Endocrinol. Japon. 1975, 22 (1), 55-60
Effect
of Triiodothyronine Inhibition
of TSH-
or and
Iodide
on
the
Dibutyryl-Cyclic-AMP
TAEKO SHIMIZU
Thyroidal
Secretion
Induced
In
Vitro
:
Endocytosis
AND YOSHIMASA SHISHIBA
Division of Endocrinology, Department of Medicine, Toranomon Hospital, and Okinaka Memorial Institute for Medical Research, Akasaka, Tokyo 107 Synopsis T3 or iodide, at the concentration of 10-3M, inhibits TSH- or dibutyryl-cyclic AMP-induced thyroidal intracellular colloid droplet formation, while leaving the TSH-induced increase in cyclic AMP levels intact. The effect of T3 appeared not to be mediated through the iodide derived from deiodination of T3. Those results clearly demonstrate that T3 or iodide acts at the point beyond cyclic AMP generation and prior to the formation of intracellular colloid droplets. This is also the site at which microtubule active reagents exert their influence. Whether T3 or iodide fits this category or acts in as yet undetermined manner remains to be established.
In our previous study it was described that the large dose of triiodo-l-thyronine (T3) or thyroxine (T4), when administered to the mice prepared for McKenzie bioassay reduced the thyroidal responsiveness to TSH or LATS as evidenced by the decrease in thyroidal intracellular colloid droplet formation and subsequent radioiodine release (Shishiba, et al., 1974). To clarify the possible site of action of thyroid hormone itself on thyroid tissue, an in vitro extention of the study was required because precise comparison of the responsiveness of thyroid tissue to those reagents was possible at a fixed concentraReceived for publication November 5, 1974. A part of this study was presented at the 47th Annual Meeting of the Japan Endocrinological Society, Tokyo, April, 1974. This study was supported in part by the Research Grant from the Ministry of Education, No. 948241, from the Hashimoto Disease Research Committee, Ministry of Health and Welfare, and also from Mitsui Life, Social and Welfare Association. Address reprint request to Yoshimasa Shishiba, M. D., Toranomon Hospital, Akasaka, Minatoku, Tokyo 107.
tion of TSH or other stimulators. For the mechanism of thyroid secretion, it was postulated that TSH stimulates adenylate cyclase activity to increase cyclic AMP levels in the thyroid tissue, the latter being responsible for the stimulation of intracellular colloid droplet formation resulting in the subsequent hormonal release (Pastan and Wollman, 1967). Taking this postulate into account, in the present study, we sought to determine whether the effect of T3 exists at the site where TSH increases cyclic AMP levels or at the site where increased cyclic AMP enhances the formation of intracellular colloid droplet.
Materials
and
Methods
The methods employed were almost the same as described by Williams and Wolff with slight modification for laboratory adoption (Williams and Wolff, 1971). The male mice of ddy atrain, purchased at the age of four weeks and kept on low iodine diet for two weeks, were employed all through the experiments. The mice were sacrificed with the dislocation of the neck. The thyroid glands were
56
SHIMIZU
removed the
with
trachea
ice-cold
bovine
serum
mg/ml,
streptomycin
and
02
5%
CO2
were
37•‹
in
with to to
gassed
taining without
medium
or
cyclic
AMP
60
min
with
In
the
tion
with
the
one from
them
in
fixed
Bouln's
counterstained
colloid
without
the
the
part
of
glands
with
cyclic
AMP Schwarz/Mann Steiner was
T3,
dissolved
in
and
ethanol,
95%
system
and
the
Iodide of
1311,
content
of
celium-arsenic contamination 1.4%,
at
The
magnitude
incubation with
the
of 125I-T3
during
was
the added
Armour
iodide
at
the
from incubation
with
was
proved of
incubation.
T3
during
of was
purchased
no
from
significant
less
the
Daiichi the
incu-
deiodination
Therefore,
the T3
not
be
examined
Under
exogenous would
to
also
Tokyo.
the
control.
beginning
125I-T3,
observed.
by water
appropriate
tissue
an
located
measured
deiodination
described,
derived
in (78:5:17) was
an
Laboratories,
condition
iodide
of
of
0.1N
a Whatman
redestilled
was with
thyroid
addition
of
on
with
iodide
of with
Radioisotope bation
least
To 100 ƒÊg
amount
portion
reaction,
than
Chemicals.
applied
eluted
of
described
acid-ammonia
overnight. addition
method
chromatographed
butanol-acetic
the
content distributed
1969).
minimum
and
and
contamination,
was
on
thyroid
blotted
kit
Sigma
iodide
the
paper
ascending
the
of
follicles slides
The
the
al.,
from
magnitude
3MM filter
The
et
intra-
instantaneously
the
to
(Steiner,
purchased
the
NaOH
al
25
the
quickly
with
for
five
liquid.
measured according
et
on of
was
PAS,
of
remaining
acetone-dry-ice
of
paper,
examined
counted
tissue
be
three
number
trachea,
The
was
by
T3
from
to thyroid
with
identity The
0.1mg.
by
check
were
for
forma-
moist filter
The
examiner.
37•‹
described.
were
and
of
excised
to
frozen
of
droplets
were
weighed
a
droplets.
knowledge the
as
incubation,
on
con-
dibutyrylat
tissue
hematoxilin
colloid
cellular
or
fixative,stained
with
intracellular
flask
contained
After blotted
then
droplet
the
flask
animals.
quickly
with
in
incubation
8
were
TSH
colloid
levels
NaOH
pre-incubation
bubbling
which
AMP
N
pre-incubato
incubated
gas
was
was
After
of and
colchi-
continuously
for
addition
continuous
cyclic
examined, tissue
CO2.
(DBC-AMP) the
0.1
pH
transferred
described
experiments
and
5%
or tyrosine
was
in
content the
exceed
of
medium 1.4%
of
T3. Thytropar
was
used
Results
prein-
described
of
medium
95%
and
or
as
a
all through the experiments. DBC-AMP was purchased from Sigma Chemicals and other reagents were purchased at the purest grade available on market.
thyroid
tyrosine T3
Endocrinol. Japon. February 1975
SHISHIBA
reutili-
with
above
whose
were
the
flasks
amount
and
glands
the
iodide,
mg/ml
Then,
the
results.
medium
95% O2
thyroid
T3,
The
5
gassed
use.
in
glucose 0.5
block
new in
minimum
7.4.
with
tion,
to
to
the
to was
min
the
the
added
adjusted
prior
with
stated
dissolved
min
M
min
:
5mg/ml,
10-3 medium
60
30
containing
penicillin
The
for
enriched as
and
(BSA)
transferred
at
medium
30
for
solution
0.5mg/ml,
iodide.
cubated
cine
salt
perchlorate
of
glands
immersed
balanced
albumin
sodium
zation and
and
Earl's
AND
source
of
TSH
Figure 1 shows the results of the experiment in which effect of concentration of TSH on the number of intracellular colloid droplets in thyroid tissue in vitro. As noted in the figure, TSH, even at the concentration of 0.005mU/ml was effective in inducing a significant rise in intracellular colloid droplets. The effect was plateaued at the concentration of TSH ranging from 0.1 mU/ ml to 2.5mU/ml, and then, decreased at the higher concentration of TSH. Table 1 summarizes the results of the experiment in which the effect of T3, iodide or tyrosine on the intracellular colloid droplet formation in vitro. As shown, TSH induced intracellular colloid droplets. to the level as much as 734. T3 or iodide, when tested at the cencenration of 10-4M or less, did not show any significant effect.. When the dose of T3 or iodide increased to 10-3M, however, significant inhibition of TSH-induced colloid droplet formation was obvious as evidenced by the decrease in the number of colloid droplet to the level approximately half of the control. This inhibitory effect of T3 or iodide on colloid droplet formation was manifest not only at the dose of TSH 0.1mU, but also at 3.3mU/ml as shown in Table 2 and 3. Tyrosine, even at the concentration of 10-3 M, was not effective at all in this aspect. Table 2 summarizes the results of the expriment in which the effect of T3 or iodide on the response of thyloid tissue either to TSH or DBC-AMP. In the former, tissue levels of cyclic AMP were concomitantly measured with the colloid droplet formation. When TSH (3.3 mU/ml) was added to the medium, intracellular colloid droplets were
Vol.22,
No.1
T3 ON THYROID
Figure
Table
* **
Mean •}
1.
SE
Statistical alone and
1.
Effect
of
the
analysis and
the
§N.S.stands
The
the
effect
of T3, iodide
value was
for
of with
not
obtained made
addition
TSH-group
of dose
on the
or tyrosine
from
for test
of TSH
the
three
or
without
significant.
colloid
droplet
formation
in vitro.
on TSH-induced
colloid
droplet
formation
in vitro.
colloid
droplets
between In
addition
intracellular
animals.
difference
substances.
57
SECRETION
all of
the test
the
number
of
experiments, substances
the is
difference
highly
significant.
between
induced no
by stimulator
TSH
58
SHIMIZU Table
* **
2.
Mean •}
SE
Mean •}SE colloid
The effect and cyclic
§N.S.stands
of T3 or iodide on TSHAMP generation.
of
the
value
obtained
from
of
the
value
obtained
from
droplet
AND
three
or DBC-AMP-induced
animals
three
Endocrinol. Japon February 1975
SHISHIBA colloid
droplet
formation
.
similar
experiments
,
one
of
which
is
composite
with
study. for
not
significant.Statistical
analysis
made
for
the
value
between
1)
and
2)
or
3)
is
listed on the table. That for colloid droplet counts between no stimulator and TSH or DBC-AMP group is also significant. That for cyclic AMP between no stimulator and TSH group is highly significant (p
Effect
of Triiodothyronine Inhibition
of TSH-
or and
Iodide
on
the
Dibutyryl-Cyclic-AMP
TAEKO SHIMIZU
Thyroidal
Secretion
Induced
In
Vitro
:
Endocytosis
AND YOSHIMASA SHISHIBA
Division of Endocrinology, Department of Medicine, Toranomon Hospital, and Okinaka Memorial Institute for Medical Research, Akasaka, Tokyo 107 Synopsis T3 or iodide, at the concentration of 10-3M, inhibits TSH- or dibutyryl-cyclic AMP-induced thyroidal intracellular colloid droplet formation, while leaving the TSH-induced increase in cyclic AMP levels intact. The effect of T3 appeared not to be mediated through the iodide derived from deiodination of T3. Those results clearly demonstrate that T3 or iodide acts at the point beyond cyclic AMP generation and prior to the formation of intracellular colloid droplets. This is also the site at which microtubule active reagents exert their influence. Whether T3 or iodide fits this category or acts in as yet undetermined manner remains to be established.
In our previous study it was described that the large dose of triiodo-l-thyronine (T3) or thyroxine (T4), when administered to the mice prepared for McKenzie bioassay reduced the thyroidal responsiveness to TSH or LATS as evidenced by the decrease in thyroidal intracellular colloid droplet formation and subsequent radioiodine release (Shishiba, et al., 1974). To clarify the possible site of action of thyroid hormone itself on thyroid tissue, an in vitro extention of the study was required because precise comparison of the responsiveness of thyroid tissue to those reagents was possible at a fixed concentraReceived for publication November 5, 1974. A part of this study was presented at the 47th Annual Meeting of the Japan Endocrinological Society, Tokyo, April, 1974. This study was supported in part by the Research Grant from the Ministry of Education, No. 948241, from the Hashimoto Disease Research Committee, Ministry of Health and Welfare, and also from Mitsui Life, Social and Welfare Association. Address reprint request to Yoshimasa Shishiba, M. D., Toranomon Hospital, Akasaka, Minatoku, Tokyo 107.
tion of TSH or other stimulators. For the mechanism of thyroid secretion, it was postulated that TSH stimulates adenylate cyclase activity to increase cyclic AMP levels in the thyroid tissue, the latter being responsible for the stimulation of intracellular colloid droplet formation resulting in the subsequent hormonal release (Pastan and Wollman, 1967). Taking this postulate into account, in the present study, we sought to determine whether the effect of T3 exists at the site where TSH increases cyclic AMP levels or at the site where increased cyclic AMP enhances the formation of intracellular colloid droplet.
Materials
and
Methods
The methods employed were almost the same as described by Williams and Wolff with slight modification for laboratory adoption (Williams and Wolff, 1971). The male mice of ddy atrain, purchased at the age of four weeks and kept on low iodine diet for two weeks, were employed all through the experiments. The mice were sacrificed with the dislocation of the neck. The thyroid glands were
56
SHIMIZU
removed the
with
trachea
ice-cold
bovine
serum
mg/ml,
streptomycin
and
02
5%
CO2
were
37•‹
in
with to to
gassed
taining without
medium
or
cyclic
AMP
60
min
with
In
the
tion
with
the
one from
them
in
fixed
Bouln's
counterstained
colloid
without
the
the
part
of
glands
with
cyclic
AMP Schwarz/Mann Steiner was
T3,
dissolved
in
and
ethanol,
95%
system
and
the
Iodide of
1311,
content
of
celium-arsenic contamination 1.4%,
at
The
magnitude
incubation with
the
of 125I-T3
during
was
the added
Armour
iodide
at
the
from incubation
with
was
proved of
incubation.
T3
during
of was
purchased
no
from
significant
less
the
Daiichi the
incu-
deiodination
Therefore,
the T3
not
be
examined
Under
exogenous would
to
also
Tokyo.
the
control.
beginning
125I-T3,
observed.
by water
appropriate
tissue
an
located
measured
deiodination
described,
derived
in (78:5:17) was
an
Laboratories,
condition
iodide
of
of
0.1N
a Whatman
redestilled
was with
thyroid
addition
of
on
with
iodide
of with
Radioisotope bation
least
To 100 ƒÊg
amount
portion
reaction,
than
Chemicals.
applied
eluted
of
described
acid-ammonia
overnight. addition
method
chromatographed
butanol-acetic
the
content distributed
1969).
minimum
and
and
contamination,
was
on
thyroid
blotted
kit
Sigma
iodide
the
paper
ascending
the
of
follicles slides
The
the
al.,
from
magnitude
3MM filter
The
et
intra-
instantaneously
the
to
(Steiner,
purchased
the
NaOH
al
25
the
quickly
with
for
five
liquid.
measured according
et
on of
was
PAS,
of
remaining
acetone-dry-ice
of
paper,
examined
counted
tissue
be
three
number
trachea,
The
was
by
T3
from
to thyroid
with
identity The
0.1mg.
by
check
were
for
forma-
moist filter
The
examiner.
37•‹
described.
were
and
of
excised
to
frozen
of
droplets
were
weighed
a
droplets.
knowledge the
as
incubation,
on
con-
dibutyrylat
tissue
hematoxilin
colloid
cellular
or
fixative,stained
with
intracellular
flask
contained
After blotted
then
droplet
the
flask
animals.
quickly
with
in
incubation
8
were
TSH
colloid
levels
NaOH
pre-incubation
bubbling
which
AMP
N
pre-incubato
incubated
gas
was
was
After
of and
colchi-
continuously
for
addition
continuous
cyclic
examined, tissue
CO2.
(DBC-AMP) the
0.1
pH
transferred
described
experiments
and
5%
or tyrosine
was
in
content the
exceed
of
medium 1.4%
of
T3. Thytropar
was
used
Results
prein-
described
of
medium
95%
and
or
as
a
all through the experiments. DBC-AMP was purchased from Sigma Chemicals and other reagents were purchased at the purest grade available on market.
thyroid
tyrosine T3
Endocrinol. Japon. February 1975
SHISHIBA
reutili-
with
above
whose
were
the
flasks
amount
and
glands
the
iodide,
mg/ml
Then,
the
results.
medium
95% O2
thyroid
T3,
The
5
gassed
use.
in
glucose 0.5
block
new in
minimum
7.4.
with
tion,
to
to
the
to was
min
the
the
added
adjusted
prior
with
stated
dissolved
min
M
min
:
5mg/ml,
10-3 medium
60
30
containing
penicillin
The
for
enriched as
and
(BSA)
transferred
at
medium
30
for
solution
0.5mg/ml,
iodide.
cubated
cine
salt
perchlorate
of
glands
immersed
balanced
albumin
sodium
zation and
and
Earl's
AND
source
of
TSH
Figure 1 shows the results of the experiment in which effect of concentration of TSH on the number of intracellular colloid droplets in thyroid tissue in vitro. As noted in the figure, TSH, even at the concentration of 0.005mU/ml was effective in inducing a significant rise in intracellular colloid droplets. The effect was plateaued at the concentration of TSH ranging from 0.1 mU/ ml to 2.5mU/ml, and then, decreased at the higher concentration of TSH. Table 1 summarizes the results of the experiment in which the effect of T3, iodide or tyrosine on the intracellular colloid droplet formation in vitro. As shown, TSH induced intracellular colloid droplets. to the level as much as 734. T3 or iodide, when tested at the cencenration of 10-4M or less, did not show any significant effect.. When the dose of T3 or iodide increased to 10-3M, however, significant inhibition of TSH-induced colloid droplet formation was obvious as evidenced by the decrease in the number of colloid droplet to the level approximately half of the control. This inhibitory effect of T3 or iodide on colloid droplet formation was manifest not only at the dose of TSH 0.1mU, but also at 3.3mU/ml as shown in Table 2 and 3. Tyrosine, even at the concentration of 10-3 M, was not effective at all in this aspect. Table 2 summarizes the results of the expriment in which the effect of T3 or iodide on the response of thyloid tissue either to TSH or DBC-AMP. In the former, tissue levels of cyclic AMP were concomitantly measured with the colloid droplet formation. When TSH (3.3 mU/ml) was added to the medium, intracellular colloid droplets were
Vol.22,
No.1
T3 ON THYROID
Figure
Table
* **
Mean •}
1.
SE
Statistical alone and
1.
Effect
of
the
analysis and
the
§N.S.stands
The
the
effect
of T3, iodide
value was
for
of with
not
obtained made
addition
TSH-group
of dose
on the
or tyrosine
from
for test
of TSH
the
three
or
without
significant.
colloid
droplet
formation
in vitro.
on TSH-induced
colloid
droplet
formation
in vitro.
colloid
droplets
between In
addition
intracellular
animals.
difference
substances.
57
SECRETION
all of
the test
the
number
of
experiments, substances
the is
difference
highly
significant.
between
induced no
by stimulator
TSH
58
SHIMIZU Table
* **
2.
Mean •}
SE
Mean •}SE colloid
The effect and cyclic
§N.S.stands
of T3 or iodide on TSHAMP generation.
of
the
value
obtained
from
of
the
value
obtained
from
droplet
AND
three
or DBC-AMP-induced
animals
three
Endocrinol. Japon February 1975
SHISHIBA colloid
droplet
formation
.
similar
experiments
,
one
of
which
is
composite
with
study. for
not
significant.Statistical
analysis
made
for
the
value
between
1)
and
2)
or
3)
is
listed on the table. That for colloid droplet counts between no stimulator and TSH or DBC-AMP group is also significant. That for cyclic AMP between no stimulator and TSH group is highly significant (p
