Vol. 63, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ORNITHINE
J.F. R i c h a r d s of B i o c h e m i s t r y , U n i v e r s i t y B.C. V6T IW5, Canada.
Department Vancouver, Received
D E C A R B O X Y L A S E A C T I V I T Y IN TISSUES P R O L A C T I N - T R E A T E D RATS
January
of B r i t i s h
OF
Columbia,
17,1975
Summary: T r e a t m e n t w i t h p r o l a c t i n caused increases of 3-100 fold in o r n i t h i n e d e c a r b o x y l a s e (EC 4.1.1.17) a c t i v i t y of kidney, adrenal gland and liver of female rats. The m a g n i t u d e of the effect v a r i e d w i t h age. In y o u n g male rats, a c t i v i t y of the enzyme was also s t i m u l a t e d m a r k e d l y in spleen, as well as those tissues m e n t i o n e d above, and there were smaller effects in thymus and heart. INTRODUCTION:
Several
lactin may be i n v o l v e d tissues
in a d d i t i o n
have b e e n
reported
(1,2,3,4). exercise
tation. mones
than
in serum o s m o l a l i t y
their effects membranes
have
that m e m b r a n e
mammary might ever
glands,
therefore
possess
correlated effect
with
fractions specific
the b i n d i n g
mary
gland has been
with
nuclei
isolated
tissues
studies,
(13).
~pyright©19~ ~ Ac~emicPress,~e. AHrights~reproductMnma~ ~ r m r e s e ~ .
in
organs,
responses
and kidney, 292
with prolactin including
sites,
(9,10,11,12).
effect
of lac-
to r e c e p t o r s
Studies
by nuclei
for
hor-
and a direct
This
role
that peptide
to the b i o l o g i c a l
Metabolic
from liver
a wider
prolactin-binding
on RNA synthesis reported
bound
to (6,7),
and m a i n t e n a n c e
from several
of this b i n d i n g
and k i d n e y
hypoglycemia
cells.
as target
is uncertain.
of p r o l a c t i n
target
responses
in response
accepted
after b e c o m i n g
be c l a s s e d
the r e l a t i o n s h i p
of the hormone
levels
in i n i t i a t i o n
of their
aorta
(8) suggest
it is g e n e r a l l y
the plasma shown
tissue,
that pro-
of n u m e r o u s
Physiological
(5), to i n s u l i n - i n d u c e d
Furthermore,
exert
gland.
in serum p r o l a c t i n
its function
suggest
the a c t i v i t y
for c a r d i o v a s c u l a r
and stress
prolactin
evidence
in r e g u l a t i n g
to m a m m a r y
Changes
and to v a r i a t i o n
lines~of
and How-
function
have not been stimulatory
isolated
from mam-
was not o b s e r v e d
yet these
tissues
Vol. 63, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
have
an a f f i n i t y
some
species
for p r o l a c t i n
the tissues
lactin
liver
tissues
of g r o w t h
hormone,
in A C T H
trophin-treated
activity
of other
phosphate
Co.
Ovine
ornithine-HCl, Tissue
preparation:
dislocation,
were
and organs
in 5 v o l u m e s
5 mM dithiothreitol,
adrenals
pooled
hand homogenizer.
Tissues
from e a c h
driven
at 0 °, and the
- L-ornithine,
reviewed on the
rats,
killed
were
pyridoxal
buffer
Searle
Corp.
of age and
and homo-
- 50 m M Tris-HCl,
using
a Servall
homogenizer.
in 1 ml of b u f f e r centrifuged
supernatant
treatment.
by c e r v i c a l
group w e r e w e i g h e d
pH 7.3,
decarboxylase
(DL-I-14C)
from A m e r s h a m
were
(16)
from the Sigma
from NIH.
glass-Teflon
293
have b e e n
by p r o l a c t i n
from H o o d e d
and h o m o g e n i z e d
Homogenates
of gonado-
that o r n i t h i n e
a gift
Animals
0.i ~ 4 EDTA,
and m o t o r
15 m i n u t e s
was
in
of p r o l a c t i n
purchased
of the f o l l o w i n g
disintegrator were
were
adrenal,
studies
affected
Materials
used.
in
rats has also b e e n r e p o r t e d
58 m C i / m m was o b t a i n e d
sex as indicated,
genized
is also
prolactin
reported
and testes
An e f f e c t
demonstrates
and d i t h i o t h r e i t o l
stimulation.
or n u t r i t i o n a l
have b e e n
and in o v a r y
AND METHODS;
ornithine
have b e e n o b s e r v e d
hormone-stimulated
liver of young
tissues
to pro-
and g l u c o c o r t i c o i d - t r e a t e d
(14,15).
investigation
MATERIALS
Chemical
and
enzyme
M a n y of these
recently
in b r a i n
The p r e s e n t
uterus,
animals.
and s u m m a r i z e d enzyme
in
was to deter-
of h o r m o n a l
by h o r m o n a l
increases
insulin
and g r o w t h
estrogen-stimulated
of this
stimulated
Hormonally-induced
animals,
tissue
response
of the enzyme,
as an i n d i c a t o r
in the a c t i v i t y
in m a n y m a m m a l i a n
investigation
show a m e t a b o l i c
The a c t i v i t y
was c h o s e n
increases
factors.
aims of this
of rats w h i c h
administration.
decarboxylase, Large
to that of m a m m a r y
(9,10,11,12).
One of the i m m e d i a t e mine
equal
fractions
The in a glass
at i0,000 used
tissue
x g for
for assay.
Vol. 63, No. 1, 1975
Ornithine
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
decarboxylase
tion c o n t a i n i n g essentially flasks
pyridoxal
as d e s c r i b e d
holding
The
a plastic
30 m i n u t e s
injected
by J~nne
0.5 ~Ci of
phosphate,
0.2 mM.
during
and their
was m e a s u r e d
30 nmoles
ornithine,
containing
experiment
included
substrate.
At this
duction
linear
and r e l e a s e incubation
contents
stoppers,
in w h i c h
substrate
COz, w h i c h shaking.
in Vials w i t h
Isocap
and radio-
300 counter.
concentrations.
concentration,
for 50-60 m i n u t e s
acid was
cocktail,
acid was
each
0.2 ml hyamine.
of 60 mins w i t h
scintillation
controls
of Land
radioactive
were placed
at two p r o t e i n
The
w e r e begun by a d d i t i o n
in a N u c l e a r - C h i c a g o
was a s s a y e d
was
Glass)
a further
i0 ml of a P P O - P O P O P - t o l u e n e
Each tissue
(DL-I-14C)
(17).
at 37 ° , 1 ml of 40% T r i c h l o r o a c e t i c
The centre wells
activity
and W i l l i a m s - A s h m a n of 1 ml,
prepara-
in 25 ml flasks
closed w i t h rubber
(Kontes
to stop the r e a c t i o n
was c o l l e c t e d
of tissue
incubated
Incubations
flasks w e r e well
Aliquots
were
in a final volume
including
of substrate.
After
0.2-6 mg p r o t e i n
contained,
ornithine,
assay:
Each
added b e f o r e
radioactive
with protein
COz pro-
concentrations
up to 7 mg. RESULTS: the a c t i v i t y young m a l e kidney,
of the o r n i t h i n e
the effect
adrenal,
liver and spleen
liver
animals effects
of this were
agree w i t h
first report
age.
found
The results
enzyme
in the tissues
effects
of p r o l a c t i n
increase
Quantitatively
in table
of female
on the activity.
294
but
is the
to p r o l a c t i n
in the
2 show the a c t i v i t y
of the
rats of d i f f e r e n t
ages,
It is a p p a r e n t
of
experi-
Our findings
but this
on
in
in m a n y
smaller,
and heart.
of this enzyme
tissues
in the a c t i v i t y
has b e e n o b s e r v e d
in thymus
treatment
in several
those of other workers,
of the response
tissues.
of p r o l a c t i n
decarboxylase
The v e r y m a r k e d
definite,
other
1 shows
rats.
ments w i t h
with
Table
and the
that the
Vol. 63, No. 1, 1975
TABLE
1.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect
of p r o l a c t i n
in tissues
of m a l e
on o r n i t h i n e
activity
rats.
Ornithine Control
Tissue
decarboxylase
decarboxylase
animals
activity
Prolactin-treated
animals
Liver
32.4
± 3.5
253.4
± 12.8
Kidney
18.2
+ 4.4
859.6
± 25.9
Adrenal
6.0 + 2.1
378.1
f 15.4
Spleen
7.8 _+ i.i
41.2
-+
4.1
-+
7.7
Thymus
39.2
± 3.1
100.6
Heart
18.4
+ 3.6
49.6
+
3.2
Lung
36.8
+ 4.8
38.4
-+
5.2
Per±renal Brown
fat
fat
265.5
+ 40.9
208.2
-+ 38.8
100.8
+ 20.2
95.6
+ 18.5
A n i m a l s w e r e g i v e n ip i n j e c t i o n of 3 mg of p r o l a c t i n in saline, or an e q u i v a l e n t volume of saline, 5 hours before sacrifice. The animals in this series of e x p e r i m e n t s were males, age 36-37 days, and each group c o n s i s t e d of 3-4 rats in each experiment. T i s s u e p r e p a r a t i o n s w e r e assayed at 2 p r o t e i n c o n c e n t r a tions. Results are e x p r e s s e d as pmoles C O 2 / m g p r o t e i n / 3 0 minutes, + S.E.M.
levels with
of a c t i v i t y
the h i g h e s t
for the 31-day
vary
values
and s i g n i f i c a n t
animals
in females,
animals.
No change tissue
with
animals
were
of the enzyme
in d e c a r b o x y l a s e
in table
3 show that
295
those
found
exception
ages, Except
activity
in spleen
activity
of female
females.
in enzyme
less than
the p o s s i b l e
or thymus
of d i f f e r e n t
in u n w e a n e d
the i n c r e a s e s
stimulation
observed
The data
occurring
old group,
prolactin-treated
in a d i p o s e
in control
in
in males, has not been
of the suckling
has b e e n o b s e r v e d
rats.
a dose of p r o l a c t i n
as small
Vol. 63, No.
1, 1 9 7 5
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
LQ .IJ
oo ,-~ +I
+I
©
Lo
L~
I.~ '~
CO 0
'm~
,--I
CO rq
~) D ~
~0
~O
.~
•
•
o ~I
m
m
N
~ U
-~ 0
O)
¢~
0
,..~0
,--I •-F.I
U
+I
+I
+i
~
kO O0 [-..
•
•
cq
ko
L}
•
~) ~J rC~ ~rq
.r-
-t-
o
00
LD
I.Q
~
o
~
+l
+t
+1
•*
o
LO
~
,-4
+i
+1
+i
~
ml
.~
N
o)
~
O
u
0
L)
,.Q ,.~
c, 0
t
.,~
~-I L~ -,-I
O~
Lr) r-I
+I
+I
(--.
I.o
g
~4
r-t
~
•
0 0
',,0 0 0
-i'-I
U
~
-,-I ~
,,-[
o,3
-,-I .,-I -,-I ~.~> ~ ~ -,.-.I
+l
+~
©
•
'..l-i
¢xl
m ,--t D
mN
.'F-I
F~
¶
(11
+1
+1
14") ~ t,l.,,l
O4
e,l
DI~4J ~ {/2+1 -rl -,-I
I
-,-i
~-I
,#
u~ 0
,-'4
~
0
0-~ ~ .,.-I ~ ~..~o
I
>-i
0
oo
On
Lo
I
,.-t © .,-I-,-i
r~ ©
I
,'-I
E~
296
I
~
-,-I ,~ ~
0
Vol. 63, No. 1, 1975
TABLE
Dose
3.
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
response
activity
relationship
to p r o l a c t i n
Activity Dosage Tissue
of o r n i t h i n e
treatment
of o r n i t h i n e
decarboxylase
of p r o l a c t i n - m g / 5 0
0
decarboxylase
g body weight 1.5
0.5
3.0
Liver
26.5
± 4.2
125.1
± 14.8
125.8
+ 20.1
220.6
± 18.8
Kidney
27.1
± 6.1
477.9
± 21.1
742.5
± 28.8
1254.3
± 51.1
6.1 ± 1.5
135.6
± 12.2
326.7
± 16.6
427.5
± 20.2
Adrenal
A n i m a l s in this series of e x p e r i m e n t s were 32-33 days of age. P r o l a c t i n or saline was a d m i n i s t e r e d by i n t r a p e r i t o n e a l i n j e c t i o n 5 hours b e f o r e sacrifice. The a c t i v i t y of the enzyme is e x p r e s s e d as pmoles C 0 2 / m g p r o t e i n / 3 0 m i n u t e s ± S.E.M.
as 0.5 m g p r o d u c e d enzyme
in the
3 tissues
the m o s t d r a m a t i c with
larger
a significant
responses.
doses. the time
the e f f e c t
is e v i d e n t
of r e s p o n s e
differed
adrenals kidney
was
still
seemed
treated other
by 2.5 hours
5 hours
increasing
in their
in this
do not show that p r o l a c t i n
various
tissues.
response
Maximal
clearly
regard,
The p a t t e r n
stimulation in the
The a c t i v i t y
in
itself
tissues,
297
show that o r n i t h i n e tissues
of rats
prolactin
resembles
on tissues.
These
acts d i r e c t l y
on the
effects
administration
in several
in-
4) show that
and a c t i v i t y
in several
studies
a metabolic
obtained
by 2.5 hours.
stimulatory
However,
showing
experiments
(table
at 7.5 hours.
increased and,
were
treatment.
in liver,
The data p r e s e n t e d
w i t h prolactin,
hormones
after
in the 3 tissues.
activity
effects
of s t i m u l a t i o n
of the
and adrenal
of a b b r e v i a t e d
to have p l a t e a u e d
DISCUSSION: decarboxylase
Increasing
course
to occur b e f o r e
on the a c t i v i t y
with kidney
The results
vestigating
seemed
studied,
effect
of the h o r m o n e and these
elicits
results provide
sup-
Vol. 63, No. 1, 1975
TABLE 4.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Time course of stimulation of ornithine decarboxylase in female rat tissues
Activity of ornithine decarboxylase Time after prolactin administration-hours Tissue
0
2.5
5.0
7.5
Liver
23.4 ± 3.6
103.8 ± 18.1
71.6 ± ii.I
22.1 ± 3.1
Kidney
20.8 ± 2.5
214.5 ± 10.6
153.3 ± 14.1
186.3 ± 8.3
Adrenal
12.6 + 3.1
30.1 ±
2.5
60.6 ±
4.8
82.6 ± 6.1
Animals were 42 day old female rats, and received prolactin or saline by ip injection. The animals were killed between ii a.m. and 2 p.m. to eliminate changes due to diurnal variation. Animals received 3 mg prolactin/50 g body weight. Activity of the enzyme is expressed as pmoles C02/mg protein/30 minutes ± S.E.M.
port for the hypothesis, previously based only on the hormonebinding studies, that prolactin may have several target tissues. The retention of sensitivity by some tissues in adult females and young males suggests too that prolactin may be involved in physiological regulation of some tissues beyond the stage of early growth and development.
The lack of effect of prolactin on the
enzyme activity in adipose tissue is difficult to reconcile with the recently noted effect of the hormone on lipoprotein lipase of mammary and adipose tissue
(18).
However,
it is possible that
the hormone may function by different mechanisms in different tissues, which would also explain the disparity of the effects on isolated nuclei mentioned previously ACKNOWLEDGEMENTS:
(13).
This work was supported by the British
Columbia Medical Research Foundation. REFERENCES i. 2.
Horrobin, D.F., Manku, M.S. and Burstyn, P.G. (1973) Cardiovasc. Res. 7:585-587. Manku, M.S., Nassar, B.A. and Horrobin, D.F. (1973) Lancet II:991-994. 298
Vol. 63, No. 1, 1975
3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lockett, M.F. and Nail, B. (1965) J. Physiol. 180:147-156. Lockett, M.F. (1965) J. Physiol. 181:192-199. Friesen, H. and Hwang, P. (1973) Ann. Rev. Med. 24:251-270. Frantz, A.G. and Kleinberg, D.L. (1970) Science 170:745-747. Copinschi, G., Virasoro, E., Vanhaelst, L., Leclercq, R., Golstein, J. and L'Hermite, M. (1974) Clin. Endocrinology 3:441-445. Buckman, M.T. and Peake, G.T. (1973) Science 181:755-757. Turkington, R.W. and Frantz, W.L. (1972) in 4th Tenovus Workshop, Prolactin and Carcinogenesis, pp. 39-47, ed. Boyns, A.R. and Griffiths, K. Alpha Omega Alpha publishin% Cardiff. Shiu, R.P.C. and Friesen, H.G. (1974) Biochem. J. 140:301-311. Posner, B.I., Kelly, P.A., Shiu, R.P.C. and Friesen, H.G. (1974) Endocrinolgoy 95:521-531. Kelly, P.A., Posner, B.I., Tsushima, T. and Friesen, H.G. (1974) Endocrinology 95:532-539. Chomcynski, P. and Topper, Y.T. (1974) Biochem. Biophys. Res. Communs. 60:56-63. Williams-Ashman, H.G., Janne, J., Coppoc, G.L., Geroch, M.E. and Schenone, A. (1972) Adv. in Enzyme Reg. 10:225-245. Morris, D.R. and Fillingame, R.H. (1974) Ann?--Rev. Biochem. 43:303-325. Roger, L.J., Schanberg, S.M. and Fellows, R.E. (1974) Endocrinology 95:904-911. J~nne, J. an--d Williams-Ashman, H.G. (1971) J. Biol. Chem. 246:1725-1732. Z-~der, 0., Hamosh, M., Fleck, T.R.C. and Scow, R.O. (1974) Amer. J. Physiol. 226:744-748.
299
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ORNITHINE
J.F. R i c h a r d s of B i o c h e m i s t r y , U n i v e r s i t y B.C. V6T IW5, Canada.
Department Vancouver, Received
D E C A R B O X Y L A S E A C T I V I T Y IN TISSUES P R O L A C T I N - T R E A T E D RATS
January
of B r i t i s h
OF
Columbia,
17,1975
Summary: T r e a t m e n t w i t h p r o l a c t i n caused increases of 3-100 fold in o r n i t h i n e d e c a r b o x y l a s e (EC 4.1.1.17) a c t i v i t y of kidney, adrenal gland and liver of female rats. The m a g n i t u d e of the effect v a r i e d w i t h age. In y o u n g male rats, a c t i v i t y of the enzyme was also s t i m u l a t e d m a r k e d l y in spleen, as well as those tissues m e n t i o n e d above, and there were smaller effects in thymus and heart. INTRODUCTION:
Several
lactin may be i n v o l v e d tissues
in a d d i t i o n
have b e e n
reported
(1,2,3,4). exercise
tation. mones
than
in serum o s m o l a l i t y
their effects membranes
have
that m e m b r a n e
mammary might ever
glands,
therefore
possess
correlated effect
with
fractions specific
the b i n d i n g
mary
gland has been
with
nuclei
isolated
tissues
studies,
(13).
~pyright©19~ ~ Ac~emicPress,~e. AHrights~reproductMnma~ ~ r m r e s e ~ .
in
organs,
responses
and kidney, 292
with prolactin including
sites,
(9,10,11,12).
effect
of lac-
to r e c e p t o r s
Studies
by nuclei
for
hor-
and a direct
This
role
that peptide
to the b i o l o g i c a l
Metabolic
from liver
a wider
prolactin-binding
on RNA synthesis reported
bound
to (6,7),
and m a i n t e n a n c e
from several
of this b i n d i n g
and k i d n e y
hypoglycemia
cells.
as target
is uncertain.
of p r o l a c t i n
target
responses
in response
accepted
after b e c o m i n g
be c l a s s e d
the r e l a t i o n s h i p
of the hormone
levels
in i n i t i a t i o n
of their
aorta
(8) suggest
it is g e n e r a l l y
the plasma shown
tissue,
that pro-
of n u m e r o u s
Physiological
(5), to i n s u l i n - i n d u c e d
Furthermore,
exert
gland.
in serum p r o l a c t i n
its function
suggest
the a c t i v i t y
for c a r d i o v a s c u l a r
and stress
prolactin
evidence
in r e g u l a t i n g
to m a m m a r y
Changes
and to v a r i a t i o n
lines~of
and How-
function
have not been stimulatory
isolated
from mam-
was not o b s e r v e d
yet these
tissues
Vol. 63, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
have
an a f f i n i t y
some
species
for p r o l a c t i n
the tissues
lactin
liver
tissues
of g r o w t h
hormone,
in A C T H
trophin-treated
activity
of other
phosphate
Co.
Ovine
ornithine-HCl, Tissue
preparation:
dislocation,
were
and organs
in 5 v o l u m e s
5 mM dithiothreitol,
adrenals
pooled
hand homogenizer.
Tissues
from e a c h
driven
at 0 °, and the
- L-ornithine,
reviewed on the
rats,
killed
were
pyridoxal
buffer
Searle
Corp.
of age and
and homo-
- 50 m M Tris-HCl,
using
a Servall
homogenizer.
in 1 ml of b u f f e r centrifuged
supernatant
treatment.
by c e r v i c a l
group w e r e w e i g h e d
pH 7.3,
decarboxylase
(DL-I-14C)
from A m e r s h a m
were
(16)
from the Sigma
from NIH.
glass-Teflon
293
have b e e n
by p r o l a c t i n
from H o o d e d
and h o m o g e n i z e d
Homogenates
of gonado-
that o r n i t h i n e
a gift
Animals
0.i ~ 4 EDTA,
and m o t o r
15 m i n u t e s
was
in
of p r o l a c t i n
purchased
of the f o l l o w i n g
disintegrator were
were
adrenal,
studies
affected
Materials
used.
in
rats has also b e e n r e p o r t e d
58 m C i / m m was o b t a i n e d
sex as indicated,
genized
is also
prolactin
reported
and testes
An e f f e c t
demonstrates
and d i t h i o t h r e i t o l
stimulation.
or n u t r i t i o n a l
have b e e n
and in o v a r y
AND METHODS;
ornithine
have b e e n o b s e r v e d
hormone-stimulated
liver of young
tissues
to pro-
and g l u c o c o r t i c o i d - t r e a t e d
(14,15).
investigation
MATERIALS
Chemical
and
enzyme
M a n y of these
recently
in b r a i n
The p r e s e n t
uterus,
animals.
and s u m m a r i z e d enzyme
in
was to deter-
of h o r m o n a l
by h o r m o n a l
increases
insulin
and g r o w t h
estrogen-stimulated
of this
stimulated
Hormonally-induced
animals,
tissue
response
of the enzyme,
as an i n d i c a t o r
in the a c t i v i t y
in m a n y m a m m a l i a n
investigation
show a m e t a b o l i c
The a c t i v i t y
was c h o s e n
increases
factors.
aims of this
of rats w h i c h
administration.
decarboxylase, Large
to that of m a m m a r y
(9,10,11,12).
One of the i m m e d i a t e mine
equal
fractions
The in a glass
at i0,000 used
tissue
x g for
for assay.
Vol. 63, No. 1, 1975
Ornithine
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
decarboxylase
tion c o n t a i n i n g essentially flasks
pyridoxal
as d e s c r i b e d
holding
The
a plastic
30 m i n u t e s
injected
by J~nne
0.5 ~Ci of
phosphate,
0.2 mM.
during
and their
was m e a s u r e d
30 nmoles
ornithine,
containing
experiment
included
substrate.
At this
duction
linear
and r e l e a s e incubation
contents
stoppers,
in w h i c h
substrate
COz, w h i c h shaking.
in Vials w i t h
Isocap
and radio-
300 counter.
concentrations.
concentration,
for 50-60 m i n u t e s
acid was
cocktail,
acid was
each
0.2 ml hyamine.
of 60 mins w i t h
scintillation
controls
of Land
radioactive
were placed
at two p r o t e i n
The
w e r e begun by a d d i t i o n
in a N u c l e a r - C h i c a g o
was a s s a y e d
was
Glass)
a further
i0 ml of a P P O - P O P O P - t o l u e n e
Each tissue
(DL-I-14C)
(17).
at 37 ° , 1 ml of 40% T r i c h l o r o a c e t i c
The centre wells
activity
and W i l l i a m s - A s h m a n of 1 ml,
prepara-
in 25 ml flasks
closed w i t h rubber
(Kontes
to stop the r e a c t i o n
was c o l l e c t e d
of tissue
incubated
Incubations
flasks w e r e well
Aliquots
were
in a final volume
including
of substrate.
After
0.2-6 mg p r o t e i n
contained,
ornithine,
assay:
Each
added b e f o r e
radioactive
with protein
COz pro-
concentrations
up to 7 mg. RESULTS: the a c t i v i t y young m a l e kidney,
of the o r n i t h i n e
the effect
adrenal,
liver and spleen
liver
animals effects
of this were
agree w i t h
first report
age.
found
The results
enzyme
in the tissues
effects
of p r o l a c t i n
increase
Quantitatively
in table
of female
on the activity.
294
but
is the
to p r o l a c t i n
in the
2 show the a c t i v i t y
of the
rats of d i f f e r e n t
ages,
It is a p p a r e n t
of
experi-
Our findings
but this
on
in
in m a n y
smaller,
and heart.
of this enzyme
tissues
in the a c t i v i t y
has b e e n o b s e r v e d
in thymus
treatment
in several
those of other workers,
of the response
tissues.
of p r o l a c t i n
decarboxylase
The v e r y m a r k e d
definite,
other
1 shows
rats.
ments w i t h
with
Table
and the
that the
Vol. 63, No. 1, 1975
TABLE
1.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect
of p r o l a c t i n
in tissues
of m a l e
on o r n i t h i n e
activity
rats.
Ornithine Control
Tissue
decarboxylase
decarboxylase
animals
activity
Prolactin-treated
animals
Liver
32.4
± 3.5
253.4
± 12.8
Kidney
18.2
+ 4.4
859.6
± 25.9
Adrenal
6.0 + 2.1
378.1
f 15.4
Spleen
7.8 _+ i.i
41.2
-+
4.1
-+
7.7
Thymus
39.2
± 3.1
100.6
Heart
18.4
+ 3.6
49.6
+
3.2
Lung
36.8
+ 4.8
38.4
-+
5.2
Per±renal Brown
fat
fat
265.5
+ 40.9
208.2
-+ 38.8
100.8
+ 20.2
95.6
+ 18.5
A n i m a l s w e r e g i v e n ip i n j e c t i o n of 3 mg of p r o l a c t i n in saline, or an e q u i v a l e n t volume of saline, 5 hours before sacrifice. The animals in this series of e x p e r i m e n t s were males, age 36-37 days, and each group c o n s i s t e d of 3-4 rats in each experiment. T i s s u e p r e p a r a t i o n s w e r e assayed at 2 p r o t e i n c o n c e n t r a tions. Results are e x p r e s s e d as pmoles C O 2 / m g p r o t e i n / 3 0 minutes, + S.E.M.
levels with
of a c t i v i t y
the h i g h e s t
for the 31-day
vary
values
and s i g n i f i c a n t
animals
in females,
animals.
No change tissue
with
animals
were
of the enzyme
in d e c a r b o x y l a s e
in table
3 show that
295
those
found
exception
ages, Except
activity
in spleen
activity
of female
females.
in enzyme
less than
the p o s s i b l e
or thymus
of d i f f e r e n t
in u n w e a n e d
the i n c r e a s e s
stimulation
observed
The data
occurring
old group,
prolactin-treated
in a d i p o s e
in control
in
in males, has not been
of the suckling
has b e e n o b s e r v e d
rats.
a dose of p r o l a c t i n
as small
Vol. 63, No.
1, 1 9 7 5
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
LQ .IJ
oo ,-~ +I
+I
©
Lo
L~
I.~ '~
CO 0
'm~
,--I
CO rq
~) D ~
~0
~O
.~
•
•
o ~I
m
m
N
~ U
-~ 0
O)
¢~
0
,..~0
,--I •-F.I
U
+I
+I
+i
~
kO O0 [-..
•
•
cq
ko
L}
•
~) ~J rC~ ~rq
.r-
-t-
o
00
LD
I.Q
~
o
~
+l
+t
+1
•*
o
LO
~
,-4
+i
+1
+i
~
ml
.~
N
o)
~
O
u
0
L)
,.Q ,.~
c, 0
t
.,~
~-I L~ -,-I
O~
Lr) r-I
+I
+I
(--.
I.o
g
~4
r-t
~
•
0 0
',,0 0 0
-i'-I
U
~
-,-I ~
,,-[
o,3
-,-I .,-I -,-I ~.~> ~ ~ -,.-.I
+l
+~
©
•
'..l-i
¢xl
m ,--t D
mN
.'F-I
F~
¶
(11
+1
+1
14") ~ t,l.,,l
O4
e,l
DI~4J ~ {/2+1 -rl -,-I
I
-,-i
~-I
,#
u~ 0
,-'4
~
0
0-~ ~ .,.-I ~ ~..~o
I
>-i
0
oo
On
Lo
I
,.-t © .,-I-,-i
r~ ©
I
,'-I
E~
296
I
~
-,-I ,~ ~
0
Vol. 63, No. 1, 1975
TABLE
Dose
3.
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
response
activity
relationship
to p r o l a c t i n
Activity Dosage Tissue
of o r n i t h i n e
treatment
of o r n i t h i n e
decarboxylase
of p r o l a c t i n - m g / 5 0
0
decarboxylase
g body weight 1.5
0.5
3.0
Liver
26.5
± 4.2
125.1
± 14.8
125.8
+ 20.1
220.6
± 18.8
Kidney
27.1
± 6.1
477.9
± 21.1
742.5
± 28.8
1254.3
± 51.1
6.1 ± 1.5
135.6
± 12.2
326.7
± 16.6
427.5
± 20.2
Adrenal
A n i m a l s in this series of e x p e r i m e n t s were 32-33 days of age. P r o l a c t i n or saline was a d m i n i s t e r e d by i n t r a p e r i t o n e a l i n j e c t i o n 5 hours b e f o r e sacrifice. The a c t i v i t y of the enzyme is e x p r e s s e d as pmoles C 0 2 / m g p r o t e i n / 3 0 m i n u t e s ± S.E.M.
as 0.5 m g p r o d u c e d enzyme
in the
3 tissues
the m o s t d r a m a t i c with
larger
a significant
responses.
doses. the time
the e f f e c t
is e v i d e n t
of r e s p o n s e
differed
adrenals kidney
was
still
seemed
treated other
by 2.5 hours
5 hours
increasing
in their
in this
do not show that p r o l a c t i n
various
tissues.
response
Maximal
clearly
regard,
The p a t t e r n
stimulation in the
The a c t i v i t y
in
itself
tissues,
297
show that o r n i t h i n e tissues
of rats
prolactin
resembles
on tissues.
These
acts d i r e c t l y
on the
effects
administration
in several
in-
4) show that
and a c t i v i t y
in several
studies
a metabolic
obtained
by 2.5 hours.
stimulatory
However,
showing
experiments
(table
at 7.5 hours.
increased and,
were
treatment.
in liver,
The data p r e s e n t e d
w i t h prolactin,
hormones
after
in the 3 tissues.
activity
effects
of s t i m u l a t i o n
of the
and adrenal
of a b b r e v i a t e d
to have p l a t e a u e d
DISCUSSION: decarboxylase
Increasing
course
to occur b e f o r e
on the a c t i v i t y
with kidney
The results
vestigating
seemed
studied,
effect
of the h o r m o n e and these
elicits
results provide
sup-
Vol. 63, No. 1, 1975
TABLE 4.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Time course of stimulation of ornithine decarboxylase in female rat tissues
Activity of ornithine decarboxylase Time after prolactin administration-hours Tissue
0
2.5
5.0
7.5
Liver
23.4 ± 3.6
103.8 ± 18.1
71.6 ± ii.I
22.1 ± 3.1
Kidney
20.8 ± 2.5
214.5 ± 10.6
153.3 ± 14.1
186.3 ± 8.3
Adrenal
12.6 + 3.1
30.1 ±
2.5
60.6 ±
4.8
82.6 ± 6.1
Animals were 42 day old female rats, and received prolactin or saline by ip injection. The animals were killed between ii a.m. and 2 p.m. to eliminate changes due to diurnal variation. Animals received 3 mg prolactin/50 g body weight. Activity of the enzyme is expressed as pmoles C02/mg protein/30 minutes ± S.E.M.
port for the hypothesis, previously based only on the hormonebinding studies, that prolactin may have several target tissues. The retention of sensitivity by some tissues in adult females and young males suggests too that prolactin may be involved in physiological regulation of some tissues beyond the stage of early growth and development.
The lack of effect of prolactin on the
enzyme activity in adipose tissue is difficult to reconcile with the recently noted effect of the hormone on lipoprotein lipase of mammary and adipose tissue
(18).
However,
it is possible that
the hormone may function by different mechanisms in different tissues, which would also explain the disparity of the effects on isolated nuclei mentioned previously ACKNOWLEDGEMENTS:
(13).
This work was supported by the British
Columbia Medical Research Foundation. REFERENCES i. 2.
Horrobin, D.F., Manku, M.S. and Burstyn, P.G. (1973) Cardiovasc. Res. 7:585-587. Manku, M.S., Nassar, B.A. and Horrobin, D.F. (1973) Lancet II:991-994. 298
Vol. 63, No. 1, 1975
3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lockett, M.F. and Nail, B. (1965) J. Physiol. 180:147-156. Lockett, M.F. (1965) J. Physiol. 181:192-199. Friesen, H. and Hwang, P. (1973) Ann. Rev. Med. 24:251-270. Frantz, A.G. and Kleinberg, D.L. (1970) Science 170:745-747. Copinschi, G., Virasoro, E., Vanhaelst, L., Leclercq, R., Golstein, J. and L'Hermite, M. (1974) Clin. Endocrinology 3:441-445. Buckman, M.T. and Peake, G.T. (1973) Science 181:755-757. Turkington, R.W. and Frantz, W.L. (1972) in 4th Tenovus Workshop, Prolactin and Carcinogenesis, pp. 39-47, ed. Boyns, A.R. and Griffiths, K. Alpha Omega Alpha publishin% Cardiff. Shiu, R.P.C. and Friesen, H.G. (1974) Biochem. J. 140:301-311. Posner, B.I., Kelly, P.A., Shiu, R.P.C. and Friesen, H.G. (1974) Endocrinolgoy 95:521-531. Kelly, P.A., Posner, B.I., Tsushima, T. and Friesen, H.G. (1974) Endocrinology 95:532-539. Chomcynski, P. and Topper, Y.T. (1974) Biochem. Biophys. Res. Communs. 60:56-63. Williams-Ashman, H.G., Janne, J., Coppoc, G.L., Geroch, M.E. and Schenone, A. (1972) Adv. in Enzyme Reg. 10:225-245. Morris, D.R. and Fillingame, R.H. (1974) Ann?--Rev. Biochem. 43:303-325. Roger, L.J., Schanberg, S.M. and Fellows, R.E. (1974) Endocrinology 95:904-911. J~nne, J. an--d Williams-Ashman, H.G. (1971) J. Biol. Chem. 246:1725-1732. Z-~der, 0., Hamosh, M., Fleck, T.R.C. and Scow, R.O. (1974) Amer. J. Physiol. 226:744-748.
299
