duced in glycerol-extracted fibers of hearts of the
untreated ovariectomized group. Tension deveIOped by
glycerol-extracted fibers of estrogen-treated groups was
significantly greater than the untreated ovariectomized
group. Nitrogen content of the glycerol-extracted

columnae carneae was not altered in any of the groups.

iii

Actomyosin was quantitatively determined by change
in viscosity in response to A.T.P. and by micro-Kjeldahl
technique. Actomyosin concentration was significantly
reduced in the ventricle of the ovariectomized groups as
compared to ventricles of the untreated intact group.
Concentration of actomyosin was significantly increased
in ovariectomized groups by treatment with estrogen. NO
qualitative change in contractile protein was found.

It can be concluded that alterations in develOped
tension in isolated surviving columnae carneae and
glycerinated columnae carneae are related directly to
quantitative changes in actomyosin concentration. These

results indicate a dependence of the cardiac contractile

system on estrogen similar to that Of the uterus. Further,

it suggests a general effect of this hormone on contractile

protein synthesis in all muscular tissue. The importance
of the gonadal steroids and the gonadal-pituitary axis
on muscle develOpment and maintenance should be investi-

gated further.

iv

ACKNOWLEDGMENTS

The author is indebted to physiologists of two
departments. In addition to the faculty of the
Physiology and Pharmacology Department of Michigan State
University, the faculty of the Physiology Department of
the University of Illinois College of Medicine must be
mentioned. Two men who have contributed unsparingly of
their time, knowledge, and research experiences must be
mentioned: Dr. w. D. Collings, who contributed his time
and knowledge generously; and Dr. W. V. Whitehorn, for
his unending help and interest, and in whose laboratory

this research was conducted.

To
Lilly

vi

TABLE OF CONTENTS

INTRODUCTION . . . . . .
LITERATURE REVIEW . . . . . .

Gonadal Steroid Effects on the Myocardium .

Estrogen Effects on Smooth Muscle .
Gonadal Steroid Effects on Skeletal Muscle

Gonadal Steroid Effects on Nitrogen and

Protein Metabolism . . .

Estrogen Effects on Enzyme Systems

Estrogen Metabolism . . .
MATERIALS AND METHODS

Experiment I.

of Isolated Left Ventricular Columnae

Carneae . . . . . .

Experiment II . . . . . .

Extraction of Ventricular Actomyosin

Studies on Glycerol-Extracted

Columnae Carneae . .
RESULTS . . . . . . . . . . .

General Data_ . . . . . .

Electrical PrOperties of Isolated

Surviving Columnae Carneae

Mechanical PrOperties of Isolated

Surviving Columnae Carneae

vii

Determination of PrOperties

ll
20

29
35
45
48

58
58

58

65

65

71

71

DevelOped Tension of Glycerol-

Extracted Columnae Carneae . . . . . . . . . 79

Nitrogen Content of Glycerol-Extracted

Columnae Carneae . . . . . . . . . . . . . . 84

Quantitative and Qualitative Determinations

of Total Ventricular Actomyosin . . . . . . . 84
DISCUSSION . . . . . . . . . . . . . . . . . . . . 87
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . 99
REFERENCES . . . . . . . . . . . . . . . . . . . . lOl

viii

LIST OF TABLES

Table Page
1. Terminal Body Weights . . . . . . . . . . . . 66
2. Wet Organ-Body Weight Ratios . . . . . . . . . 68
3. Water Content of Ventricular Myocardium . . . 7O

4. Refractory Periods of Isolated Surviving
Columnae Carneae . . . . . . . . . . . . . . . 72

5. Strength-Duration Values for Series A,

Experiment I . . . . . . . . . . . . . . . . . 73
6. Chronaxie and Rheobases for Experiment I . . . 75
7. Maximum DevelOped Tension of Isolated

Surviving Columnae Carneae . . . . . . . . . . 82
8. Maximum DevelOped Tension and Nitrogen Content

of Glycerol-Extracted Columnae Carneae . . . . 83
9. Quantitative and Qualitative Study of

Ventricular Actomyosin . . . . . . . . . . . . 85

ix

LIST OF FIGURES

Figure

l.

10.

11.

Photograph and Diagram of Rat Heart Muscle
Preparation . . . . . . . . . . . . . . . .
Drawing of the Experimental Apparatus . . .
Record Of an Isometric Contraction,
Stimulus, and Time Line . . . . . . . . . .
A Typical Refractory Period Determination .
Actomyosin Calibration Curve . . . . . . .
Strength-Duration Curves for Intact Males
and Females of Series A, Experiment I . . .
Passive-Tension Curves for Intact Males

and Females of Series A, Experiment I . .
Passive-Tension Curves for Female Groups of
Series B, Experiment I . . . . . . . . . .
Passive-Tension Curves for Male Groups of
Series B, Experiment I . . . . . . . . . .
DevelOped-Tension Curves for Female Groups
of Series B, Experiment I . . . . . . . . .
DevelOped-Tension Curves for Male Groups of

Series B, Experiment I . . . . . . . . . .

Page

51
52

55

57
62

74

76

77

78

80

81

INTRODUCTION

This work is concerned with the physiologic effects
of estrogen on rat myocardium. In Selye's Textbook of
Endocrinology published in 1947 he states, ”heart action
is not significantly influenced by ovarian hormones":
however, no references are given to support this state-
ment. In a literary survey, only one reference was found
in which sex steroids were administered to animals for a
period of time and the functions of the heart studied. A
number of papers may be found in which these steroids
have been added to cardiac muscle baths and effects on
tension studied. One certainly cannot relate such results
to in‘zigg functions for a number of reasons, outstanding
among which is the fact that the heart is not exposed to
such steroids in a one-shot manner, but rather is continu—
ously eXposed to varying concentrations of hormones.

Biologists know well that the entire female repro-
ductive tract is significantly influenced by estrogen, as
well as certain general characteristics including body
shape, character of the voice, and pubic hair distribution.
Knowledge concerning the less obvious actions of estrogen
is lacking. A need certainly exists for eliciting more
information on the mechanism of action of these steroids
at the cellular level. A second and probably more urgent
reason for knowing the various effects of estrogen, par-

ticularly on the cardiovascular system, is the sex

difference in mortality from cardiovascular disease.
Heart-disease statistics compiled by the Metr0politan
Life Insurance Company for the years 1951-1953 are
impressive. In the age range from 40-74 years, heart
disease kills twice as many men as women in the United
States: the incidence is 872 per 100,000 men versus
437 women. In all countries heart disease kills more
men than women at all ages. Regardless of diet and
standard of living, the ratio is never reversed. A
third reason is the widespread use of estrogen and its
derivatives in clinical medicine. This use includes
treatment of adolescent acne, inhibition of metastasis
of breast and prostatic cander, relief of pruritus
associated with advanced liver disease, and attempts to
decrease the incidence of myocardial infarction. If
estrogen is effective in these listed clinical conditions,
it must indeed have diversified and subtle effects.
Review of the literature will include the relation
of estrogen to muscle function, including skeletal, smooth
and cardiac, and to protein anabolism and specific enzyme
systems. This writer found that a review of these areas
was necessary for understanding the possible effects of
estrogen on cardiac muscle. Such a knowledge gives one
indications of possible mechanisms of estrogen action.
If one compares the literature of skeletal muscle to that

of cardiac muscle, he will immediately be aware of the

lack of knowledge available on steroid action in cardiac
muscle. A second reason of importance to have knowledge
of estrogen"s action on skeletal and smooth muscle is the
possibility of arriving at a unified concept of steroid
action on all muscle at a cellular level. No attempt
will be made to include a review of the voluminous
literature on the well known reproductive action of

estrogen and its relation to the pituitary-ovarian axis.

LITERATURE REVIEW
GONADAL STEROID EFFECTS ON THE MYOCARDIUM

The literature concerning gonadal hormone influences
on the myocardium may be divided into observations dealing
with effects on energy—generating systems and observations
dealing with effects on functional activity and contrac-
tile protein systems.

In the first group, one may include effects on
glycogen, phosphocreatine (P.C.) and adenosinediphosphate
(A.D.P.) content of the myocardium.

Schumann (1939) determined the glycogen content of
the rat myocardium. He found a mean value of 478 mg.$ in
intact male rats. Determinations completed 21 days and
54 days following castration gave values of 402 and
318 mg.$ respectively. The mean concentration 54 days
following castration was significantly reduced as compared
to the intact animal. Glycogen content of 484 mg.%,

215 days following castration, was not unlike the average
for intact animals. He suggested that castration resulted
in a defect in energy metabolism of the male heart related
to the absence of testosterone. This function is then
taken over by some other anabolic principle. No comment
was made on the specific nature of this physiologic

adjustment.

Schumann (1940) published investigations on effects
of castration on A.D.P. and P.C. concentration. Fifty-
four days after castration, P.C. had decreased to 63% of
the normal value, while glycogen decreased to 66% of
control values. Adenosinediphosphate showed a signifi-
cant decrease from 19.6 i 0.5 mg.%. Administration of
5 mg. of testosterone daily from the 54th to the 60th day
resulted in a return of P.C. and A.D.P. to normal values.
Glycogen showed a more striking response in that it
increased to 777 mg.% as compared to 478 mg.% for intact
animals.

Schumann (1942) found similar effects in hearts of
ovariectomized and estrogen—treated female rate.

It has therefore been shown that castration of either
females or males alters the energy metabolism of the
myocardium and, further, that the changes induced are
corrected by the specific hormone in question. It is of
interest that in adrenalectomized rate no changes are
observed in A.D.P. or P.C. concentration in the myocar-
dium (Schumann, 1942) and that administration of
testosterone does not increase these substances above
normal (Schumann, 1942). Myocardial glycogen content is
markedly reduced in adrenalectomized male rats, and
testosterone is effective in returning this to normal
intact values (Schumann, 1940). Further, testosterone

administered to intact male rats results in an increase

in myocardial glycogen content to 819 mg.% as compared to
control values of 478 mg.%. It appears that carbohydrate
metabolism in the myocardium is markedly influenced by the
presence of testosterone, even in the absence of cas-
tration.

In summary, testosterone and estrogen have a role in
maintaining glycogen and high-energy phosphate metabo-
lites of the myocardium. Since these phosphate compounds,
along with glycogen, provide energy for muscle contrac-
tion, one would expect alterations in functional capacity
in gonadectomized rat hearts.

In the second area of investigation, namely function-
al activity and protein systems, one finds the observa-
tions of Blasius, Kafer, and Seitz (1956) on the effect
of testosterone on contractile proteins of the heart.

They isolated proteins of the myocardium and fractionated
them by micro-electrophoresis. Three large fractions
were found: myoalbumin: a myosin fraction including
alpha, beta, and gamma groups: and a third fraction
including myogen, myoglobin, and water soluble protein
elements. No quantitative or qualitative difference was
observed between hearts of intact males, intact females,
or castrate male rabbits. In hearts of castrate male
rabbits, treatment with 1 mg., 25 mg., and 50 mg. of
testosterone prepionate for 90 days following castration

resulted in an increase in the myosin fraction. This

increase was prOportional to the quantity of testosterone
given, and the increase was most marked in the alpha
myosin curve.

Blasius 31 g. (1957) reported on the efficacy of
various synthetic steroids, including testosterone-
phenylprOpionate, a combination of different testosterone
esters with prolonged depot action (prOpionate, phenyl-
prOpionate, isocapronate, caprinate): nor-androstenolon—
phenylpropionate: and one synthetic steroid, methyl-
estrenclone, that combines the protein anabolic effect
with a so-called 'gestative-luteinizing" effect. As in
their original experiment these androgenic steroids
resulted in an increase in the myosin fraction of heart
from castrate rabbits: however, the response of alpha,
beta, and gamma components of myosin varied. Methyl-
estrenolone resulted in a marked response in all three
components of the myosin fraction. They concluded that
this protein anabolic effect is not necessarily limited
to androgenic steroids, but may also be mediated by a
steroid with eetrogenic activity.

This discussion has demonstrated that estrogen and
testosterone have a role in maintaining the energetics
of the myocardium and further, that testosterone and
possibly estrogen mediate an increase in myosin content
of the myocardium. This demonstrates that both the
energy supply mechanism and the contractile protein

system may be altered by the gonadal steroids. One would

thus expect altered myocardial activity when these
steroids are absent or administered.

One paper was found in which steroids were admin-
istered for a period of time and then myocardial function
was studied. Korenchevsky, Hall, Burbank and Cohen (1941)
completed a long-term experiment noting gonadal steroid
relation to capacity of the myocardium for work. They
investigated the action of androsterone and testosterone
prOpionate on hearts from castrate male rats. These
steroids were administered for periods up to two months
following castration. Work measurements were made by
attaching 10 and 15 gram weights to a lever attached to
the heart. All contractions were recorded on a kymograph.
Amplitudes of contraction were measured and their number
counted before, during, and after suspension of the
weights. They found on calculation of work performed
that hearts of uninjected castrates were significantly
weaker than either those of the uninjected normal con-
trols or hearts of castrated rats treated with androsterone
or testosterone. These men made no conclusion as to the
significance of their work. Increased work capacity of
treated castrate males could be explained by known
increases that occur in energy supply (Schumann, 1959,
1940) and in the contractile proteins (Blasius'gt‘gl.
1956) on testosterone administration. However, the de-

creased capacity of castrate animals cannot be related to

a decrease in "myosin" content (Blasius 33 El. 1956),
but rather only to a defect in the energy-supplying
system. No results were reported for female rats, and
no further work by these authors could be found.

A difference in response of male and female hearts
to stress of treadmill exercise has been reported.

Van Liere and Northup (1957) noted a significantly
greater increase in heart-body weight ratio in exercised
albino female rats than in males. No significant dif-
ference between the ratios of male and female albino
control hearts was observed. They concluded that females
show a more efficient response to severe exercise, sug-
gesting that female sex hormone might be responsible for
the observed difference. No such difference was observed
in similar experiments on hooded rate. It should be
observed that the albino females lost weight on exercise
while hooded females did not.

In addition to changes in mechanical activity,
electrical activity has been reported to be influenced by
altered gonadal function. Electrocardiogram (3.0.6.)
changes have been reported in response to deficiencies of
estrogen. Ovarian insufficiency, as observed in young
women with underdeveIOpment of the ovaries and in women
at the menopause has been reported to be associated with
typical changes in the E.O.G. (Scherf and Boyd, 1940).
These changes include a depression of the S-T segment in

all leads, being maximal in lead 2. In addition, the
T wave is frequently reduced. These changes are reversed
by treatment with synthetic or natural estrogens.

One area of functional activity not yet mentioned is
the ingzitgg cardiac muscle studies in which steroids
were added to the perfusing fluid of a bath chamber.

This work, in the Opinion of the author, has limited
application to the understanding of the inmzizg role of
gonadal hormones on the myocardium. This Opinion is

based on: (1) the difficulties in solubilizing the
involved steroid, (2) the exposure of muscle for rela-
tively short periods of time to fixed quantities of one
hormone, (3) the question of state of circulating hormones;
that is, the importance of protein binding of ig;zgzg
circulating estrogen for its activity (Szego, 1953).

[In 13352 cardiac muscle studies include the use of
testosterone, progesterone, and their metabolites, as
well as estrogen. Gowdey, Loynes and Ward (1950) investi-
gated effects of a large number of steroids on isolated
frog and rabbit heart. Estrone, estradiol, and testosterone
were found to augment contractility but showed little ef-
fect upon heart rate. Progesterone, however, caused a
definite reduction in cardiac contractility. Rubin (1950)
made similar observations on the action of progesterone
on isolated papillary muscle of the cat. Nahum, Geller,
Levine, and Sikand (1951) studied effects of progesterone

10

and pregnolene on isolated cat papillary muscle and
hearts of intact dogs. Pregnolene was found to decrease
contractility and lowered the threshold of isolated
papillary muscle. In intact dogs the threshold was
found to be unchanged with 100 mg.% of pregnolene.
PrOgesterone was found, as reported before, to decrease
contractility of isolated preparations, and to increase
the threshold in intact animals. It is of interest that
neither of these steroids produced an effect until after
15 minutes of infusion.

Szent-Gygrgyi (1953) observed that progesterone was
one of the steroids that abolished the treppe response
in frog ventricles. The ventricle regained its former
strength more rapidly. Utilizing his concept of
"favorable state", he assumes this to mean that progest-
erone regulated the change in K+ from one contraction to
the next, such that the outflow of K+ was kept in balance
with its inward flux, within the muscle fiber.

In reviewing the literature on estrogenic effects on
the myocardium, past work has been limited to effects on I
glycogen and other energy-supplying substances. No study
on tension production in ovariectomized or ovariectomized,

treated females was found.

ESTROGEN EFFECTS ON SMOOTH MUSCLE
In considering muscle that is obviously regulated by

endocrine factors, one thinks first of uterus.

ll

G. W. Corner (1923) was one of the early workers who
demonstrated that uterine activity was mediated by hor-
mones of the ovary. Reynolds (1931) noted in rabbits
that estrogen affects motility of the uterus only in 1112
and only after a definite latent period. He observed,
following ovariectomy and before regressive changes in
histology occurred, that motility was markedly decreased.
Frank 22.2l- (1925) observed that with administration of
estrogen to ovariectomized rats, the excised uterus,
which is ordinarily inactive as a result of castration,
underwent rhythmic contractions.

It was only a question of time before the techniques
of Szent-Gygrgyi (1949) were utilized to determine the
presence of actomyosin in uterus, and further to attempt
to find the mechanism by which estrogen affects motility
of uterus.

Csapo (1948), a student in the laboratory of Szent-
Gygrgyi, demonstrated the presence of actomyosin in the
uterus. With a viscosimetric technique for determination
of actomyosin, he demonstrated that uterine muscle has a
lower actomyosin concentration than skeletal muscle.

Csapo (1950 a) employed actomyosin threads prepared
from uterine muscle and observed a difference in their
contraction as compared to skeletal muscle extracted
actomyosin threads. Threads extracted from uterus were

characterized by a longer ”latency period" and slower and

12

smaller contractions, similar in behavior to the differ-
ences in contraction observed for the whole muscle.

Csapo (1948) determined uterine actomyosin in ad-
vancing pregnancy and found that actomyosin increased as
pregnancy advanced. He then attempted to find the effect
of ovariectomy on actomyosin concentrations (Csapo, 1950b).
Using virgin female rabbits, he found that ovariectomy
produced a decrease in uterine weight, On determining
uterine actomyosin concentration, he observed, two months
following ovariectomy, an 80% decrease from control
levels. Adenosine triphosphate activity was 15 times
less than that for tissues of estrous rabbits. Adminis-
tration of estrogen caused a marked increase in actomyosin
formation within twelve hours. Actomyosin continued to
increase for the duration of estrogen treatment. Adenosine
triphosphate activity increased 10 times within 12 hours
and estrus values were obtained within 4 days of estrogen
treatment. This work was substantiated by Blasius and
Schuck (1955) and Cretius (1957).

Blasius and Schuck (1955) investigated the uterine
muscle protein electrophoretically, employing uteri of
immature rabbits before and after estrogen administration.
They demonstrated four components in uterine muscle
proteins: myogen, B myosin, actomyosin, and myoalbumin.
The only pattern change observed after estrogen treatment
was an increase in the actomyosin component. This work

has demonstrated that the concentration of uterine

l3

actomyosin, the contractile protein of the myometrium, is
under control of ovarian estrogenic hormone. Actomyosin
concentration changes during physiological conditions
such as pregnancy and the menOpause, which involve alter-
ations in estrogen output of the ovaries. It would
therefore appear that the quantity of actomyosin present
at any time is consistent with the work required of the
uterus in a given physiological condition.

Cretius (1957) determined the contractile proteins
separately in the corpus and in the cervix of human
uteri. These uteri included non-pregnant, pregnant, and
puerperal uteri. His method of actomyosin determination
was that of Szent-Gygrgyi. Pregnant uteri contain ed more
contractile protein per weight than non-pregnant ones,
and these contained more during sexual maturity than
during the menOpause. The cervix was found to contain
less contractile protein than the fundus. Cretius postu-
lated that decrease of contractile proteins in the fundus
causes,clinically, a primary inertia. Finally Cretius
demonstrated that administrations of estrogen produced an
increase in contractile proteins in the uterine muscula-
ture of climacteric women.

Csapo and Corner (1953) demonstrated that the concen-
tration of contractile substance would determine the
maximal force which the muscle is able to develop. They
measured maximal isometric tension develOped by uterine

strips. These were removed from virgin mature, female

14

white rabbits under controlled hormonal conditions in
which the concentration of uterine actomyosin had previ-
ously been determined, i.e., in natural estrus, after
ovariectomy, and after ovariectomy followed by estrogen
treatment. Average maximal tension of uterine strips
from estrous rabbits was 9.6 gm. per mm.2 cross-sectional
area. The muscle contained 7.6 mg. actomyosin per gram
of tissue. Values for uterine muscle three weeks after

bilateral ovariectomy decreased to an average of 2.3 gm.

2

of tension per mm. and 1.7 mg. of actomyosin per gram of

tissue. Comparing estrus and the castrate state as ex-
tremes, the ratio of tension in the two contrasting
states is 9.6/25514.2, and that of actomyosin 7.6/1.7:
4.5. The figures are therefore in agreement. On ad—
ministering estrogen to ovariectomized animals, maximal
tension returned to the estrus value of 9.55 gm., with
a proportionate increase in actomyosin.

Administration of progesterone to castrate animals
(Csapo and Corner, 1953) Produced no significant changes
in tension developed by their uteri. When estrogen and
progesterone were combined, maximal tension of the
uterus was not significantly different from that occur-
ring with estrogen alone. It would appear that proges-
terone does not significantly alter actomyosin content
of uterus.

Without question, it has been demonstrated that the

contractile system of the uterus is subject to the

15

influence of estrogen. With increasing estrogen levels,
actomyosin increases and the magnitude of developed ten-
sion increases.

Of particular interest is progesterone's role in
membrane excitability. Csapo 21 El. (1953) observed
that progesterone had no function in maintaining or in-
creasing the actomyosin-contractile system or the high-
energy phosphate supply. Rather, progesterone's role
in motility is related to holding in abeyance a chemi—
cal structure that is capable of maximal contractile
capacity. He found when the uterus is dominated by
progesterone that normal electrical or pharmacological
stimuli are relatively ineffective. However, if estro-
gen alone is present the uterus is readily and effective-
ly stimulated electrically. It would appear that
progesterone acts only to block conduction within the
muscle and prevent the typical all-or-none response.
This work is the foundation for Csapo's theory (1956)
of control of onset of labor; that is, the prime cause
of labor is withdrawal of progesterone.

Effects on Other Smooth Muscle.

A question of interest to this writer is whether
the uterine myometrium is a unique form of smooth muscle
or whether it is a question of sensitivity of anabolic
reactions to endocrine stimuli. Could the same observa-

tion be made on smooth muscle of blood vessels?

16

In discussing effects of steroids on circulatory
status, one is instantly reminded of vasomotor insta-
bility observed at the natural menOpause and with surgi-
cal castration. This syndrome involves a combination of
a variety of symptoms and signs that include both cardio-
vascular and autonomic nerve manifestations. The signs
and symptoms of "change of life" include: "hot flushes",
palpitations, cardiac pain, sighing-dyspnea, sweating,
vertigo, and "splitting headache" (Scherf, 1940).

One cannot explain the presence of such difficulties
on the basis of an altered cardiovascular system alone.
It is obvious that these changes are observed in men as
well as in women who are in anxiety-producing situations.
In addition, frequency of these symptoms is greatest in
those women who experience clinical difficulty at man-
strual periods. For this reason, it is difficult to
evaluate effectiveness of estrogen therapy in this condi-
tion. However, it has been universally reported that
estrogen is effective. The only objective finding on
estrogen treatment is the correction of the S-T segment
depression in the E.C.G. (Scherf and Boyd, 1940). Sub-
jective findings include disappearance of "hot flushes",
palpitations, and paresthesias. Even though estrogen is
effective, one must question the site of action and
whether this effect is a direct one or merely serves to
correct some hormone imbalance including the pituitary-

adrenal and pituitary-thyroid axis.

17

Reynolds (1941) contributed much toward the study
of the effect of estrogen on smooth muscle by his obser-
vations on local effects of estrogen on arteriole tone.
In man, he found that intramuscular estrogen was capable
of inducing appreciable increases in finger volume for
as long as two hours. In addition to this work, Reynolds
gt 5;. (1940) applied estrogen intradermally to ears of
ovariectomized rabbits. A substantial vasodilation oc-
curred when the results were quantitated according to
percent of light transmission: a decrease of 15-20% was
noted following estrogen injections. Congestion of mu-
cosal surfaces, especially the nasal mucosa, during
estrus in animals is well-known. Mortimer (1936) found
he could induce vasodilation.with local application of
estrogen to nasal mucosa of monkeys.

It would appear that estrogen has an effect on the
functional status of arterioles, at least in skin and
mucosal surfaces. The physiological significance in
vascular homeostasis is yet to be established. The lit-
erature is not consistent: Liebhart (1934) and Crainicianu
(1932) observed a prolonged lowering of blood pressure
in women with estrogen therapy, while Steinkamm and
Giesen (1936) and Wallis (1936) found no depression of
blood pressure in women with even larger dosages of
estrogen. In rats, Oster (1950) reported a significant
effect induced by castration. With a tail plethys-

mography method for blood pressure determination, he

18

found an increase in mean systolic pressure in castrate
males of 16 mm. Hg, and a 13 mm. Hg decrease in castrate
females. He did not, however, indicate his variation.

Depressant action of estrogen on blood pressure has
been observed in one other species, the chicken. Weiss
'gt‘gl. (1957) found systolic pressure of adult male
Leghorns to be 15-25% higher than that of females. No
significant difference existed between sexes up to 8
weeks of age. However, between the 8th and 13th week,
pressures began to diverge with a resultant 26-30 mm.

Hg sex difference. This was due to a rise in male sys-
tolic pressure, whereas the female systolic pressure re-
mained relatively stable. It is of interest that the
diastolic pressure rose while heart rate decreased, sug-
gesting that an increase in cardiac output occurred.
These changes occurred at the same time as sexual
maturation.

In summary, estrogen has little, if any, effect on
blood pressure in man. There is indication that it may
have a hypotensive effect in rats and chickens, as sug-
gested by its vasodilating action on cutaneous and muco-
sal blood vessels.

Pertinent to the blood pressure problem is the role
of estrogen in prevention of coronary and aortic athero-
sclerosis (Katz‘gt‘gl. 1953). This work has been done
primarily in cholesterol-fed rabbits and chickens.
Hyperlipemia and aortic atheromata of cholesterol-fed

l9

intact female rabbits can be markedly inhibited by ad-
ministration of estradiol (Ludden.gtԤ1. 1942). Athero-
matosis of coronary arteries of cholesterol-fed chickens
can be prevented, and a regression induced, by use of
estrogen (Pick, 1952). This has been related to alter-
ations in circulating B-lipOproteins and, hence, to de-
generative changes of the intima. It is known that
atherosclerosis with hypertension forms a vicious cycle
terminating in arteriosclerotic heart disease. Although
estrogen appears to have a definite vascular morphogenic
effect, this author will make no attempt to review the
voluminous literature concerning estrogen's relation to

experimental atherosclerosis.

GONADAL STEROID EFFECTS ON SKELETAL MUSCLE

A. Androgenic Effects

Investigation of the action of androgenic steroids
on striated muscle was stimulated by the report of
Papanicolaou and Falk (1938) on the hypertrOphy of skele-
tal muscle induced by androgenic hormones. In adult fe-
males the temporal muscles are comparatively small and
flat, while in the adult male they are much larger.
They found, during the course of an experiment with
gonadotrophic hormones, that the temporal muscles of
adult females underwent considerable hypertrOphy. In
addition, they reported that growth was not limited to

the temporal muscle, but other muscles of the body were

20

 

 

similarly affected, indicating that the effect is on

the muscular system in general. They further observed,
in adult male guinea pigs castrated before sexual ma~
turity, that the temporal muscles remain small as in the
adult female. 0n treatment with gonadotrOphic hormones
the muscles of such castrated males did not respond.

In castrated females such treatment was also ineffective,
indicating that the presence of gonads is necessary for
the production of muscular hypertrOphy. 0n administer-
ing testosterone prOpionate to castrate immature males
and spayed, as well as normal, adult females, a definite
hypertrOphy of the temporal and somatic muscles occurred.
They therefore assumed that the trOphic action of gonado-
trophic hormone in intact females was mediated indirectly
through androgenic hormones released from interstitial
cells of the ovary. Ovaries from these treated animals
showed hyperplasia and hypertrOphy of interstitial tis-
sue. No data on muscle weights were given in this publi-
cation. Dosages of gonadotrOphic hormone, testosterone,
estradiol, and progesterone were not given. The question
of purity of gonadotrOphic hormone seems rather academic
in the absence of any reported data. Kochakian, Humm

and Bartlett (1948), as well as Scow and Roe (1953) were
successful in confirming atrophy of temporal muscle in
adult male guinea pigs following castration and its cor-

rection by androgen administration.

21

Kochakian and Tillotsen (1955) investigated effect
of castration on 49 individual skeletal muscles of
guinea pigs and substantiated Papanicolaou's and Falk's
(1938) findings of a generalized effect on the muscular
system. In general, degree of weight loss induced by
castration was most marked in cephalad muscle groups and
least in caudal muscle groups.

In noting the effect of age and castration on total
skeletal muscle mass, Kochakian and Tillotsen (1955) i
found that the total muscle mass increased progressively
with age in the normal guinea pig. In this experiment
the two groups of animals were not pair~fed and no men—

tion of any change in food intake was noted. In addition,

 

the spontaneous activity of the castrate animals might
well be decreased, which would effect the growth incre-
ment. Nevertheless, it is apparent that the muscular
system has varying degrees of sensitivity to the cas-
trate state.

Scow and Hagan (1955) determined the effects of
testosterone on myosin, collagen, and other protein frac-
tions of the temporal and the rectus femoris muscle in
castrate guinea pigs. It was found that all nitrogenous
fractions of the temporal muscle were reduced by cas-
tration: this includes myosin, collagen, non-protein
nitrogen, a "water-soluble" protein (corresponds to the
sarcoplasmic protein fraction and includes myoalbumin,

most of the glycolytic cycle enzymes, and all particulates)

22

and an alkali—soluble stroma fraction. The greatest
change was in myosin formation, which was reduced to 7%
of that of intact animals. In the rectus femoris muscle,
myosin, "water—soluble" protein, and non-protein nitrogen
were not affected by castration, whereas formation of
alkali-soluble stroma and collagen fractions were reduced
about 50%. Testosterone administration to castrate
guinea pigs returned the altered nitrogenous fractions of
both muscles to normal.

The difference between temporal and rectus femoris
muscle in the response of myosin and actomyosin to
testosterone is quite striking. Scow and Hagan (1955)
suggest two possibilities; the first is that these frac-
tions in the two muscles are not identical and such iden-
tification would require electrOphoretic or ultraoentri-
fugation studies. The second possibility suggested is
that formation of the major part of proteins in the tem-
poral muscle of normal male guinea pigs is under direct
control of testosterone. He suggests that this muscle
should be classified as a secondary sexcharacteristic of
the male guinea pig. To support this, Scow and Hagan
(1957) observed that fierce fighting occurs between males
when placed with estrous females. Hypertrophy of head and
neck muscles is an important factor in this sexual be-
havior pattern in the guinea pig.

Collagen formation in the rectus femoris and tempo~

ral muscle of the guinea pig was decreased by castration

23

and restored to normal by testosterone. Scow and Hagan
(1957) repeated their studies on thigh muscles in cas-
trate rats and found no effect on formation of various
protein fractions or on weight gain. Their observation
of collagen reduction in thigh muscles of guinea pigs
was not confirmed in the rat.

Wainman and Shipounoff (1941) observed, in male rats,
a stimulating effect of testosterone propionate on the
perineal complex: bulbocavernosus, ischiocavernosus,
and levator ani muscles. The profound atrOphy of castra-
tion was prevented by the administration of testosterone.
Comparable changes were not seen in other striated mus-
cles. Scow (1952) substantiated this work. He reported
a weight increase of 6-7 times in the levator ani muscle
of castrate rats given testosterone. Eisenberg and
Gordan (1950) noted effects of various hormones on the
levator ani. They observed that the levator weighed
17:1.0 mg. in the 30-day-old male rat. The castrate male
rat levator weighed 33:2.9 mg. at 60 days, while intact
normal muscle weighed 101:6.3 mg. Thus, even in the cas-
trate animal there is growth of the levator ani. They
observed that pituitary growth hormone, unesterified
testosterone, testosterone propionate, and methyl-
testosterone are potent myotrOphic agents, all restoring
the weight from 23% to 49% of normal levator ani weight.

Stewart (1955) induced the levator ani muscle of the

rat to hypertrOphy by subcutaneous implantation of

24

testosterone pellets 30 days prior to sacrificing the
animals. Determinations of total protein nitrogen and
protein fractionation were made. Analysis of cytoplasm,
connective tissue, and contractile protein showed no
significant alteration in relative distribution of pro-
teins. He concluded that the hypertrophy induced was a
true growth.

Leonard (1952) determined glycogen levels in the
rectus femoris, abdominal and cremaster muscles on the
seventh day following castration of male rats. Castra-
tion was found to decrease glycogen content of all three
muscles, but only in the cremaster muscle was this dif-
ference statistically significant. On determination of
glycogen 32 days after castration, all three muscles
showed a significant decrease. Testosterone adminis-
tration to castrate males resulted in a significant in-
crease in glycogen in all three muscles within 6 days.
Spayed females showed the same response to testosterone
in abdominal and thigh muscle. This work demonstrates
the importance of the time-interval between castration
and observation of expected changes.

The growth effects of androgenic hormones have been
extensively investigated in the male rat and guinea pig.
It has been successfully demonstrated that growth of the
masticatory muscles of the male guinea pig is under the
primary influence of the male sex hormone. In male rats

the analogous muscle is the levator ani group and the

25

cremaster. This effect has been localized to an action
on nitrogen metabolism, such as affecting the quantity
of contractile or non-contractile proteins including
collagen. In addition, glycogen content of cremaster,
rectus femoris, and abdominal muscles of the male rat
can be increased with testosterone administration.

The obvious question after observing alterations
in growth of these muscles is: What is the effect of
androgens upon tension production? To the writer's
knowledge, such work has not been done in the male rat
or male guinea pig. Simonson, Kearns, and Enzer (1941)
found in four men, two castrates and two eunuchoids,
that methyltestosterone was capable of inducing marked
improvement in work capacity. However, no adequate
controls were provided and possible effects of learning
and training make the results difficult to evaluate.
Samuels, Henschel, and Keys (1942), studying male medical
students, found no improvement in physical ability on
administering 50 mg. of methyltestosterone daily for a
two-week period. This was a double blind study with
each man serving as his own control and receiving first
a placebo for two weeks, then receiving testosterone.
Physical ability was measured by determining grip
strength. This finding in itself hardly negates the re-
sults in castrate males mentioned above.

The effect of testosterone on growth of muscle and

tension production might well be similar to effects of

26

somatotrOphin on skeletal muscle. Bartoli, Reed and
Struck (1937) found a small, but definite,increase in
weight of the quadriceps muscle of male rats treated
with pituitary extracts. Greenbaum and Young (1950)
confirmed this observation with growth hormone. They
found that the rate of growth of tissues was not uni-
form throughout the body. Differences in rates of
growth of muscles were noted. Plattener and Reed (1939)
studied the twitch tension of the gastrocnemius muscle
during long-continued stimulation and found that this
hypertrOphied muscle did not develop more tension than
did muscles from control rate. All of their eXperiments
were performed on the gastrocnemius, a muscle in which
increase in weight under growth hormone treatment is
relatively small. Bigland and Jehring (1952) employed
the quadriceps muscle of female hooded Norway rats, after
21 days of treatment with 0.5 mg. of "pure" growth hor-
mone. They completed their studies by using ig‘gitu a
femoral-nerve-quadriceps preparation, in which the nerve
was stimulated through electrodes. The animals were
pair-fed with untreated controls. Isometric records of
single twitches, summated twitches, tetanus, and fatigue
were obtained. In spite of differences in weight of up
to 40% between the two groups of muscles, the treated
muscle gave less tension per gram of muscle weight than
the controls. Histological comparison of treated muscle

with untreated controls showed an increase of fiber

27

cross-sectional area of 6-12% associated with an increase
in muscle weight of 20-30%. Because of size of the quadri-
ceps, contractions could not be measured when the muscle
was stimulated directly. The possibility of defects in
transmission at the neuromuscular junction or of changes
in the propagation of the excitation wave might account
for these findings. Nevertheless, there is a possibility
that with increased growth of muscle there is not a direct
1:1 ratio with the ability to develOp tension on stimu-
lation. For this reason, the response to stimulation of
the masticatory muscles of guinea pigs and of the levator
ani of rats should be investigated.

B. Estrogen Effects

Effects of estrogen on skeletal muscle growth or
tension production have not yielded the consistently
trOphic effects noted in testosterone investigations.
Papanicolaou and Falk (1938) in their original work found
no definite effect on any voluntary muscles on adminis-
tering progesterone or estradiol for periods of 6-8 weeks.
They attributed the response of intact females to gonado-
trOphic hormones to the release of androgenic substances
from interstitial tissue of the ovary.

As a result of this early work, most investigations
of estrogens are limited to application of estrogenic
preparations to muscles of males being investigated for
their response to androgens. Wainman and Shipounoff

(1941) found that progesterone increased the weight of

28

the levator ani of castrate male rats 15% above untreated
castrates, while estradiol dipropionate and desoxycortico-
sterone acetate produced no significant increase in the
weight of the muscle.

A recent paper was found in which estrogen effects
on striated muscle were investigated. Bajusz (1959) noted

effects of various hormones on regression of muscle

atrophy following denervation. He used female rats and
crushed the obturator, sciatic, and femoral nerves intra-
pelvically. Such a procedure resulted in a marked atrOphy
of the triceps surae muscle. Methyltestosterone and alpha
estradiol were most effective in preventing this denerva-
tion atrOphy, maintaining 89.8% and 85.4% of preOperative
weights respectively. No tension studies were done.

No study is known in which tension production per
gram of skeletal muscle is compared for males and females.
As already mentioned, the response to growth is not al-
ways directly related to tension production. Even though
estrogens are incapable of inducing skeletal muscle
growth in ovariectomized females, there may be an effect

on work capacity of specific muscles.

GONADAL STEROID EFFECTS ON NITROGEN AND PROTEIN METABOLISM
A. Androgenic Effects

The role of androgens in metabolism of protein was
first investigated by Kochakian and Merlin (1955). They

found that "male hormone" extracted from urine of male

29

medical students produced a marked reduction in urinary
nitrogen excretion of "thin" and "fat" castrate dogs
maintained on a constant diet. The maximal rate of ni-
trogen retention was attained in two to three days, and
continued injections or increased dosages did not in-
crease the rate of retention. The maximal rate of
retention was 5-6 mg. per kg. of body weight per day.

On cessation of injections some nitrogen was lost, and
it was assumed that this had not been incorporated into
proteins. The "male hormone" employed in this experiment
was identified as androsterone. Following this experi-
ment testosterone was employed in hypophysectomized-
castrate dogs (Kochakian, 1957), castrate rats
(Kochakian, 1944), hypOphysectomized male rats (Rupp and
Paschkis, 1953). and sunuchoids (Knowlton at 21,, 1942)
with the same results. It was observed that treatment
of normal dogs in the same manner as castrate dogs re-
sulted in no nitrogen retention (Kochakian and Merlin,
1956). In none of these experiments was fecal nitrogen
decreased.

The fate of retained nitrogen has been the subject
of investigation by Kochakian and Merlin (1955). In
their original experiment, they found urine urea de-
creased, likewise blood N.P.N. and urea were also
decreased. It was suggested by these workers that the

retained nitrogen was utilized in skeletal muscles and

30

secondary sex organs. No alteration was noted in plasma
protein concentration.

Kochakian, Robertson and Bartlett (1950) investi-
gated the site and nature of protein synthesis in cas-
trate rats treated with testosterone. Castrate adult
male rats brought into body weight equilibrium and in-
jected with testosterone propionate deposited protein
in the carcass, seminal vesicles, prostates, liver, and
kidney, in decreasing order. On continued treatment for
46 days, loss of carcass fat and protein occurred. This
protein apparently was diverted to accessory sex organs
and to the kidney, since these organs continued to grow.
Appetite changes were observed with testosterone admini—
stration to castrate males, whereas no effect was noted
in intact male rats. Determination of amino acids of
the gastrocnemius, liver, kidney, seminal vesicles, and
prostate indicated no qualitative or quantitative change
per unit weight of tissue. They assumed, therefore, that
true growth of tissue occurred. Total protein, non-
protein nitrogen, and amino acid nitrogen of plasma from
treated castrate rats were identical to those of control
rats.

In summary, androgens have an anabolic action in
castrate animals but not in intact animals. This is
manifested by retention of urinary nitrogen and reversal
of a negative nitrogen balance, but unassociated with any

elevation of plamma nitrogen. The retained nitrogen is

31

utilized in specific organ-tissue growth. The anabolic
effect of androgens on skeletal muscle has been des-
cribed in detail in the skeletal muscle section of this
literature review.

The mechanism of the anabolic effect remains to be
determined. It is effective in the hypOphysectomized
animal (Rupp and Paschkis, 1953), but it is known that
androgens lose their nitrogen-retaining ability in the
absence of insulin (Gaebler and Tarnowski, 1945). The
exact cellular mechanisms remain to be ascertained.

B. Estrogenic Effects

 

The action of estrogen was first investigated by
Thorn and Engel (1958). They found a marked decrease in
nitrogen excretion of five normal dogs after a single
subcutaneous injection of 5 mg. of alpha estradiol or
15 mg. of estrone, but no effect was noted after 20 mg.
of progesterone. This implies that estrogen is active
in nitrogen metabolism in a different manner or at a site
different from testosterone, since testosterone or
androsterone is not effective in intact males but only in
castrate animals while estrogen is effective in non-
Operated animals. Gaebler and Tarnowski (1945) found
that administration of estrone to normal dogs did not in-
duce elevation of N.P.N. or blood urea, in the presence
of reduced urinary nitrogen. Knowlton 23 a1. (1942) ad-
ministered 5 mg. per day of alpha estradiol benzoate to

two eunuchoids and one hypogonadal woman for 4~2O days

52

and noted decreased nitrogen excretion comparable to that
obtained with 5 mg. per day of testosterone prOpionate.
In studies of a 19-year—old normal girl, they found the
characteristic decrease in urine nitrogen on adminis—
tering 5 mg. per day of alpha estradiol; but as reported
above in dogs, progesterone did not decrease urine nitro-
gen.

Workers have found increases in plasma protein of
birds on estrogen administration. Common gt 21. (1948)
demonstrated that estrogen administration to chickens in-
creased serum calcium, phosphOproteins, lipids, and liver
proteins. Mandel, Clavert and Mandel (1947) demonstrated
in pigeons that the increase in total plasma proteins
following estrogen administration was mainly in the al-
bumin fraction. Sturkie (1951) studied the effect of
estrogen on plasma protein and plasma volume in chickens.
Using 20 mg. of dienestrol per day in their feed for 52
days, he found no significant difference in total plasma
protein. Continuing injections for 14 more days at 40
mg. per day per bird, plasma proteins increased from
5.14 gm.% to 8.50 gm.%. Globulins increased from 2.11
gm.% to 5.20 gm.%, with no change in the A/G ratio. Thus,
in chickens, plasma proteins are significantly increased
by estrogen as much as 65% within 14 days. After ceasing
treatment, plasma proteins returned to pretreatment

levels within two weeks. No significant change in plasma

55

volume or hematocrit was found by Sturkie with either
dosage of estrogen.

It may therefore be concluded that estrogens, like
androgens, are active in preparing a milieu favorable
for protein synthesis. Further, it has been demonstrated
in birds that one may induce synthesis of plasma pro-
teins. This has not been found to be true in mammals
(Gaebler and Tarnowski, 1945) (Knowlton.g£ al., 1942),
even though nitrogen retention has been demonstrated.

Estrogens have not been found to be uniformly anam
bolic hormones. Glasser (1954) studied the influence of
stilbestrol on nitrogen metabolism in adult male rats.
Injection of 0.1 mg. of stilbestrol daily for 20 days
resulted in a body weight loss and a negative nitrogen
balance. The latter was not due to an increased urinary
1itrogen, but rather to a reduction in food intake with~
out conserving body nitrogen. Honnone withdrawal re-
sulted in an immediate recovery of body weight and a
return to positive nitrogen balance.

Telfer (1953) noted the influence of estradiol on
nucleic acids, respiratory enzymes, and nitrogen dis»
tribution in rat uterus. Her work demonstrates a meta"
bolic system which is exceedingly sensitive to estrogen.
Nitrogen content of the uterus determined within 48 hours
of the first estrogen injection was markedly increased.
This increase was primarily in the mitochondria fraction

with the expected increase in ribonucleic acids.

34

 

Progesterone administration to ovariectomized rats did
not increase uterine nitrogen.

In summary, estrogen administration induces the re-
tention of urinary nitrogen in mammals in the absence of
any blood nitrogen change. Since testosterone results
in nitrogen deposition in the carcass, one might expect
estrogens to induce similar changes. As discussed in
the skeletal muscle section of this review, estrogen
has no effect on skeletal muscle weight. The most logical
remaining site of nitrogen utilization is in the uterus.
As already discussed, there is a rapid and marked re-
sponse of the uterus to estrogen administration. This
reminds one of the negative nitrogen balance and marked
urinary nitrogen loss in post-partum women associated
with involution of the uterus in the presence of low
post-partum estrogen.

The changes in plasma proteins and minerals in birds
is consistent with requirements of these animals for egg

laying, a process directly influenced by estrogen.

ESTROGEN EFFECTS ON ENZYME SYSTEMS
The ultimate explanation of biological actions of
estrogens will probably be found in an investigation in
the realm of enzymatic phenomena. The essential specific
enzyme sysiems in which estrogen is active as a substrate
or as a coenzyme must be elucidated. A number of enzyme

systems have been investigated.

55

These include: B-glucuronidase, alkaline phosphatase,
cholinesterase, and succinic dehydrogenase.
B-glucuronidase was one of the first to be investi-
gated, primarily because of the finding of the conjuga-
tion of steroids with glucuronic acid. A relationship
between estrogen activity and B—glucuronidase was first
established by Fishman and Fishman (1944). They exam-
ined influences on the enzyme of repeated injections of
ovariectomized mice with estrone, l7-B estradiol,
estriol glucuronide, stilbestrol, grogesterone, and
pregnanediol. The estrogens, natural or synthetic,
caused a rise in B-glucuronidase activity in the uterus,
but not liver, kidney, and spleen. Progesterone and
pregnanediol had no effect on uterine enzyme activity.
Ovariectomy was found to cause a fall in enzyme activity
in the uterus compared with intact controls. They ex-
plained these results in terms of adaptation of enzyme
concentration to quantity of substrate present, estrogen
being the substrate. Lack of response of non-reproductive
organs such as the liver, kidney, and spleen to estrogen
was explained by Fishman (1947) in one of two ways.
Either a specific glucuronidase for estrogens exists in
the uterus; or physicochemical factors, such as permea-
bility of tissue to estrogens, were responsible. The
findings for non-reproductive organs have not always been
consistent with Fishman's results. Kerr £3 31. (1950)

found stimulation of liver B-glucuronidase activity by

56

estrone in ovariectomized mice; and this effect was
antagonized by testosterone and progesterone, although
neither of the latter had any effect by themselves. This
action of estrone was also seen in normal and castrate
males, but was absent in intact female mice. Estriol and
estradiol did not affect liver B—glucuronidase activity.
No changes in the kidney enzyme were found. They related
these results to the stimulated mitotic activity of es-
trone in these livers; this was not seen in intact females.
Other tissues known to have alterations in B-
glucuronidase include proliferated human breast tissue in
pregnancy, with even higher values in carcinoma of breast
(Fishman and Anlyan, 1947). Blood plasma B-glucuronidase
activity increases during pregnancy and drops to pre-
partum levels after parturition (Fishman gt_§l., 1950).
This fall is prevented by administering stilbestrol
(Fishman gt gl., 1950). There is no sex difference in
human serum B-glucuronidase activity, suggesting that
the change found in the plasma activity during pregnancy
is related to spillage from tissue such as endometrium,
which is one of the richest sources of B-glucuronidase in
humans (Odell and Fishman, 1950). This work suggests
that B-glucuronidase activity is under endocrine control.
Whether it has the nature of an adaptive response to the
growth stimulation by estrogens remains to be deter—

mined.

57

Alkaline phosphatase of the uterus has been the
object of interest. Atkinson and Elftman (1946) found
that in castrated female mice large amounts of alkaline
phosphatase are present in the longitudinal muscle of
the uterus, whereas much smaller amounts are found in
the uterine glands and epithelium. On injection of
estradiol benzoate into these animals, a marked increase
in alkaline phosphatase was observed in uterine glands,
epithelium, and in circular muscle. In monkeys,

Atkinson and Engle (1947) found phosphatase increased in
endometrial glands following estrogen administration, and
subsequent reduction was noted on injecting progesterone.
In addition, these men found alkaline phosphatase activi-
ty was highest in endometrium of women during the pro—
liferative phase, reduced during the secretory phase, and
absent several days before the menses. Atkinson and
Elftman (1946) proposed that increase in alkaline phos-
phatase of the uterus after estradiol administration could
be related either to appearance of glycogen or to disap-
pearance of lipids. Stafford 33 El. (1947) observed in»
creases in acid and alkaline phosphatase in the corpora
lutea as pregnancy advanced and also during lactation.

In support of Atkinson's work, Li EI.§£° (1946) found

a decrease in alkaline phosphatase of rat plasma in
hypOphysectomy.

Birkhause and Zeller (1940) found that the livers

of mature female rats possessed 5-5 times as much capacity

58

for hydrolyzing acetylcholine as male rat livers. Sawyer
and Everett (1946) observed that serum cholinesterase
levels parallel estrogen activity, but not that of
progesterone. The enzyme levels were low in estrogen-
poor conditions, such as the first half of pregnancy and
the post-partum state; whereas they were high in condi-
tions characterized by high estrogen activity, such as
estrus and the last half of pregnancy. On castration of
female rats, the nonspecific cholinesterase of serum
dropped to a level of about that found in males. On
castration of male rats, this enzyme level became greater
than control male values. In the castrate female, ad-
ministration of estradiol restored blood cholinesterase
to normal while testosterone administeration antagonized
this estrogen action. Progesterone was found by these
men to have no effect on blood cholinesterase in cas-
trate female rats.

Succinic dehydrogenase and succinoxidase systems
have been investigated by the Meyer, McShan group. Meyer
31 2l° (1945) found lutein tissue of the ovaries of preg—
nant and pseudOpregnant rats rich in succinic dehydro-
genase. They observed a return to normal values in
corpora lutea following parturition (Meyer gt gl., 1947).
In addition, cytochrome oxidase concentration was much
higher in corpora lutea of pregnancy than in diestrus.
Investigation in zitrg of this enzyme system isolated

from liver and pituitary tissue of rats revealed that the

59

synthetic estrogen, stilbestrol, and estrone both pro-
duced inhibition (McShan and Meyer, 1946). In a study

of in 2113 effects of estrogen, liver and uterine tissues
were assayed for succinic dehydrogenase and cytochrome
oxidase, following treatment of adult female rats with
various estrogens daily for 10 days. They found inhi-
bition in these tissues when the animals had been treated
with estrogen containing the phenolic group, whereas no
inhibition was found associated with the alcoholic group
of androgens. The inhibition was shown to be through the
cytochrome oxidase. They suggested that phenolic groups
of estrogenic compounds combine with the active centers
and remain attached to the enzyme.

In this review of estrogen's action on enzymes, the
papers so far discussed have been mostly limited to ob-
servations on concentration of enzymes in tissue follow-
ing estrogen administration. This is far from deter-
mining the primary site of estrogen activity, since this
is a manifestation of the general growth-promoting action
of estrogen. The findings that estrogen inhibits certain
enzyme systems both in 31332 and in 3113 contributes
little to the answer of the basic mechanism of activity,
since it is highly unlikely that estrogen produces its
marked changes in metabolism by inhibiting synthesizing
reactions.

In contrast to the papers previously discussed is

the work of Hagerman and Villee (1955). They investigated

40

the mode of action of estrogen on human endometrium.

They found that small amounts of estradiol added‘in 32332.
increased the rate at which oxygen and pyruvic acid are
utilized. They found that in addition to endometrium,
placenta contained an estrogen-sensitive system, and this
was later extended to include the mammary gland, ventral
prostate gland, and the pituitary gland (Villee and
Gordan, 1955). Liver and kidney did not contain such an
estrogen-sensitive enzyme system (Villee, 1955). On the
basis of this work, Villee postulated the existence of a
specific DEN-linked isocitric dehydrogenase that was
activated by minute quantities of estradiol.

Talalay and Williams-Ashman (1958) confirmed exis-
tence of a soluble enzyme from human placenta that pro-
motes transfer of hydrogen between two pyridine nucleotide
coenzymes, and that this reaction is activated by minute
quantities of certain steroid hormones. The overall

reaction that is catalyzed:
TPNH + DPN+ —9 TPN+ + DPNH

TPN and DPN being the oxidized form of tri- and di-
phosphOpyridine nucleotides, with DPNH and TPNH being
the reduced forms. It was found by Talalay that the re-
sponse of crude placental extracts to steroids was vari-
able and that stimulation of reduction of DPN by certain
hormones disappeared upon fractionation of the extracts.

They observed that addition of catalytic quantities of

41

TPN to the reaction mixture increased markedly the ability
of different placental preparations to respond to steroids,
and even-restored the effect in inactive preparations.
Apparent stimulation of a DPN-linked isocitric dehydro-
genase can be accounted for in terms of coupling of TPN-
specific isocitric dehydrogenase of placenta with action

of a soluble trans-hydrogenating system described here:

+ \ alpha-keto
Isocitrate + TPN , glutarate (A)

+002 + TPNH + H

 

 

 

 

 

TPNH + mm“ - 1 ; TPN++ DPNH (B)
alpha-keto glut ate
Isocitrate DPN‘ 9 + 002 + DPNH + :3

 

In step B, the transhydrogenase system is activated by
steroids. Partially purified fractions from placenta
which catalyze hydrogen transfer between the two forms of
pyridine nucleotide also catalyze oxidation of steroids
by DPN and TPN. Of the steroids examined, only those
which can undergo oxido-reduction by pyridine nucleotide-
linked hydorxysteroid dehydrogenase are active. This is
consistent with the idea that proteins catalyzing dehydro-
genation are identical.

In further studies, Talalay 21 El: (1958) demon-
strated the estrogen sensitive transhydrogenase to be the
same enzyme as the l7-B estradiol dehydrogenase. Evi-
dence for this included the following findings: the two

enzymatic activities are not separated by purifying

42

 

fractionation procedures; and secondly, only those
steroids which can undergo enzymatic oxidation can be
active in transhydrogenation. Talalay's work led to the
following conclusions: (1) The enzymatic basis of
steroid-mediated transhydrogenation is ascribed to the
reversible oxidation of steroid by hydroxysteroid de—
hydrogenase which react at comparable rates with both
DPN and TPN. (2) Hydroxysteroid dehydrogenase with dual
nucleotide specificity may function as a pyridine nucleo-
tide transhydrogenase. Thus, it is suggested that
steroids may be more prOperly regarded as hydrogen car-
riers and pyridine nucleotides as substrates in these

reactions. As an example of such a coupled reaction:

 

 

 

 

 

Estradiol + DPN+ es Estrone + DPNH + H+
V
H+ + Estrone TPNH 6 } Estradiol + TPN+
Sum: TPNH + DPN+ > DPNH + TPN+

 

 

Hurlock and Talalay (1958) found that purified rat
liver 5-alpha hydroxysteroid dehydrogenase in the presence
of minute amounts of androsterone or other 5-alpha
hydroxy or 5-ketosteroids catalyze the transfer of hydro-
gen between DPN and TPN. They have therefore succeeded
in extending their original observations in support of

the idea of steroids being coenzymes of hydrogen transfer

between pyridine nucleotides.

45

The importance of these findings in explaining the
observations of other workers is easily seen. There is
evidence that DPN and TPN serve different metabolic
functions and that the natural occurrence of these two
different nucleotides is of great functional signifi-
cance. TPNH is active as a reducing agent in synthetic
reactions that take place outside the mitochondria and
in which DPNH cannot participate. An example of such a
reaction is the entry of l—C fragments into serine and
into purines catalyzed by a series of folic acid-
dependent enzyme systems which utilized TPNH as a specif-
ic hydrogen donor. Mueller and Herranen (1956) found
that shortly after administering 17-B estradiol to
ovariectomized rats, the incorporation of l-C fragments
into serine and purines of nucleic acids in the uterus .
was greatly increased.

In accessory organs of reproduction in the male, a
number of biochemical changes induced by testosterone
could have as their basis a change in TPN or DPN. These
biochemical changes include the synthesis of fatty acids
from acetate in the prostate gland, which is most sensi-
tive to the presence of testosterone (Nyden 33 21., 1955).
Androgenic steroids initiate and support the accumulation
and secretion of fructose and citric acid in some male
accessory sex tissue (Williams-Ashman 23 31., 1954).
Synthesis of fructose by these organs involves reduction

of glucose to sorbitol by TPNH, followed by DPN—linked

44

oxidation of sorbitol to fructose (Hers, 1956). Overall
conversion of glucose to fructose simulates the action of
pyridine nucleotide transhydrogenase, in that there is a
stoichiometric transfer of hydrogen from TPNH to DPN.
There appears to be ample evidence that many of the
observed biochemical changes induced by the presence of
estrogens may well be explained on the basis that estra-
diol is acting as a coenzyme in transhydrogenation.
There are, however, many compounds with high estrogenic
activity that are structurally incapable of participating
in hydrogen transfer systems. The second consideration
of importance is that certain compounds that are highly
estrogenic in one species are inactive or, at best,
weakly active in another; it remains to be found where

their biochemical action resides.

ESTROGEN METABOLISM

A thorough discussion of the metabolism of estrogen
has not been included in the literature review. However,
a few statements for completeness can be made here. The
exact chemical precursor of natural estrogens is not
known. Three possibilities exist: the first two concern
cholesterol. Levin and Jailer (1948) observed a decrease
in ovarian cholesterol following gonadotrOphic stimu-
lation. Direct evidence of transformation of cholesterol.
to l7—B estradiol, the true ovarian estrogen (Heard and

O'Donnell, 1954), is lacking. Cholesterol could be

45

converted first to progesterone, then through estrone to
l7-B estradiol or directly to l7-B estradiol. The third
possibility is based on finding labeled estradiol and
estrone in the perfusate of ovaries of the sow following
addition of labeled acetate (Werthessen.gtԤl., 1955).
It would appear to this writer that active 2—C fragments
are serving as the precursor of cholesterol and that
cholesterol could either be converted to progesterone or
directly to l7-B estradiol, depending on the presence of
the specific gonadotrOphin. This point in the steroid
cycle might well be the metabolic site of action of
follicle-stimulating hormone and luteotrophin.

The site of production of 17-B estradiol is believed
to be cells of the theca interna (McKay and Robinson,
1947). The question of interconversion of estrogens is a
complex one. Ryan and Engel (1955) demonstrated that
17-B estradiol and estrone are readily inter-converted in
human tissues including placenta, liver, adrenal cortex,
pregnancy breast, and hyperplastic endometrium. The im-
portance of this conversion in terms of transhydrogena—
tion (Talalay £1 31., 1958) has been discussed in the
enzyme section of the literature review. While both
estradiol and estrone are quite active estrogens, estriol
is the least potent and is considered a degradation prod-
uct (Gallagher, 1944).

The metabolism of estrogens appears to center, at

least in part, in the liver. The liver appears to be

46

the magor site of estrogen inactivation (Biskind, 1941).
The appearance of hyper-sstrinism has been well documented
in partially hepatectomized rats (Schiller, 1944) and in
human cirrhotics (Glass, 1950). It appears that the liver
carries out this function in two ways. Heller (1940)
found that incubation of l7-B estradiol or estrone with
liver slices induces a marked loss of estrogenic activity.
A second method in contrast to inactivation is the ex-
istence of an entero-hepatic circulation, in which
estrogens are excreted into the biliary tract (Cantarow
‘33 $1., 1942). The kidney also plays a role in estrogen
metabolism by conjugating estrogen with glucuronic acid
and sulfuric acid (Fishman, 1951).

This is a superficial review of the metabolism of
estrogens and this writer is not attempting to discuss all
areas of estrogen metabolism nor to infer that all the

questions are answered in any of the areas discussed.

47

MATERIALS AND METHODS

In these experiments the influence of gonadectomy
and steroid administration on male and female albino rat
hearts were investigated. Myocardial function was studied
in isolated surviving columnae carneae (trabeculae carneae)
of the left ventricle in preparations of glycerol-extracted
columnae carneae and further, by extraction of ventricu-
1ar actomyosin. Although the bulk of the experiments
were on female animals a few preliminary studies were made

on castrate and estrogen-treated males.

Experiment I. Determination of PrOperties of Isolated
Left Ventricular Carneae ‘
Sixty-six albino rats of the Sprague—Dawley strain
weighing between 75 and 100 grams at the start of the
experiment were employed in two series of experiments.

The animals were divided into the following groups:

Series A. ' No.
l) Intact females. 15
2) Intact males. 15
Series B.
1) Sham operated females given sesame oil 5
injections.
2) Ovariectomized females given sesame 6

oil injections.

5) Ovariectomized females given 0.1 gamma 8
alpha estradiol/day

No.
4) Sham Operated males given sesame 4
oil injections.

5) Orchiectomized males given sesame 7
oil injections.

6) Orchiectomized males iven 1.0 8
gamma alpha estradiol day.

In series B, treatment was initiated one day post-
Operatively and continued for a thirty-day period. In-
jections were made daily in varying subcutaneous areas.

On the final day of treatment, the animals were
weighed and killed by decapitation. The hearts were
immediately excised and placed in bicarbonate buffered
Ringer's solution described by Fcigen 23 El. (1952).
The composition of the solution was as follows: In
1 liter of distilled water:NaCl, 9.0 grams, KCl, 0.42
grams, CaC12, 0.62 grams, NaHCOB, 0.60 grams and glucose,
1.0 gram.

In addition to the heart, adrenals and uterus, or
instead of uterus,semina1 vesicles, were removed and
weighed wet on a torsion balance. After removal of the
columnae carneae, the heart with the above-mentioned
organs was placed in an oven at 100°C. and dry weights
determined after 24-48 hours.

Properties of left ventricular columnae carneae were
determined according to the method of Ullrick and
Whitehorn (1956). The heart was removed from the buf-
fered Ringer's solution and the right ventricle was cut

away, exposing the interventricular septum. The septum

49

was excised, exposing the chamber of the left ventricle.
On the posterior wall of this chamber, the paired col-
umnae carneae were found. These consist of rounded
muscle columns extending from the apex of the left ven-
tricle to the atrio-ventricular junction (Figure 1).

0n dissection, these muscle columns are cylindrical and
in a lOO-gram rat have a diameter of approximately one
millimeter and a length of six to eight millimeters.
The length of the columnae carneae nearest the inter-
ventricular septum was measured to the nearest 0.25 mm.
with a millimeter rule. This length is termed the "i2
gitg length".

Figure 2 is a diagram of the eXperimental apparatus;
numbers in parenthesis refer to this figure.

The bundle was dissected free and the atrial end (10)
fastened in a stainless steel muscle spring (9). The
apical end (10) was placed in a plastic clamp (7) which
contained within it a pair of chlorided silver elec-
trodes (ll). The electrodes were freshly cleaned and
chlorided for each experiment. The stainless-steel spring
(9) was attached by means of a connecting wire (4) to a
rigid spring arm (5) and a Statham strain gauge (5),
model 0.7-0.5-8000. This spring arm in turn was con-
nected to a micrometer screw (1) so that the arm could be
lowered or raised in relation to the muscle. In this
manner the muscle length could be adjusted to the measured

in situ length. The resistance to stretch, or resting

50

H/ax‘
; Trabecu/a \
Cornea Poster/hr waI/ of
' ‘ V _ _Leff Venin‘c/e

 
       
   

    

lnferventn‘cu/ar Sepfum

 

 

Figure 1. Photograph and diagram of rat heart muscle
preparation.

51

 

 

 

 

 

 

‘_ l . M i c r o m e i e r
to amplifier 2. Clamp
8. Strain gauge
4. Connecting wire
5. Rigid spring wire
f‘“ 6. Scale (or reading
resting tension

_— 7. Plastic muscle clamp with

F. incorporated stimulating

E electrodes

fi L :— 8. Metal tube
I _—_ 9. Muscle spring clamp
:_ 10. Muscle column
I: l l . Stimulating electrodes
l2. Rubber bands
,1,
A?
to stimulator
*.
. 2 n -4
l

 

 

 

 

 

 

Figure 2. Drawing of the experimental apparatus.

52

tension, of the muscle could be read in grams from a
calibrated scale (6), indicated by noting the position of
the arm of the spring (5) at the point of connection to
the muscle. The muscle connection was in alignment with
the spring arm and the strain gauge. Contractions of the
muscle on stimulation were essentially isometric.

Output of the strain gauge was amplified and recorded
on one channel of a Grass ink-writing oscillograph. The
system was calibrated by placing known weights on the
stainless steel spring clamp (9) and measuring the pen
deflection from the baseline on the oscillograph; thus,
changes from the baseline represent changes in tension of
the isometric contracting muscle. In this way the scale
for passive tension could be calibrated.

The strain gauge and muscle clamp assembly was
mounted on a vertical adjustable carriage which allowed
the unit to be lowered into a glass muscle bath. This
inner chamber was fixed in a Sargent constant temperature
water bath, so that the temperature of the muscle bath
was kept constant at 57.7° i 0.25°C.

The inner chamber, which held the muscle, contained
45 ml. of the buffered Ringer's previously described.
Fresh Ringer's solution was constantly added to the bath
at a rate of 5-6 ml. per minute by a steady drip. The
solution in the bath was oxygenated by means of oxygen

entering the bottom of the bath through a gas dispenser

55

tube. Volume of the bath was maintained by means of an
automatic overflow siphon.

After the muscle bundle was placed in the bath, it
was immediately stretched to igwgitg length and allowed
to equilibrate to the length adjustment (setting) for
45 minutes. Threshold to a square wave stimulus of
6 milliseconds' duration was determined, and active
contractions were induced atwa frequency of l per second.
The strength of the stimulus used was read from the cali-
brated screen of a cathode ray oscilloscOpe, which was
placed in series with the output of a square wave genera-
tor. Stimulus strengths were recorded in milliamperes.

A second channel of the Grass oscillograph received
a separate output from the stimulator which signalled the
generation of each pulse. A Franz timer was attached to
the oscillograph. In this way stimulus, muscle tension,
and time were simultaneously recorded. A sample record
of an isometric contraction, stimulus, and time line
are shown in Figure 5.

At the end of the 45 minute equilibration period
at in.§itg_length, refractory period, strength-duration
curve, and active and passive length-tension relationships
were determined.

Refractory period determinations were carried out by
applying a square wave stimulus of 6 milliseconds' duration
immediately following the driving stimulus. The strength
of both stimuli was approximately 10 times threshold

54

 

 

Record of an isometric contraction, stimulus

Figure 5.

and time line.

55

intensity. The Grass oscillograph was set to run at its
fastest speed while the interval between the driving and
second stimulus was gradually increased. This period
between stimuli was gradually increased until a second
response of the muscle was first seen. The time interval
between the two stimuli was then measured on the oscil-
lograph paper with the aid of the Franz timer and recorded
in milliseconds as the absolute refractory period.

Figure 4 is a typical refractory period determination.

Strength-duration curves were obtained by applying a-
stimulus of varying duration 500 milliseconds after the
driving stimulus. The strength of the second stimulus
was obtained from the calibrated oscilloscope. This pro-
cedure was repeated for stimuli of 10, 6, 4, 2, 1, 0.8,
0.6, 0.4, and 0.2 milliseconds' duration. Figure 6 is a
strength-duration curve.

Following this group of determinations, the strain
gauge was disconnected; and by means of the micrometer
screw on the spring arm mount, all resting tension was
removed from the muscle. The micrometer was then ad-
justed to the point where resting tension just began to
develop, and the strain gauge was reconnected to the
system. The muscle was then,allowed to equilibrate for
50 minutes. At the end of the 50 minute equilibration
period, that length, at which resting tension just began
to develop, was recorded as the equilibration length. A
record was then taken of the tension developed by the

56

 

 

 

 

 

Figure 4. A typical refractory period determination.

57

muscle. Muscle length was measured with a calibrated
binocular dissecting microscOpe, set beside the transpar-
ent bath. The muscle throughout this procedure, as be-
fore, was stimulated at a l per second frequency with

4-6 milliampere stimuli of 6 milliseconds' duration. The
muscle was then stretched in 10% increments of equili-
bration length, by increasing the tension on the muscle
by adjustment of the micrometer screw. At each new
length, and therefore new resting tension, a ten—minute
period of equilibration was allowed in order that the
length would become constant before a record of active
tension was obtained. Length-tension diagrams were pre-
pared by plotting the resting and active tension of the
muscle against the percent increase of equilibrium length.
Figure 10 is a length-tension diagram.

After length-tension data were obtained, the muscle
was removed from the bath and the active contracting por-
tion between the clamps was out free, weighed, dried at
100°C. for 24 hours, and then reweighed. The portions of
the muscle within the clamps were added to the remainder
of the ventricles and weighed wet, dried, and reweighed.

Experiment II: Extraction of Ventricular Actomyosin and
Studies on Glycerol-Extracted Columnae
Carneae '
Forty—six albino rats weighing between 80-100 grams

at the start of the experiment were used. The animals

58

were divided into the following groups:

1) Sham Operated, given sesame oil 8.
injections.

2) Sham Operated, iven 100 gamma 8
alpha estradiol day.

5) Ovariectomized, given 0.1 gamma 8
alpha estradiol/day.

4) Ovariectomized, given 1.0 gamma 8
alpha estradiol/day.

5) Ovariectomized, given 10.0 gamma 6
alpha estradiol/day.

6) Ovariectomized, given sesame oil 8

injections.

Treatment was initiated one day postOperatively and
continued for a thirty-day period.

On the final day of treatment, the rats were killed
as described in the first experiment. The hearts were
excised, Opened, and placed in ice-cold de-ionized water.
The columnae carneae were tied to applicator sticks at
in gigg length and glycerinated according to the tech-
nique described by Szent-Gygrgyi (1949) and Benson 3}; g_l_.
(1958). This consists of placing the bundles, tied to
applicator sticks, in 50% ice-cold glycerol for 72 hours.
At the end of this time the bundles were transferred to
fresh 50% glycerol and placed in a freezer at 0°C. for a
period of 2 weeks before use.

The remainder of the ventricles were weighed on a
‘Roller-Smith torsion balance and placed on a glass plate
chilled with ice. This tissue was minced fine with a

59

razor blade and transferred to a glass homogenizing
vessel surrounded by ice. Five m1. of cold 0.6 M KCl
buffered with 0.4 M NaHCO3 and 0.01 M Na2003 were added
and homogenation carried out for 6 minutes, using a tis-
sue grinder of the Potter-Elvehjem type driven by an
electric motor at high speed.

The homogenate was transferred to a 50 ml. centrifuge
tube. The homogenizing vessel was washed thoroughly with
2 ml. of ice-cold 0.6 M K01 and this was added to the
homogenate. Two-hundredths m1. of a 10% solution of the
disodium salt of A.T.P. was then added to the homogenate.
Following thorough mixing, the homogenate was placed in
a refrigerator at 6 i 2°C.

After 24 hours the homogenate was centrifuged for
10 minutes at 2,000 g. in a refrigerated centrifuge at
0°C. The supernatant was decanted. It appeared viscous
red-brown and moderately turbid. The volume of superna-
tant was measured and sufficient amounts of ice-cold
glass-distilled water were added to bring the molarity
of the 0.6 M KCl solution to 0.1 M. Actomyosin appeared
as a fine slightly flocculant precipitate. This solution
was allowed to stand in ice for 1 hour to allow maximal
actomyosin precipitation. It was then centrifuged at
0°C. at 2,000 g. for 10 minutes and the supernatant was
discarded. The sediment was then redissolved in 7 m1. of

buffered 0.6 M KCl.

60

One ml. of solution containing the redissolved pro-
tein was withdrawn for nitrogen determination by the
micro-Kjeldahl technique. The remainder of the solution
was used for determination of relative viscosity (0stwald
viscosimeters at 20°C.) before and after addition of
A.T.P. Determination of relative viscosity was performed
by measuring the outflow time of solvent without protein
and then determining the outflow time of the solution
containing protein before and after the addition of
0.02 ml. of a 5.0% solution of the disodium salt of A.T.P.

__’E.
n rel — t

0
where t = the measured outflow time of the protein-
containing solution.
t

0 = the outflow time of the medium without
protein.

n rel = relative viscosity.

Change in viscosity in response to A.T.P. has a direct
relationship to the concentration of actomyosin in solu-
tion. This is seen in Figure 5 and is another means of
quantitatively measuring actomyosin. This relationship
was first reported by Mommaerts (1950) and by Balenovic
and Straub (1942) for actomyosin of rabbit striated
muscle.

Viscosity data may also be expressed in terms of

"viscosity number" and "A.T.P. sensitivity" of Portzehl

61

 

ACTOMYOSIN
CALIBRATION
CURVE

 

 

+‘7250

 

 

O 02 0.4 0.6 0.8 |.O l2
PROTEIN CONC. Gm/L.

Figure 5. Actomyosin calibration curve.

62

33 El: (1950). "Viscosity number" relates viscosity to

protein concentration and is defined by the equation:

Viscosity number = Zn = g:2.l2€.2.£fll

Where 0 = concentration of protein in mg./ml. "A.T.P.
sensitivity" is a percentage and is used to characterize
actomyosin qualitatively as to actin content. This is

defined as:
A. To Po sensitivity 8 W X 100

Where Zn = viscosity number before addition of
A.T.P.

Z A. T. P. = viscosity number after addition
of A. T. P.

In summary, data obtained by this procedure allow a
quantitative and qualitative characterization of isolated
actomyosin. Isolation and purification of actomyosin was
completed by the utilization of Benson's gt‘gl. (1955)
modification of Szent-Gygrgyi's (1949) procedure.

DevelOpment of tension by A.T.P.-treated glycerinated
strips was determined as follows. After a two-week
period of storage in a freezer at 0°C., the glycerinated
bundles were transferred to 15% glycerol for one hour.
Each bundle was teased into two or more strips 0.2-0.5 mm.
in diameter and lOOps of fine silk thread were tied to
the ends of each strip. Each strip was then placed be-
tween two hooks. The tOp hook was fixed to a Statham

65

strain gauge (G-7-O.5-800): the bottom hook was adjusted
by means of a micrometer so that 50 mg. of tension was
placed on the strip. The strip was then lowered into a
muscle bath containing a perfusion solution of 0.05 M KCl,
0.005 M Mg012 and 0.0008 M CaCl2 buffered at ph 8.2 with
tris (hydroxymethyl) amino methane. Temperature of the
bath was maintained at 25°C. After equilibration for ten
minutes, the perfusion fluid was removed and replaced with
the same fluid, but with added disodium salt of A.T.P.

(5 x 10'3) at ph 7.0. The isometric tension developed by
the fiber was recorded by the Statham strain gauge and
suitable amplifiers on a Grass recorder. The bundle was
then removed, dried at 100°C., and nitrogen was deter-

mined by a micro-Ejeldahl technique.

64

RES ITS

In the 1956-1957 year a preliminary investigation
was completed. This consisted of a comparison Of myo—
cardial function of intact male and female rats. This
preliminary work will be referred to as series A in
Experiment 1. Series B of Experiment I consists of a
study of the influence of castration in both males and
females, including groups treated with two different
dosages of alpha estradiol. This work was completed in
the summer of 1957. Experiment II, completed in the
summer of 1958, involved studies on glycerol-extracted
columnae carneae and total ventricular actomyosin ex-
traction of intact,ovariectomized and estradiol-treated

ovariectomized and intact rats.

General Data

Terminal body weights

The mean terminal body weights for EXperiment I
and II are listed in Table 1. In series A the intact
males have continued to grow while the intact females
reached a plateau. Series B is consistent with series
A, intact females weigh significantly less than intact
males. Ovariectomy or ovariectomy with 0.1 gamma alpha
estradiol does not produce a significant difference in
terminal body weights from those of the intact female.
Growth appears to be decreased by castration of male

rats, but the difference is not statistically significant.

65

Table 1.

TERMINAL BODY WEIGHTS. MEANS i S.D.
Body Weight

 

 

Exper. I Groups No. in Grams
Series A l. Intact females 15 206.4 : 5.2
2. Intact males 15 247.2 i 55.0
Series B 1. Intact females 5 207 i 25.2
2. Castrate females 6 222 i 24.7
5. Castrate females 8 216 i 21.7
0.l‘{'<estradiol
4. Intact males 4 501 i 44.2
5. Castrate males 7 254 i 68.1
6. Castrate males 8 227 i 28.8**
1.0‘re4estradiol
Exper. II 1
l. Intact females 8 215.2 - 12.8
2. Castrate females 8 215.1 i 25.1
5. Castrate females 8 208.2 i 17.1
0 . 1 Yo< estradiol
4. Castrate females 8 187.7 i 7.6*
1.0‘724estradiol
5. Castrate females 6 172.7 i 12.4*
10.0‘XEQestradiol
6. Intact females 8 159.6 i 15.5*
100.0r{e(estradiol

 

*Significant at<1 0.01 level from intact females of
Experiment II.

**Significant at.410.01 level from intact males of
Series B, Experiment 1.

(Analysis of data by Unpaired Rank Analysis of F. Wilcoxon
1949

66

The mean of the castrate male groups with 1.0
gamma alpha estradiol is significantly less than that
of the intact male group.

In Experiment II the intact group and the ovariecto-
mized groups do not have different terminal body weights.
It would appear that treatment with alpha-estradiol in
sufficient dosage in ovariectomized rats results in a
decrease in mean body weights. This decrease in body
weight is statistically significant in 1.0, 10.0, and

100 gamma—treated groups as compared to the intact group.

Organ Weights: Heart, Adrenal, Uterine, and Seminal

 

Vesicles

With the observed changes in body weights, absolute
organ weights would give misleading impressions. In an
effort to avoid this, wet organ weight-terminal body
weight ratios have been calculated. Table 2 lists
these data for Experiments I and II.

The wet heart-body weight ratios are not different
in Series A or Series B of Experiment I or of Experi-
ment II. The hearts of animals in Experiment II are
larger than those of Experiment I, but no significant
difference exists between groups in Experiment II.

Adrenal-body weight ratios of the intact male and
intact female Of Series A and Series B are signifi—
cantly different. Castration of the male rat does not

result in a significant increase in adrenal weight.

67

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68

 

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Castrate male rats treated with alpha-estradiol are
significantly different from intact males. Ovari—
ectomy does not result in significant adrenal weight
changes. One or 10 gamma of estradiol per day in
ovariectomized rats results in a significant adrenal
weight increase as compared to intact females,

whereas 0.1 gamma or 100 gamma-treated females are not
significantly different from untreated intact females.

Uterine weight was significantly reduced in the
ovariectomized groups of Series B and Experiment II.
Estradiol administration significantly increased
uterine weights in the ovariectomized groups. This
increase is directly prOportional to the quantity of
estrogen administered, up to a limit.

Seminal vesicle weights of the castrate male rats
are significantly reduced as compared to the intact
males. Seminal vesicles of castrate males receiving
estrogen were not significantly different from the
non-treated castrate rats.

Water content of the heart

Water content of the myocardium was not altered

in any of the groups in Series A or Series B. Table 3

lists mean group values for Series A and B of Experi-

ment 1.

69

Table 5.

WATER CONTENT OF VENTRICULAR MYOCARDIUM. MEANS i S.D.

 

Groups N2; Percent Water
Exper. I
Series A 1. Intact females 15 76.40 i 0.62
2. Intact males 15 77.64 i 0.75
Series B 1. Intact females 5 78.05 i 0.52
2. Castrate females 6 77.89 i 0.54
5. Castrate females 8 77.89 i 0.41
0.1% «Lestradiol
4. Intact males 4 77.80 i 0.68
5. Castrate males 7 78.76 i 1.20
6. Castrate males 8 77.97 i 0.20

l . 0 X ocestradi 01

 

(Analysis of data b Unpaired Rank Analysis of
F. Wilcoxon (1949) .

70

Electrical PrOperties of Isolated Surviving Columnae

Carneae

Refractory period

Table 4 shows the results of the refractory
period determinations for series A and B of Experiment I.
There was no significant difference between intact
groups and eXperimental groups.
Strength-duration curve

The data for the strength—duration curves for
series A are found in Table 5. No difference between
the male and female groups exists. Figure 6 confirms
this, since the curves are exceedingly close. Table 6
lists the chronaxm and rheobase determinations for
series A and the female groups of series B. No differ-
ence was found in any of the groups. Chronaxie and
rheobase determinations were not completed for the male

groups of series B.

Mechanical PrOperties of Isolated Surviving Columnae
Carneae

Passive-tension curves of isolated surviving columnae
Carneae

Figures 7, 8, and 9 show passive-tensions of the

groups of series A and B of Experiment I, plotted

against percent of equilibrium length. The passive-

tensions develOped for intact male and females of

series A are not different. Comparison of the intact

71

Table 4.

REFRACTORY PERIODS 0F ISOLATED SURVIVING COLUMNAE CARNEAE
Means : S.D.

Experiment I.

 

Series Groups No. Milliseconds
A 1. Intact females 15 58.2 i 5.6

2. Intact males 15 61.4 i 7.0

B l. Intact females 5 56.5 i 11.2

2. Castrate females 6 61.6 i 8.6

5. Castrate females 8 54.5 i 8.5

0.1Y°< estradiol

4. Intact males 4 57.6 i 8.0

5. Castrate males 7 54.6 i 7.4

6. Castrate males 8 56.7 i 5.8

1.0‘{3<estradiol

 

(Analysis offidataby Unpaired Rank Analysis of F. Wilcoxon
1949

72

Table 5.

STRENGTH~DURATION VALUES FOR SERIES A

Means : S.D.

Experiment I.

 

 

Duration of Threshold
Stimulus in in
Milliseconds Np; Microamperes
Females ‘Males
10 14 152 i 19.4 121 i 20.0
6 14 158 i 20.8 155 i 22.2
4 14 185 i 22.2 159 i 28.0
2 14 216 i 45.5 184 i 54.4
1 14 274 i 52.2 221 i 40.8
0.8 14 519 i 69.0 275 i 45.6
0.6 14 565 i 69.4 555 i 56.1
0.4 14 478 i 92.7 456 i 88.8
0.2 14 725 i 110.0 845 i 155.5

 

Anal sis of data by Unpaired Rank Analysis of F. Wilcoxon
1949

75

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l
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Table 6.

CHRONAXIE AND RHEOBASE FOR EXPERIMENT I. MEANS : S.D.

 

 

Chronaxie Rheobase
in in
Groups Milliseconds Microamperes
+
Series A Intact females 0.80 i 0.14 152 m 17
Intact males 0.90 i 0.19 121 I 19
Series B Intact females 0.86 i 0.16 156 i 16
Castrate females 0.92 i 0.18 142 i 20
Castrate females 1.10 i 0.20 128 i 19

0.1‘{e<estradiol

 

(Analysis of data by Unpaired Rank Analysis of F. Wilcoxon
(1949 )

75

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77

 

PASSIVE TENSION "" MALES
7.- INTACT 0—0 u
CASTRATE 0—-—O
61- CAST RATE 'I' --—n
ESTROGEN “ x/
p”
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IOO IIO I20 I30 I40 I50 I60 I70
PERCENT EQUILIBRIUM LENGTH

78

Passive—tension curves for male groups of Series B,

Experiment I.

Figure 9.

and experimental curves for females and males show similar
shapes and slepes in all six groups of series B.
Developed Tension of isolated surviving columnae carneae
Figures 10 and ll show tension develOped as a func-
tion of equilibrium length for the female and male groups
of series B. It is clear that muscles from ovariectomized
untreated animals deveICp less tension at a given length
than do control muscles. Values for mean maximal tension
develOpment shown in Table 7 also indicate a reduction
in femalesT due to castration. The ovariectomized treated
group is not significantly different from the intact
group. The male rat curves (Figure 11) show no difference
between intact and experimental groups. Curves for intact
male and female rats are likewise similar. Table 7
lists the maximal tension in grams per milligram of wet
weight for series A and B of Experiment I. The results of
series A show no difference between intact males and fe-
males. Maximal tension develOped by the male experimental
and control groups are not different.
DevelOpedeTension of GlycerolaExtracted Columnae Carneae
Table 8 lists the maximal isometric tension den
veloped in the presence of A.T.P. Tension is expressed in
milligrams per milligram of protein per millimeter of
fiber length. This is an attempt to relate tension to
crossasectional area. Tension developed was significantly
reduced in fibers of the untreated ovariectomized group.

Treatment with increasing levels of estradiol resulted in

79

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Table 7.

MAXIMUM DEVELOPED TENSION OF ISOLATED

SURVIVING COLUMNAE CARNEAE

4.
Means 1 S.D.

Groups
_‘—

 

 

 

Series A
l. Intact females
2. Intact males
Series B
l. Intact females
2. Castrate females
3. Castrate females
0.3364 estradiol
4. Intact males
5. Castrate males
6. Castrate males
1.0 Yokestradicl
Y - Y
*t test = p 1 2
2 2
E1 1 52.
n1 n2

12(9) 7: 2035

Max1mum Tension

 

grams mg. wet weight

4-

0.256
0.253 I

l+

I+

0.156

[H-

0.253

II+

0.258

fi+

0.294

1+

0.284

00096
0.080

0.081
0.056*
0.034

0.091
0.117
0.058

where Y. and Y9, and

2

SI and SS are

the respective

means and variances of the

two series of n- and 112

individuals.

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increasing the mean maximal developed tension; however,
there was greater variability in these groups. The un-
treated ovariectomized group and the 100 gamma treated
intact group are significantly different. The intact
treated group is not different from the untreated intact
.groups.
Nitrogen Content of Glycerol-Extracted Qolumnae Carneae

Total nitrogen of glycerol-extracted bundles was
determined. These data are listed in Table 8. No
significant difference in total nitrogen content was
observed for the control or the experimental groups.
Quantitative and Qualitative Determinations of Total

Ventricular Actomyosin

Actomyosin content of ventricular muscle was de-
termined by precipitation with determination of nitrogen
content and by change in viscosity on addition of A.T.P.
Values found by these two methods are listed in Table 9.
The mean values for each group obtained by the two methods
agree well. The quantity of contractile protein isolated
is markedly reduced in the ovariectomized group. The
mean actomyosin isolated increased as the quantity of
estrogen administered increased; however, the variability
also increased. There is no significant difference be-
tween the estrogen-treated groups.

Qualitative information about actomyosin was also
derived from viscosity studies. These data are also

listed in Table 9.

84

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'75

relative visctsity for protein content. No difference
between groups wa. found when this correction was made.
As an index of actin content. expressed in viscosity
response to A.T.P., "A.T P- sensitivity" was calculated.
The mean values for the ovariectomized group are somewhat
lower than for the intact group. and there was some ing
crease on estrogen administration; however, variability

is large and differences are not statistically significant.

(1)
O\

Study of isilated surviVing columnae carneae of

ovariectomized rats demérstra:

)
I'D

imprired ability to
develop tension upon stimulation. Developed tension of
columnae carneae of estrogenetreated ovariectomized fee
males was not unlike control females. Changes in ability
to develop tension on stimulation was unaccompanied by
any alteration in heart—body weight ratios, indicating
that no change in work requirements was induced by the
experimental conditions. Explanation of such an observa»
tion should be found in a consideration of physiology of
muscle contraction. SzenteGygrgyi (1953) states:
"contraction in mu ole is essentially a reaction of
actomyosin, A.T.P., and ions". Since columnae carneae

studies were completed in vitro in a controlled milieu.

 

free of extrinsic nerves and humoral agents. an intrins c
change in cardiac muscle must have occurred. This alterm
aticn can be placed in one or more of three areas: First.
changes in membrane and electrolyte composition of cells;
second, changes in the energy~supplying systems; and
lastly, changes in the contractile protein, actomyosin.
There were no changes in percent water of the
myocardium and electrical properties of columnae carneae
in experimental and control groups. suggesting that sigu
nifioant change in cellular electrolytes did not occur.

However. determinations of transmembrane potentials and

87

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tension can be obtained only by stimulating the membrane

simultaneously at several points suggesting that with

progesterone, propagation of depolarization fails to occur.
Such a phenomenon has been observed by Csapo (1959) when
an estrogenwdominated uterus was exposed in vitro to a
modified Krebs solution containing excess potassium and
low sodium. The inference is that progesterone reduces
both potassium and sodium gradients across excitable
membranes. Marshall (1959) demonstrated existence of foci
in estrogenmdcminated uteri whose electrical potentials
are analogous to those of the sinus node of hearts, i.eo.
slow membrane depolarization culminating in the upstroke
of an action potential. These observations suggest an
important functional site of steroid activity on cell
membranes with a direct role on electrolyte transfer.

It was thought necessary, because of the work of
SzentnGygrgyi and Csapo, to evaluate the role of the cell
membrane and electrolyte metabolism to the altered response
of the experimental female groups. This was accomplished
by use of a model of muscle9 namely, glyceroleextracted
muscle. SzenteGycrgyi (1949) reported on extraction of
rabbit psoas with 50% glycerol. Such a preparation repree
sents a contractile system in which membrane effects have
been eliminated. Small molecular weight compounds and
electrolytes are eluted. Contraction is then induced by
perfusion of glycerinated fibers in an electrolyte solu»

tion in the presence of A.T.P. Observations on such a

89

preparation therefore immediately implicate or rule out
the importance of tell membrane and electrolyte change.
The results of glycerol~extracted fiber studies completed
by this writer paralleled those of isolated surviving
columnae carneae studies. Glycerinated fibers of hearts
of ovariectomized animals develOped significantly less
tension than those of intact females. Groups receiving
estrogen were intermediate in their response, with in»
creasing tension production as estrogen administration
increased. One can conclude that altered ability to de-
velop tension in these experiments must be due to changes
other than at the cell membrane or in electrolyte cone
centration.

In the preparation of glycerolmextracted fibers,
small molecular weight compounds are eluted, including
highuenergy phosphate compounds (SzentmGygrgyi, 1949).
Among these, A.T.P. or some nucleotide is required to
induce contractions. Lacking this, contraction fails.
Schumann (1940) demonstrated reduced quantities of A.D.P.
and P.C. in hearts of castrate male rats. One would ex-
pect altered concentrations of such compounds in hearts
of ovariectomized rats; however, quantitative determina»
tions of these compounds were not performed. Since
glyceroleextracted fibers in the presence of A.T.P.
sufficient to induce contraction failed to achieve cone
trol tension levels, one would question the functional
importance of reductions in these highwenergy phosphate

compounds. 90

The finding that reduced develOped tension in relation
to presence or absence of estrogen in the glycerol—extracted
preparation infers there is either a change in configura-
tion of actomyosin and/or a change in quantity of acto-
myosin present. The literature furnishes several examples
of functional and endocrine states in which actomyosin is
altered either quantitatively or qualitatively. Benson
(1955) reported that dogs with experimental heart failure
showed a decrease in concentration of ventricular acto-
myosin, reduced viscosity per unit of actomyosin, and de-
creased viscosity response to the addition of A.T.P.

Benson suggested that a defect exists in longitudinal
orientation of actomyosin produced by increased resting
tension characteristic of hearts in failure. Ellenbogen
gt_gl. (1959) found a molecular weight change in acto-
myosin isolated from dog myocardium in failure; molecular
weight increased from 223,000 to 740,000. Blasius gt‘gl.
(1956) observed no change in quantity of actomyosin iso-
lated from hearts of castrate male rabbits as compared to
hearts of intact males. However, the concentration of
actomyosin in cardiac muscle of castrate male rabbits

was increased by testosterone treatment. Endocrine effects
on actomyosin of skeletal and uterine muscle have been
reported. Testosterone is known to play a.regulatory role
on concentration of actomyosin of head and neck muscles

of guinea pigs (Scow'gtlgl., 1955), and on the levator

ani group of muscles of male rats (Stewart, 1955).

91

Estrogen has been said to have such a function in only
one muscle, namely, uterus. Csapo (1948, 1950 a, 1950 b)
demonstrated that the primary hormone involved in metabo-
lism of contractile proteins of the uterus is estrogen.
In this writer's work, actomyosin of the myocardium
was studied both quantitatively and qualitatively in
intact, ovariectomized, and estrogen-treated groups.
The results demonstrate that estrogen has a fundamental
role in function of the myocardia of female rats. Altera-
tions in tension in isolated surviving columnae carneae
and glycerinated columnae carneae are related directly to
quantitative changes in actomyosin concentration.
Qualitative changes in the actomyosin molecule may
be ascertained by determination of "A.T.P. sensitivity"
(as described in "Materials and Methods") and for intrin-
sic viscosity. Measurement of viscosity response of iso-
lated actomyosin in the presence of A.T.P., calculated as
"A.T.P. sensitivity", has been considered to be a measure
of the actin content (Portzehl gt‘al., 1950). "A.T.P.
sensitivity" values for the ovariectomized group are some-
what lower than for the intact group; and there was some
increase on estrogen administration, but variability was

large and differences are not statistically significant.

n rel—1

ofprotein/ml ) relates relative

Intrinsic viscosity (mg

viscosity (see pagefil) to protein concentration. When
thus expressed in terms of protein concentration, no dif—

ferences in viscosity were observed in any group.

92

i those obtained

f91'71terts Cfi"7 it? il.5&; '13"F), viva feund tin: low-esi
qiicti': s a t‘.v*s;n when G Tcuiating estrogen was
l*w CY atse 1 Ir pregnan v or with increasing quantities
of estrogen. ectomy’ei: irrreased. This was also substan~

tiated by Blasius e: at 579??) and Cretius (1957). With

respect it a:t m csfin content: cardiac and uterine muscle

respond Similarly to estrogen. However, estrogen results

in a marked increase in uterinesbody weight ratios, where«
as heartrlvwiv weighf'ivi‘ioc rmfle not irfl“rmnmced by the

9““? If estrogen The conclusion is that

resen e vr
estrogen has, in addition to the classical growth‘promoting
action on uterus, a prominent role in specific contractile
protein synthesis.

Several questions immediately arise if one postulates
an estrogen funrticn in maintenance of myocardial acto-
myosin. First, what maintains or regulates actomyosin
concentration in the myocardium of male rats? In EXperi~
ment I, three male groups were investigated. No differences
in tension production of isclated surviving columnae carneae

of intact, crehiectcmized9 or estr- en treated crchiecto~

0)
gr;

mized males were faund. The re nits in orchiectomized and

U)

intact male groups are consistent with Blasius7 et 1.
(1956) findings. Lack of effect in hearts of males re»
ceiving 1.0 gamma alpha estradiol could be interpreted in

a number of ways. Since Blasius a: a1. (1,56) found that

increasing quantities of test-sterone induced increasing

93

quantities of actomyosin in hearts of castrate male rabe

its, it could be postulated that estrogenis influence on
actcm,csin is limited to hearts of females, while testos~
terone s activity would be limited to hearts of males.
Such an idea is not consistent with our knowledge of here
mone action. Rather, one could speculate and state that
there is a difference in the sensitivity of the involved
metabolic systems; that is, to produce an effect in males,
the quantity of estrogen needed is many times greater than
that required to produce similar effects in females.

The lack of effect of castration on develOped tension
of male hearts does not rule out the effectiveness or im*4
portance of estrogen in males. The adrenal glands could
well be altering their output of estrogenic principles,
in response to elevated gonadotrophins. The most likely
reason for a difference in response to orchiectomy and
ovariectomy on tension production is that they do not
produce equivalent alterations in endocrine balance.

The question as to whether the increased actomyosin
found with estrogen administration is mediated directly
by estrogen or indirectly through the pituitary should be
considered. Experiments reported in this thesis have not
taken into account effects of the pituitary. No data are
reported on hypOphysectomized rats. The literature shows
that hypOphysectomized rats have a reduced heartmbody
weight ratio, and that administration of growth hormone

results in an increase in heart weight (Beznak, 1952).

94

Growth hormone, as well as specific trOphic hormones,
thyroidwstimulating hormone, and the adrenocorticotrOphic
hormone may well be involved in cardiac muscle metabolismo
As in all endocrine studies, ablation of one gland results
in changes in the entire endocrine constellationo In com-
parison of intact females to intact male rate, no differ—
ence in tension production of isolated surviving columnae
carneae was foundo This is consistent with Blasiusc 33 El'
(1956) work, where no difference was found in actomyosin
content in hearts of intact males and femaleso

The next question of importance is: If ovariectomy
produces such pronounced changes in actomyosin concentra-
tion, does it also produce in 3132 alterations in cardiac
function? None of the ovariectomized animals gave any
indication of cardiac failure, ioeo, edema, pulmonary con~
gestion, or excessive weight gainsa A possible eXplanation
for the failure to observe a reduction in cardiac reserve
is the reduced physical activity reported for ovariectomized
females (Pincus, 1950)o Operated animals were not exercised
or stressedo If they had been, it is possible that there
would have been a demonstration of reduction in cardiac
reserveo

Finally, if cardiac actomyosin increase is a direct
effect of estrogen, how is this mediated? A search of
the literature reveals no studies of estrogen effects upon
cardiac enzymeso Villee (1955) observed an estrogen~

sensitive enzyme system which increased utilization of

95

oxygen and pyruvatev This observation was first made in
the endometrium but was later extended to the mammary
glands, ventralwprostate gland, and pituitary; while

liver and kidney did not contain such an estrogenmsensitive
systemu No statement of investigation of heart muscle was
made. The possible regulatory role of estrogens or andro~
gens upon enzyme systems of heart needs investigation.

Certain aspects of the studies in this thesis require
special comment. This will be limited to the glycerol»
extracted fibers and actomyosin studies.

Variability of glycerolwextracted fiber data was
great. In part, it is believed to be due to the intrinsic
nature of the preparation. Briggs“ (1958) data shows a
variability in the standard deviation of the means as high
as 55%, while Benson°s gt Ely (1958) work has a variability
in standard deviation of 16%. Furthermore, in the present
study, fibers were not tied prior to removal from the
hearts but rather in situ measurements were made; and
after removal, the bundles were stretched to lfl.§li§
length and then tiedo Some bundles could not be stretched
to lfl.§l:2 length and some were stretched to lengths

greater than in situ length° Since the magnitude of conw

 

traction is dependent on initial length of the prepara~
tion, one may see an obvious reason for variability.

With respect to the actomyosin study, one should men;
tion literature reports on quantities of actomyosin isow

lated from different muscles. It has been demonstrated

96

that actomyosin is found in decreasing quantities in
skeletal, cardiac, and uterine muscle (Csapo, 1950 a).
The quantity of actomyosin isolated per unit weight of
the heart depends on the chamber from which the sample
is taken. Left ventricle has more than right ventricle
(Kovats, 1949). Similarly higher concentrations of
actomyosin are found in the fundus of the uterus as com-
pared to the cervix (Cretius. 1957). Values reported
for dog (Benson gt al., 1955), pig (Kovats, 1949), and
rabbit (Kovats, 1949) hearts eXpressed in percent wet
weight of tissue are, 5.4 u 5.7%; 2.9 _ 3.5%; and 2.0 u
2.7%, respectively. Rat heart actomyosin concentration
I first reported here was 0.5 ~ 1.0%. This value is ap-
proximately half that reported for a closely related
species, the rabbit. Total prorein determinations on t
ventricular myocardium were not done; but rather, total
protein was determined on the extracted fibers. No
change in total protein in any of the female groups was
found. It is easily seen that actomyosin could be
greatly reduced before any change in total protein would
be noted. ’

The difficulty in extracting actomyosin from cardiac
muscle should be mentioned. SzentaGygrgyi (1949) first
attempted this, and encountered such problems that he
selected the rabbit psoas as his source of actomyosin.
The first investigator to isolate and quantitate cardiac

actomyosin was Kovats (1949).

97

This writer realizes that the study reported here
is incomplete, Data on hypOphysectomized rats and an
extensive study of hearts of male rats is necessary.

In addition to tension and actomyosin studies on the
above groups, females with higher dosages of estrogen
should also be investigatedo It would be interesting
to note the effects of these steroids on smooth muscle
other than the uterus and on skeletal muscle. If con-
sistent results could be found for the mentioned groups
and smooth muscle, such as the taenia coli, a more
unified theory of muscle development and maintenance by

steroids would be possible.

98

SUI‘JI‘IARY AI‘ID COUCTUSI 038

l. The influence of castration and estrogen
administration on the prOperties of the myocardium of
the albino rat was studied.

2. Heart weight, heart~body weight ratios, and
water content of the myocardium were not altered by
ovariectomy, orchiectomy, or estrogen treatment of oas-
trate males and females. No differences in these para-
meters existed between intact male and female groups.

3. Expected changes in organ~body weight ratios
for adrenal, uterus, and seminal vesicles were found in
the experimental groups.

4. in vitro studies of isolated surviving columnae
carneae revealed no differences in excitability curves
or refractory periods between intact females, intact
males, and experimental female groups. Study of mechani-
cal prOperties revealed a significant reduction in mean
maximal developed tensions in castrate females, as
compared to intact females. DevelOped tension in estrogen-
treated castrate females was not different from intact
females. No significant difference in develOped tension
was observed in any of the male groups. Passive tension
curves were not altered in any of the male or female
groupso

5. Maximal A.T.P.-induced tension of glycerol-

extracted fibers of hearts of untreated castrate females

99

was significantly reduced from that of the intact females.
Tensions developed by fibers of estrogen treated castrate
groups were not different from intact controls. Total
nitrogen content of the glycerolaextracted columnae
carneae was unaltered by ovariectomy or estrogen treatment.

6. Total ventricular actomyosin content was signim
ficantly reduced in ventricles of castrate female groups,
as compared to ventricles of intact females. Actomyosin
content increased in the estrogen treated castrate groups.
No qualitative change in actomyosin was found.

7. It is concluded that there exists a dependence
of the cardiac contractile system on estrogen, similar to
that known for the uterus. This suggests a general effect

of estrogen on contractile protein of all muscle.

100

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