Transient increase in prostaglandin production as an acute response of thyroid isolated follicles to thyrotropin

BIOCHEMICAL

Vol. 99, No. 4,198l April

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RESEARCH

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30, 1981

TRANSIENT

INCREASE IN PROSTAGLANDIN PRODUCTION AS AN ACUTE

RESPONSE OF THYROID ISOLATED FOLLICLES Yasuyuki Department

Received

Igarashi

of Biochemistry,

Biochemistry,

Institute

March

1045-1050

School

TO THYROTROPIN

and Yoichi

Kondo

of Medicine

of Endocrinology,

and Department

Gunma University,

of Physical

Maebashi

371,

Japan

4,1981

SUMMARY 1. Isolated follicles obtained from pig thyroid were incubated in KrebsRinger bicarbonate buffer containing glucose. Following an immediate increase, immunoreactive prostaglandin E in the reaction system reached a plateau. When 80 mu/ml of thyrotropin was added 30 min after the onset of incubation, the prostaglandins abruptly increased within 1 min and returned to the original level by 10 min, while no appreciable change was observed in the absence of TSH. 2. The minimal effective dose of TSH for this induction of prostaglandin increase was between 1 and 5 mu/ml under the present conditions. 3. The specificity of the stimulant was tested by using the same number of units of two thyrotropin preparations and the same weight of other hormones. ACTH was not active. Lutropin and follitropin were considerably less active than, and epinephrine was as active as, thyrotropin. 4. The phenomenon may reflect the TSH-induced transient accumulation of diacylglycerol (B.B.R.C. 97, 759 and 765) as a possible source of prostaglandins. INTRODUCTION The involvement has been

the subject

prostaglandin has not et al. release initial

phase

release

in

isolated plasma

investigators

Toccafondi

et al.

the first they

did

not

membranes. was a major

of prostaglandin L/ Abbreviations DG, diacylglycerol;

and focussed

studies

in which (7,

linked

8),

o b served

The results production

of TS&'

a high

level

of thyroid

on the TSH dependent an acute

depression

effect

as an acute

that

the enzymes

and led response

degradation

1045

of PGE concomitantly. in thyroid

lipases

produced follicles

used are: TSH, thyrotropin; PGE, prostaglandin adenosine 3'5'-monophosphate. CAMP, cyclic 0006-291

the

of TSH on the

us to examine of the

TSH. in

was increased

phosphatidylinositol

suggested

with

TSH action

of DG and monoacylglycerol

of prostaglandins

on

of prostaglandin

slices

to this

CAMP production

we found

with

and the existence source

effect

functions

and Haye et -- al. (2, 3), but (Boeynaems et (41, Margotat - al -*

pay much attention

period

DG accumulation follicles

(6)

of thyroid

(1)

5 min of the incubation

of incubation previous

by Burke

by other

the following

In our

which

Recently hand,

The stimulatory

was reported

been confirmed

On the other

in the regulation

of controversy.

synthesis

(5)). during

transient

of prostaglandins

X/81/081

in the

arachidonate the possibility to TSH. E; 045-06$01.00/0

Copyrighf 0 I981 by Academic Press, Inc AN riphts of renroduction in nnv form wwnxvi

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Vol. 99, No. 4,198l

In the

present

PGE production

paper,

in the

AND

we describe

first

10 minutes

BIOPHYSICAL

RESEARCH

the TSH-specific

induction

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of transient

of incubation.

MATERIALS AND METHODS 1) Preparation and incubation of thyroid follicles Pig thyroid follicles were prepared by the method reported previously (7). Briefly, the glands were perfused with collagenase solution and incubated. Follicles were liberated by mechanical squeezing. The isolated follicles were practically pure and not contaminated with blood cells and other tissue cells. About 0.2 g of packed follicles were incubated with 0.8 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM of glucose. Before starting the incubation, the follicle suspension was kept at 0 C. In some experiments, a TSH solution was added (80 mu/ml in the vessels, reaction medium) after 30 min preincubation at 37 C. In the control the TSH solution was replaced with the Krebs-Ringer bicarbonate buffer containing 1% albumin which was used to dissolve TSH. 2) Extraction of prostaglandins After the incubation of follicles, prostaglandins were extracted by the method of Yaffe and Behrman (9). 3 ml of ethylacetateisopropanol-0.2 N HCl (3:3:1) was added into each reaction mixture and vortexed for 30 sec. 3 ml of water and 2 ml of ethylacetate were then added and the mixture was shaken vigorously. After centrifugation at 1,200 x g for 5 min, 2 ml of the upper phase was pippeted out and evaporated. The dried residue was dissolved in 0.1 ml of methanol, diluted with an appropriate volume of 10 mM potassium phosphate buffer (pH 7.4) containing 0.15 M NaCl, 0.1% NaN3 and 0.1% BSA and subjected to radioimmunoassay of PGE. The efficiency of the extraction procedure was determined by adding 3H-PGE2 to the incubation mixture and 87.5% of the total radioactivity was recovered in the final extract. All procedures except evaporation were carried out at 0 to 4 C. By this procedure, both extracellular and intracellular prostaglandins were extracted. 3) Radioimmunoassay factory (Miles-Yeda

of prostaglandin Ltd., Rehovot,

E Radioimmunoassay was performed Israel) recommended method.

by a

4) Hormones and chemicals Whale TSH (1.6 U/mg) was kindly prepared and supplied by Drs. H. Takahashi and N. Ui of the Institute of Endocrinology, Gunma University were purified by the method of Yora and Ui (11). (i0). Bovine TSH and lutropin This TSH preparation was practically pure on the molecular basis. The biological activity was estimated to be between 10 and 20 U/mg, by comparison with whale TSH TSH contamination of the lutropin in terms of morphogenetic activity (12). preparation was estimated by a radioimmunoassay to be 2 to 3 % of the total protein. Follitropin was NIH-FSH-S5 Ovine. Epinephrine was purchased from Merck (Darmsadt, ACTH (synthetic ACTHI(Cortrosin)) from Daiichi Pharmaceutical Co. Germany), (Tokyo, Japan). [5,6,8,11,12,14,15(n)-3H] PGE2 (160 Ci/mmol) was obtained from Radiochemical Center (Amersham, England), rabbit anti-PGE -BSA serum (a product of Miles-Yeda Ltd. (Rehovot, Israel)) was a gift of Dr. H. Okazaki of Seikagaku Kogyo Ltd., PGE2 was purchased from Sigma Chemical Co. (St. Louis, MI) and Collagenase II was obtained from Worthington Biochemicals (Freehold, NJ). Other chemicals were reagent grade. RESULTS Pig bicarbonate abrupt

thyroid buffer

increase

The level no further

follicles in

stored

containing

glucose.

the amount

of prostaglandins

of PGE decreased production

at 0 C were

slightly

of PGE by the

Five during

incubated minutes

at 37 C in a Krebs-Ringer incubation

measured a further

incubation

IO46

at 37 C caused

as immunoreactive

60 min of incubation,

of follicles

in the buffer

an

PGE. indicating (Fig.

1).

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Vol. 99, No. 4,1981

01 Ot

AND

BIOPHYSICAL

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Tlme(mln)

Tlme(mln)

Change in PGE content of thyroid follicles incubated The chilled thyroid isolated follicles were incubated bicarbonate buffer (pH 7.4) containing 10 ml4 glucose. prostaglandins in the reaction system were extracted ethylacetate-isopropanol-0.2 N HCl and measured by for PGE. x indicates the mean of two assayed values Fjg.

2.

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without TSH. in Krebs-Ringer Total by adding the radioimmunoassay (01.

Effect of TSH on PGE content in thyroid fol.licles. The incubation and the PGE determination were performed as described in the legend to Fig. 1, except that at 30 min (indicated by an arrow), TSH dissolved in an albumin solution was added in the experimental medium or the albumin solution containing no TSH in the control medium. The incubation was continued for the time ir.dicated until 30 min after the addition of the hormone. x indicates the mean of two assayed values a, with TSH; o, without TSH. (*, 0).

Therefore,

in further

experiments

the agents

were

into

added

to see the effect

the follicle

suspension

of agents

on PGE production,

after

30 min preincubation

at

the amount

of PGE was observed

37 c. As shown in after

the

several

minutes,

TSH solution Fig. reach purified

increase

the addition

change

as 1 to 5 mu/ml did

U/mg) did

which

was contained

(Fig.

1 min for

in

the

of PGE.

PGE increase.

As shown in the

and the effect

seemed to

10 to 20 mu/ml.

affect

TSH (10 to 20 U/mg)

TSH (1.6

was maintained

the amount

of the acute

above not

in

level

of TSH was effective

at concentrations

bovine

in

of serum albumin

no appreciable

serum albumin

whale

protein

while

caused

a plateau Since

crude

2, a sharp

of 80 mIJ/ml of TSH and the higher

3 shows TSH dose dependency as little

figure,

Fig.

addition

PGE production

showed 4),

(Fig.

the same extent the TSH action

2) and a highly of stimulation

was not

as the

a nonspecific

action. In the experiment

hormones and lutropin epinephrine

were

tested.

shown in Fig.

and follitropin was slightly

4, the stimulating

Among the pituitary were

hormones

much less

more active

active

than TSH.

1047

activities tested,

than

TSH.

of some other

ACTH had no activity On the other

hand,

Vol. 99, No. 4,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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d

i LH

Fig.

3.

Fig.

Adr

Effect of TSH concentration on PGE content. The incubation and the PGE determination were performed as described in the legend to Fig. 2, except that the reaction was stopped 1 min after the addition of TSH. x indicates the mean of two assayed values

4.

Effect of various hormones on PGE content. The incubation ccnditions and PGE determination described in the legend to Fig. 3. Column of two assayed values (0). A dotted line wTSH, whale TSH (1.6 U/mg); bTSH, bovine follitropin; LH, lutropin; Adr, epinephrine. all hormones was 1.25 ug/ml in the incubation concentration of wTSH which was 12.5 ‘sg/ml.

(0).

were performed as indicates the mean the control level. FSH, to 20 U/mg); The concentration of medium, excepting the

height indicates TSH (10

DISCUSSION The

study

present

TSH resulting in the response

the

in a transient

introduction, of the

tissue in

in prostaglandin

--et al. (4) reported However, they did

and therefore

our

between

one to show an acute

increase

was observed.

reported

discrepancy

first

Boeynaems

of prostaglandin those

is

present

their

experimental

not

is

results

production

that

Fiirstenberg

production

of PGE in mouse epidermis

stimulated

the

transient

the case,

and ours.

short

found

by --in vivo

term such as

may be no

Regarding

and Marks

production

phenomena there

the

transient

a similar

treatment

last

two papers

in the present

study

(7.

8),

induced

we reported

that

the immediate

the

transient

with

phorbol

same dose of TSH as that

degradation

and the transient accumulation of arachidonate-containing membranes contained DG and monoglyceride specific lipases arachidonate only

As mentioned

no TSH-dependent examine

to

(13). In our

used

noted

this

of PGE, it

ester

is

If

of thyroid

production. that

may have missed

paper.

response

as a source

a minute

several

(7),

minutes

TSH was estimated inconsistent with degraded

while

(Fig.

2).

of prostaglandins. the TSH-induced In addition

even

PGE increase to these,

if

DG and that which could

The TSH-induced

we considered

1048

that

the thyroid produce

DG increase

continued

the amount

to be much more than that of PGE. These the idea that the PGE produced is derived

phosphatidylinositol,

of phosphatidylinositol

for

lasted

at least

of DG produced findings mainly

by

were not from the

a considerable

part

of

Vol. 99, No. 4,198l

BIOCHEMICAL

AND

BIOPHYSICAL

the arachidonate-containing

DG may be recovered

turnover

to prostaglandins

cycle

or converted

On the other

hand,

hypothetical

TSH-dependent

results

in the

ruled

at this

formation

immediately

after

A similar

the initiation

via

PGE and other

an alternative phospholipids

a stimulant not

its

metabolites.

possibility that by phospholipase

of PGE is

not

an A2

completely

we do not

On the other activity

might

total

using

release

from

stimulated

the

stimulation study,

thyroid

it

slices

phenomenon

two systems,

although

is noted

that

was not

is

In a preliminary

experiment

the epinephrine

adrenergic

effect

action, (data

not

prostaglandin

1 h incubation,

a transient

suggesting

phenomenon.

the relationship

between may work

At this

the epinephrine

by means of other

that

the

stage

of the

actions

in

mechanism

of TSH. The role

controversial

of prostaglandins (14,

15).

of prostaglandins

TSH-specific prostaglandins

production

the thyroid

secretion

system

provide

direct

study.

However,

seems to indicate

of thyroid

evidence

had been

of a physiological

the occurrence a role played

of the by

regulation

to refer to an earlier assumption of prostaglandins in the regulation the amylase

not

in the present

prostaglandin in

in the regulation

We could

induced

system. In this connection, we wish by Boeynaems -et -*al (15) on the involvement of hormone secretion and by Hokin's group

by cholinergic

ACKNOWLEDGEMENTS We are grateful to Prof. S. Yamashita and discussion and to Dr. K. Hosaka of our

1049

of

in progress.

to be sensitive

after

the stimulant

to a few percent

by Boeynaems -et -*al This (4). to phentolamine.

was found

was observed

active.

Such TSH-like

the epinephrine-induced

reported

of the

TSH was fully

possibility

inhibited

was an alpha

dependent PGE

PGE production.

only weak activity.

TSH.

phentolamine

had been

by epinephrine

than

was not

stage

study,

this

in a buffer

of increased

corresponding

to examine

effect

release

we couldnotexplain

the part

of the spontaneous showed

more active agents,

this,

prostaglandin

However,

study

the epinephrine

Concerning

any addition.

incubation

At this

in the present

by TSH contamination

blocking

that

shown).

tested

was slightly

without

PGE increase

for

by the TSH stimulation.

A further

adrenergic

suggesting

the

spontaneous

and follitropin

be explained

protein.

Epinephrine

this

during

the precursor

know the mechanism

lutropin

increased

by Boeynaems --et al. who used thyroid slices cases, such an abrupt PGE increase was

Since

hormones

hand,

follicles

in the buffer

was observed

of follicles,

Among pituitary

thyroid

In both

increase

be the DG produced

investigation,

the

of incubation

was added.

TSH stimulation

should

of PGE in the

preparation.

and no further

unless upon

in

than

as a source

had been reported

of a follicle

transient

role

other

of other

1, the amount

phenomenon

instead

the

in phosphatidylinositol

of the study,

of arachidonate

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out. As shown in Fig.

that

stage

degradation

RESEARCH

stimulation

(16).

of our University for University for advice

encouragement and discussion.

than

Vol. 99, No. 4,1981

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RESEARCH

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We thank Dr. H. Takahashi and Prof. N. Ui of our University for supplying whale Kogyo Co. for the gift of antiTSH. We thank Dr. H. Okazaki of Seikagaku prostaglandin E serum. This study was supported in part by a grant from the Ministry of Education, Culture and Science of Japan. REFERENCES 1. Burke. G.. Chang. L-L. and Szabo. M. (1973) Science, -180, 872-875 Haye,.B.,'Champion, S. and Jacquemin, C. (1973) FEBS Lett. 30, 253-260 2. Haye, B., Champion, S. and Jacquemin, C. (1973) FEBS Lett. 41, 89-93 3. M. and Dumont, J.E. (1979) Endocrinology, 105, 4. Boeynaems, J.M., Woebroeck, 988-995 5. 6.

7. a. 9.

10. 11. 12. 13. 14. 15. 16.

Margotat, A., Rolland, P-H., Charrier, B. and Mauchamp, J. (1978) FEBS Lett. 95, 347-351 Toccafondi, R.S., Patrono, C., Pinca, E., Aterini, S., Rotella, C. and Tanini, A. (1980) in Thyroid Research VIII (Stockigt and Nagataki eds.) Australian Acad. Sci. Camberra, Australia p 77-80 Igarashi, Y. and Kondo, Y. (1980) Biochem. Biophys. Res. Commun. 97, 759-765 Igarashi, Y. and Kondo, Y. (1980) Biochem. Biophys. Res. Commun. 97, 766-771 Jaffe, B.M. and Behrman, H.R. (1974) in Methods of hormone radioimmunoassay (Jaffe and Behrman eds.) Academic Press, New York p 22 Takahashi, H. and Ui, N. (1972) J. Biochem. 2, 531-541 Yora, T. and Ui, N. (1978) J. Biochem. 83, 1173-1190 Kondo, Y., Horiuchi, R., Inoue, K., Ui, N. and Sho, K. (1980) in Thyroid Research VIII (Stockigt and Nagataki eds. ) Australian Acad. Sci. Camberra, Australia p 88-91 FUrstenberg, G. and Marks, F. (1980) Biochem. Biophys. Res. Commun. 92, 749-756 Wolff, J. and Moore, W.V. (1973) Biochem. Biophys. Res. Commun. 11, 34-39 Ketelbant-Ballase, P., Van Sande, J. and Dumont, J.E. (1976) Boeynaems, J.M., in Thyroid Research (Robbins and Braverman eds.) Excerpta Medica, New York, p 76-77 Marshall, P.J., Dixon, J.F. and Hokin, L.E. (1980) Proc. Natl. Acad. Sci USA II, 3292-3296

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