BIOCHIMICAET BIOPHYSICAACTA
727
BBA 75060
T H E E F F E C T OF E S T R O G E N ON AMINO ACID T R A N S P O R T IN RAT
UTERUS
R O B E R T ROSKOSKI, JR.* AND DONALD F. S T E I N E R
Department of Biochemistry, University of Chicago, Chicago, Ill. ( U.S.A .)
(Received April 3rd, 1967)
(Revised manuscript received May 26th, 1967)
SUMMARY
I. Some characteristics of amino acid transport in the isolated rat uterus have
been studied using the non-metabolized amino acid analogue a-aminoisobutyric acid.
The effect of estrogen injection into ovariectomized rats on the rate of transport of
a-aminoisobutyTic acid was also determined.
2. A single injection of estrogen increased the initial rate of a-aminoisobutyric
acid transport into uterine cells. The effect was evident b y I h and maximal at
8 h; it persisted for more than 24 h. Control values were reached again b y 48 h.
The rate of a-aminoisobutyric acid uptake in vitro was the same in the presence or
absence of glucose.
3. Several natural amino acids inhibited transport of a-aminoisobutyric acid
to a greater or lesser extent, whereas others were without effect.
4. I t is concluded that estrogen increases the rate of uptake of a-aminoisobutyric acid by the isolated rat uterus, and that it m a y increase the rate of transport
of a number of the natural amino acids.
INTRODUCTION
In I957 NOALL eta/. x demonstrated that the concentration of the non-metabolized amino acid analogue, a-aminoisobutyric acid, in the immature rat uterus was
increased 280% 20 h after estrogen injection 1. DANIELS AND KALMAN* reported
that this effect of estrogen was detectable after 2 h and was maximal at I2 h. NOALL
AND ALLAN8 were able to demonstrate an increased rate of a-aminoisobutyric acid
uptake in the rabbit uterus in vitro within 30 rain after hormone treatment was
given in vivo. HALKERSTON et al. ~ reported that a-aminoisobutyric acid uptake
in the uterus of the ovariectomized rat was unchanged 1. 5 h after estrogen injection,
but was increased after 6 h. RIGGS and co-workers s found t h a t uptake in vivo of
the model amino acid I-aminocyclopentanecarboxylic acid b y the immature rat
" Presently Captain, USAF, MC, Wilford Hall USAF Hospital, Aerospace Medical Division
(AFSC), Lackland Air Force Base, Texas, U.S.A.
Biochim. Biophys. Acta, i35 (1967) 727-731
728
R. ROSKOSKI,Jr., D. Y. STEINER
uterus was increased after estrogen injection. In addition, KALMAN AND LOMBROZEe
demonstrated an increase in the concentration of m a n y of the free amino acids in
the rat uterus 4 h after estrogen injection.
Uterine cell membrane transport has been discussed in several recent reviews ~-9.
The data from all of the studies mentioned, except those of NOALL AND ALLAN,
demonstrate the ability of the uterus to develop and/or maintain a concentration of
the amino acid but do not necessarily define a maximal rate of uptake. In the present
study, the rate of transport of a-aminoisobutyric acid was measured during relatively
short periods of incubation in vitro in an effort to approximate an initial, maximal,
value for the rate of uptake of the analogue.
METHODS AND MATERIALS
Chemicals
The a-amino[IJiC2isobutyric acid (I mC/mmole) obtained from the New
England Nuclear Corp. was kindly supplied b y Dr. I. G. WOOL. Unlabeled amino acids
were purchased from Calbiochem Corp. The sources of the other materials were
listed previously 1°.
Incubation medium
Uterine horus were incubated in Krebs-Ringer bicarbonate buffer ll equilibrated with O2-CO 2 (95:5, v/v) (pH 7.4) at 37 % Each ml of incubation medium contained o.Io #C of a-aminoEI-14C:isobutyric acid (IoopM) and 0.05 pC of D-[I-SH]sorbitol. Carrier D-sorbitol was added to make its final concentration I.O mM.
Additions to the medium were made in Io-pl aliquots to give the final concentration.
L-Aspartate, L-giutamate, L-tyrosine, and L-tryptophan were added to the buffer as
crystalline solids to give the desired final concentrations. Each uterine horn was incubated in 0.5 ml of medium for the specific time period.
[ I ntr acellular a-aminoisobutyric acid J /[ E xtr acellular a-aminoisobutyric acid]
Uterine 14C and 3H uptake was measured as described previously 1°. The extracellular distribution of a-aminoisobutyric acid was assumed to equal the distribution
of h-sorbitol.
[intracellular a-aminoisobutyric acid]
total a-aminoisobutyric acid -- extracellular a-aminoisobutyric acid
=
[extracellular a-aminoisobutyric acid]
extracellular a-aminoisobutyric acid
Details pertaining to the preparation of animals and procedures for incubation of the
uteri, measurement of radioactivity, calculation of results, and administration of
hormone have been described elsewhere 1°. The dose of estrogen employed was I.O pg
of I7/~-estradiol per animal.
RESULTS
Uptake of a-aminoisobutyric acid by uterine horns from untreated and estrogen-treated
(2 h) ovariectomized rats
The upper curve (Fig. I) shows the rate of a-aminoisobutyric acid uptake b y
uteri from rats that received estrogen 2 h before sacrifice and the lower curve, the
Biochim. Biopkys. Acta, 135 (1967) 727-731
AMINO ACID TRANSPORT IN UTERUS
729
rate from rats which received only the vehicle. The same values are obtained in the
presence or absence of 8 mM glucose; therefore, the increased uptake of a-aminoisobutyric acid is independent of sugar transport. In both cases, the ratio of intracellular to extracellular a-aminoisobutyric acid after incubation exceeds I.O. Since
a-aminoisobutyric acid is not metabolized b y this tissue, this ratio indicates that it is
concentrated in the cells. It follows that entry occurs, at least in part, by means of an
active process, rather than by simple diffusion.
Time course of the a-aminoisobutyric acid transport response to estrogen
Animals were sacrificed at various intervals after estrogen injection (Fig. 2).
The control group received vehicle 2 h before sacrifice. The a-aminoisobutyric acid
uptake was measured after incubation for 30 min in vitro. The results show a 50 %
increase after I h of hormone treatment (P < 0.02). The m a x i m u m rate occurs at
8 h; the rate remains elevated for at least 24 h and returns to the control level b y
48 h.
Effect of amino acids on a-aminoisobutyric acid uptake
Uterine a-aminoisobutyric acid uptake was measured for 30 min in the presence of 3.5 mM concentrations of the specified amino acids (Fig. 3)- a-Aminoisobutyric acid was present in a concentration of IOO/~M. The concentration of the
test amino acid is approx, lO-2O times that of plasma 1~. L-Methionine, L-alanine,
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30
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Time
in minutes
90
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Fig. I. (AIB) u p t a k e in u t e r i f r o m e s t r o g e n - t r e a t e d (z h) a n d control o v a r i e c t o m i z e d rats. T h e
i n c u b a t i o n m e d i u m c o n t a i n e d a i o o ~uM c o n c e n t r a t i o n o f a-amino[~4C]isobutyric acid a n d i.o m M
D-[I-SH]sorbitol. T h e calculations were carried o u t as described in ~ T H O D S AND MATERIALS.
E a c h p o i n t r e p r e s e n t s t h e m e a n of f o u r d e t e r m i n a t i o n s . Solid s y m b o l s are d e t e r m i n a t i o n s f r o m
t h e uteri o f 2-h e s t r o g e n - t r e a t e d a n i m a l s a n d o p e n s y m b o l s d e t e r m i n a t i o n s f r o m t h e u t e r i of
control a n i m a l s . T h e s t a n d a r d error for each p o i n t is less t h a n (:k o.I5). 0----0, glucose-free
m e d i u m ; A - - A , 8 m M glucose.
Fig. 2. T i m e course o f t h e e s t r o g e n - i n d u c e d a - a m i n o i s o b u t y r i c acid (AIB) t r a n s p o r t increase.
E x p e r i m e n t a l a n i m a l s received I.o/~g of IT~-estradiol in z o % e t h a n o l a t zero t i m e . T h e y were
sacrificed a t t h e t i m e s t a t e d , a n d t h e r a t e of u t e r i n e a - a m i n o i s o b u t y r i c acid t r a n s p o r t w a s determ i n e d d u r i n g a 3 o - m i n i n c u b a t i o n period. Controls received vehicle o n l y for 4 h. E a c h p o i n t
r e p r e s e n t s t h e m e a n o f t h e r e s u l t s f r o m five a n i m a l s . T h e vertical lines r e p r e s e n t 2 s t a n d a r d
errors o f t h e m e a n .
Biochim. Biophys. Acta, i35 (z967) 727-73 z
73°
R. ROSKOSKI, JR., D. F. STEINER
L-serine and L-cysteine inhibit a-aminoisobutyric acid uptake by about 50% (P <
o,ooi). L-Glycine, L-proline, L-leucine, L-isoleucine, t-phenylalanine and L-valine
inhibit uptake by lO-3O% (P < o.oi). L-Arginine, L-threonine, L-aspartate, L-glutamate, and L-lysine inhibit a-aminoisobutyric acid uptake by approx. IO% (P < 0.05).
The remaining amino acids do not inhibit a-aminoisobutyric acid uptake significantly.
DISCUSSION
Glucose has no effect on the initial rate of a-aminoisobutyric acid uptake in
uteri from animals receiving estrogen, or in the controls (Fig. I). One might suppose
that glucose would stimulate a-aminoisobutyric acid uptake, for the process is
energy dependent. However, in the case of the isolated rat diaphragm KIPNIS AND
NOALL12 report that glucose decreases a-aminoisobutyric acid uptake in non-fasted
and 72-h fasted animals in the presence and absence of insulin. The rate of a-aminoisobutyric acid uptake is stimulated by insulin in either case.
The stimulation of a-aminoisobutyric acid uptake by estrogen reported here
is in accord with the findings of NOALLand co-workers 1, who showed that the hormone increased the concentration of a-aminoisobutyric acid in immature rat uteri
20 h after injections of estrogen and a-aminoisobutyric acid. In the present study,
it was found that the rate of a-aminoisobutyric acid uptake (measured in isolated
uteri) was increased by estrogen for at least 24 h after hormone iniection. However,
their studies reflect the ability of the uterus to develop and/or maintain a greater
concentration of a-aminoisobutyric acid. The results reported here corroborate those
of NOALLAND ALLAN, who reported that estrogen injection into the ovariectomized
rabbit increased the rate of a-aminoisobutyric acid uptake in the isolated uterus.
HALKERSTON and co-workers 4 were able to demonstrate an increase in a-
400
-
35O
÷
:57 43 44 51-"I2-7-2~79 80 86 87 89 90 91
I00 I01 102 I00
Fig. 3- T h e effect of v a r i o u s L-amino acids on u p t a k e o f a - a m i n o i s o b u t y r i c acid (AIB) b y t h e isolated r a t u t e r u s . T h e t e s t a m i n o acids were p r e s e n t a t a c o n c e n t r a t i o n of 3.5 raM. T h e c o n c e n t r a tion o f a - a m i n o i s o b u t y r i c acid was ioo/~M. T h e i n c u b a t i o n period w a s 30 rain. E a c h b a r r e p r e s e n t s
t h e m e a n o f f o u r d e t e r m i n a t i o n s . T h e vertical line r e p r e s e n t s 2 s t a n d a r d errors of t h e m e a n .
T h e n u m b e r r e p r e s e n t s p e r c e n t a g e of control value.
Biochim. Biophys. Acta, 135 (1967) 727-731
AMINO ACID TRANSPORT IN UTERUS
731
a m i n o i s o b u t y r i c acid u p t a k e in the u t e r u s of t h e o v a r i e c t o m i z e d r a t 6 h after hormone iniection (1. 5 h after a - a m i n o i s o b u t y r i c injection). T h e y r e p o r t t h a t there
was no d e t e c t a b l e change 1.5 h after h o r m o n e a n d a - a m i n o i s o b u t y r i c acid injection.
I n t h e present s t u d y t h e increased a - a m i n o i s o b u t y r i c acid u p t a k e was discernible
I h after h o r m o n e iniection. The reasons for this d i s c r e p a n c y b e t w e e n t h e u p t a k e
r a t e m e a s u r e d in vitro as a g a i n s t in vivo are n o t a p p a r e n t .
Several n a t u r a l l y occurring a m i n o acids i n h i b i t e d a - a m i n o i s o b u t y r i c acid
u p t a k e to a g r e a t e r or lesser e x t e n t in this s t u d y . I n h i b i t i o n here p r o b a b l y d e p e n d s
u p o n the affinity of t h e a m i n o a c i d s for the m e m b r a n e carrier(s) responsible for
a - a m i n o i s o b u t y r i c acid t r a n s p o r t . A c o m p o u n d w i t h high affinity for the carrier
(low Kin) w o u l d inhibit a - a m i n o i s o b u t y r i c acid u p t a k e to a greater e x t e n t t h a n a
c o m p o u n d w i t h low carrier affinity (high Kin). The inhibition of a - a m i n o i s o b u t y r i c
acid u p t a k e b y several of the a m i n o acids t e s t e d provides p r e s u m p t i v e evidence t h a t
a - a m i n o i s o b u t y r i c acid is t r a n s p o r t e d b y a physiologically i m p o r t a n t m e m b r a n e
carrier(s), a n d the increase in a - a m i n o i s o b u t y r i c acid u p t a k e i n d u c e d b y estrogen
m a y be a reflection of increased t r a n s p o r t of some of the n a t u r a l l y occurring a m i n o
acids from t h e e x t r a c e l l u l a r to t h e intracellular space u n d e r the anabolic s t i m u l u s
of t h e hormone.
REFERENCES
I" M. W. NOALL, T. R. RIGGS, L. M. WALKER AND H. N. CHRISTENSEN, Science, 126 (1957) lOO2.
2 J. R. DANIELS AND S. 1VL KALMAN, Federation Proc., 20 (1961) 2.
3 M. W. NOALL AND W. M. ALLEN, J. Biol. Chem., 236 (1961) 2987.
4 I. D. K. HALKERSTON, J. EICHHORN, M. FEINSTEIN, E. SCULLY AND O. HECNTER, Proc. Soc.
Exptl. Biol. Med., lO3 (196o) 796.
5 T. R. RIGGS, R. B. SANDERSAND H. K. WEINDLING,Endocrinology, 73 (1963) 789 •
6 S. M. KALMANAND M. E. LOMBROZO,J. Pharmacol. Exptl. Therap., I3i (1961) 265.
7 0 . HECHTER AND G. LESTER, Recent Progr. Hormone Res., 16 (196o) 139.
8 0 . HECHTER AND I. D. Z. HALKERSTON, in G. PXNCUS, Hormones, Vol. 5, Academic, New
York, 1964, p. 697.
9 T. R. RIGGS, in G. LITWACK AND D. KRITCHEVSKY, Actions of Hormone on Molecular Processes,
Wiley, New York, 1964, p. i.
Io R. RosxosxI, Jr. AND D. F. STEINER,Biochim. Biophys. Acta, 135 (1967) 717 .
I I I. G. WOOL,in G. LITWACKAND D. KRITCHEVSKY,Actions of Hormones on Molecular Processes,
Wiley, New York, 1964, p. 422.
12 D. M. KIPNIS AND M. W. NOALL, Biochim. Biophys. Acta, 28 (1958) 226.
Biochim. Biophys. Acta, 135 (1967) 727-731