Effects of prostaglandins Fα on dog thyroid cyclic AMP level and function

Biochimica et Biophysica Acta, 716 (1982) 53-60 Elsevier BiomedicalPress

53

BBA21109

EFFECTS OF PROSTAGLANDINS Fa ON DOG THYROID CYCLIC AMP LEVEL AND FUNCTION J. VAN SANDE, P. COCHAUX, C. DECOSTER, J.M. BOEYNAEMS and J.E. DUMONT

Institute of Interdisciplinary Research, Erasmus Hospital, B~t. C Route de Lennick 808, 1070 Brussels (Belgium) (Received August 24th, 1981) (Revised manuscript received December 23rd, 1981)

Key words: Prostaglandin Fa; Cyclic AMP; (Dog thyroid)

Prostaglandins Fla and F2., at high concentrations ( ~ 2 8 / z M ) enhanced cyclic AMP accumulation in dog thyroid slices. At lower concentrations, they inhibited the cyclic AMP accumulation induced by thyrotropin (TSH), prostaglandin El, and cholera toxin. This effect was rapid in onset and of short duration, calcium-dependent and suppressed by methylxanthines. Prostaglandin F~ also inhibited TSH-induced secretion and activated iodide binding to proteins. These characteristics are similar to those of carhamylcholine action, except that prostaglandins F did not enhance cyclic GMP accumulation. The effect of prostaglandin F,, was not inhibited by atropine, phentolamine and adenosine deaminase and can therefore not be ascribed to an induced secretion of acetylcholine, norepinephrine or adenosine. It is suggested that prostaglandins F act by increasing influx of extracellular Ca2+. Arachidonic acid also inhibited the TSH-induced cyclic AMP accumulation. However this effect was specific for TSH, it was enhanced in the absence of calcium and was not inhibited by methylxanthines or by indomethacin at concentrations which completely block its conversion to prostaglandin F~. Arachidonic acid action is sustained. This suggests that arachidonic acid inhibits thyroid adenylate cydase at the level of its TSH receptor and that this effect is not mediated by prostaglandin F~ or any other cyclooxygenase product.

Introduction It is well-known that prostaglandin E I stimulates thyroid adenylate cyclase, increases cyclic AMP concentration in the thyroid gland and reproduces most of the effects of TSH on thyroid intermediary metabolism and specialized functions [1-5]. As compared to prostaglandin El, the actions of prostaglandins F (prostaglandin Fl~ and prostaglandin F2~) on the thyroid have not been well characterized so far. Stimulatory effects of

Abbreviations: TSH, thyroid stimulating hormone; IBMX, isobutylmethylxanthine; EGTA, ethyleneglycol bis(fl-aminoethylether-N,N'-tetraacetic acid. 0304-4165/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

prostaglandin Fi,~ on the thyroid in vitro, similar to those of prostaglandin E I have been reported by Burke [3]. Shenkman et al. [6] have observed that the in vivo administration of prostaglandin F2, to pregnant women stimulated the secretion of thyroid hormones. On the contrary, Champion et al. [7] and Yamamoto et al. [8] did not detect any increase of thyroid cyclic AMP level in response to prostaglandin Fi,, or prostaglandin F2a. Moreover, Champion et al. [7] have observed that prostaglandin F2a decreased the accumulation of cyclic AMP induced by TSH in the pig thyroid and we [9] have shown an inhibition by prostaglandin F~,, of omithine decarboxylase induction in the dog thyroid, a cyclic AMP-dependent effect. In order to clarify this controversial matter, we have studied

54 in detail the effect of prostaglandins F on the thyroid cyclic AMP system and compared it to previous work on known inhibitors of cyclic AMP accumulation in the thyroid, carbamylcholine and arachidonic acid. Materials and Methods

Thyroid slices were prepared with a Stadie Riggs microtome (Arthur Thomas, Philadelphia, PA) from dogs pretreated with thyroid extract for 1 day (100 mg/10 Kg, Thyranon, Organon Oss, The Netherlands). Within 30 min after the thyroid resection, the slices (30-60 mg wet wt.) were incubated at 37°C under an atmosphere of O2/CO 2 (95:5, v/v) in 2ml Krebs-Ringer bicarbonate buffer (pH 7.4) enriched with 8 mM glucose. The experimental protocol involved a preincubation of 60 min to achieve steady-state of the cells in vitro. The slices were then transfered to fresh medium supplemented with the tracer and chemical required for the measurement of the metabolic variable under study i.e. 2 mM methimazole and 1 mM NaC104 for secretion and KlalI (40/~M; spec. act. 12.5 Ci/mol) for protein iodination. The duration of the test incubation was chosen optimally for the agents tested and the variable studied. It lasted 45 min for the measurement of [ t311]iodide binding to proteins, 4h for secretion and 5-180 min for cyclic nucleotides determination as notified in the text. Evaluation of thyroid hormones secretion by the release of butanol-extractable radioiodine from prelabeUed tissue [10] and of thyroid protein iodination by the incorporation of radioiodine in the trichloroacetic acid-precipitate of the slices [4] were performed as previously described. For cyclic nucleotides assays the slices were immediately dropped into 1 ml of boiling deionized water for 4 min, homogenized and centrifuged. The supernatant was lyophilized and resolubilized in water (10-20 pl H20 per mg wet wt.). The cyclic AMP was measured by the method of Gilman [11]. All the controls of the method have been reported previously [12]. The cyclic GMP concentration was measured by the radioimmunoassay method of Cailla et al. [13] with minor modification. Succinylation was performed after dilution of the samples and separation of bound from free cyclic GMP was carried out by precipi-

tation of the bound nucleotide with isopropranol 1.1 ml for 0.3 ml medium (Merck, Schuchardt, F.R.G.). This method allows detection of 2 fmol cyclic GMP. The antibodies were provided by the Centre d'Immunologie of Luminy, Marseille, France. The tracer (iodinated 2'-O-succinyl-cyclic GMP-tyrosine methyl ester) was prepared and purified in our laboratory. In each experiment, the activity of the reagents and the reactivity of the slices were checked using the binding of 131I to proteins as an index [4]. Results are expressed as mean-+ range of duplicate or mean + standard deviation of the mean of triplicate sets of slices in one typical experiment. Prostaglandins E l, Fl,, F2~ were gifts of Dr J. Pike of the Upjohn Company (Kalamazoo, U.S.A.). TSH as thytropar was obtained from the Armour pharmaceutical company (Chicago, U.S.A.). Carbachol (carbamylcholine) was obtained from K and K (Plain View, NY) and arachidonic acid from Sigma Chemical Company (St Louis, MO). Cholera toxin (prepared by Dr. R.A. Finkelstein) was provided by Schwarzmann (Division of B.ecton-Dickinson and Co, Orangeburg NY). The phosphodiesterase inhibitor Ro 20-1724 was a gift from Hoffman-La Roche (Nutley, New Yersey, U.S.A.) isobutylmethylxanthine (IBMX) was purchased from Aldrich Chemical Co (Milwaukee, WI) and theophylline from Sigma Chemical Co. Cyclic AMP, cyclic GMP, 2'-O-succinyl-cyclic GMP-tyrosine methyl ester, were obtained from Boehringer Pharma (Mannheim, F.R.G.), cyclic [3H]AMP from Amersham International (U.K.). All of the other reagents were of the highest purity commercially available. Results

Prostaglandins FI, and F2~ increased cyclic AMP levels at high concentrations but prostaglandin Faa was more potent. 28 /tM prostaglandin Faa elicited a cyclic AMP response while prostaglandin F~, was ineffective at that concentration. At 70 /~M the two prostaglandins induced a cyclic AMP response which was higher for prostaglandin F2~ (Table I). Prostaglandin Fl~ decreased the cyclic AMP

55 TABLE I EFFECT OF PROSTAGLANDIN F1,, AND PROSTAGLANDIN F2~ ON CYCLIC AMP LEVELS IN DOG THYROID SLICES Results are expressed as pmol cyclic AMP± S.E. per 100 mg wet wt. tissue. The test incubation in the presence of the prostaglandin F and IBMX (0.1 mM) lasted 30 min. Prostaglandin F concentration (pM)

Prostaglandin F la Prostaglandin F2a

0

0.28

2.8

28

70

44 ± 2 44±2

39 --+3 45---2

48 ± 2 45±2

44 ± 4 69---4

58 ± 5 126-- 15

a c c u m u l a t i o n i n d u c e d b y TSH. This was o b s e r v e d when T S H a n d p r o s t a g l a n d i n F]a were a d d e d together to the m e d i u m or when p r o s t a g l a n d i n F]a was a d d e d 30 m i n after TSH. A s shown in Fig. 1 the effect of p r o s t a g l a n d i n Fla was the most p r o n o u n c e d at the shortest time s t u d i e d i.e., 5 min. F o r a p e r i o d longer than 60 min, the cyclic A M P

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levels of p r o s t a g l a n d i n F l ~ - t r e a t e d slices progressively a p p r o a c h e d the values of c o n t r o l slices (without p r o s t a g l a n d i n Fla ). T h e effect of higher c o n c e n t r a t i o n s o f T S H (10 a n d 50 m U / m l ) on cyclic A M P levels was also d r a m a t i c a l l y i n h i b i t e d b y 2.8, 5.6 a n d 28 # M p r o s t a g l a n d i n Fl~. T h e a c t i o n of p r o s t a g l a n d i n F2a was similar b u t for p r a c t i c a l reasons m o s t of o u r w o r k was p e r f o r m e d with p r o s t a g l a n d i n Fla. W e have shown previously that c a r b a m y l c h o line t h r o u g h a c t i v a t i o n of a m u s c a r i n i c r e c e p t o r also r a p i d l y decreases cyclic A M P i n t r a c e l l u l a r levels in s t i m u l a t e d d o g t h y r o i d slices [14]. This effect is relieved b y m e t h y l x a n t h i n e t y p e phosp h o d i e s t e r a s e inhibitors. A s shown in T a b l e II, the i n h i b i t o r y effect of p r o s t a g l a n d i n F I , , on cyclic A M P levels was s u p p r e s s e d in the presence of 1 m M t h e o p h y l l i n e or 0.1 m M I B M X . 0.1 m M R o 20-1724 a n o n - m e t h y l x a n t h i n e t y p e of phos-

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CO5 2030/.0 60

90

120

LL___J:

150 180 0 [O 3Oleo 60 rain 35

90

rain

120

Fig. I. Kinetics of cyclic AMP accumulation in 1 mU/ml TSH-stimulated dog thyroid slices incubated in presence or absence of 2.8 pM prostaglandin Fi,,. Panel A: TSH and prostaglandin 1::1,,were added together. Panel B: prostaglandin F,° was added 30 re.in after TSH. The arrows indicate the time of prostaglandin F~. addition to the medium. (O O) pmol cyclic AMP per 100 nag wet wt. tissue. ( × - - - ×) intraceHular cyclic AMP levels in prostaglandin Fna-treated slices expressed as percent of the values obtained in control slices incubated without prostaglandin Fi.. Phosphodiesterase inhibitor present in the medium was 0.1 mM Ro 20-1724.

Results are expressed as pmol cyclic AMP per 100 mg wet wt. tissue. The test incubation with TSH (1 mU/ml) and the phosphodiesterase inhibitors lasted 30 min. 5.6 /~M prostaglandin F,° was added 5 min before the end of the incubation. Phosphodiesterase inhibitor

Control

Theophylline (I mM) 82---+5 IBMX (0.1 mM) 75±5 Ro 20-1724 (0.1 mM) 86±7

TSH

TSH + prostaglandin Fn~

454± 47 1814±261

530± 79 2015± 10

2467± 70

1 149± 117

56

phodiesterase inhibitor allowed the expression of prostaglandin F1,, as well as carbamylcholine [14] inhibitory effect. As carbamylcholine, prostaglandin Fi,~ and prostaglandin F2~ markedly enhanced the binding of iodide to proteins and depressed the TSHinduced secretion in the dog thyroid slices (Fig. 2). Prostaglandin El, which by itself activates both functions [4,5], did not inhibit the TSH action (data not shown). Arachidonic acid, a fatty acid of C20, and the precursor of prostaglandins, inhibits cyclic AMP accumulation in stimulated thyroid tissue [15]. This effect is marked at 32 #M but % BE

1311

% PB 1311

4.

r20

t' 1

L

/' /

.....

,I

~15 ,

LIO

increases up to 160 #M. It is relatively specific for arachidonic acid as linoleic and linolenic acid fail to reproduce it under standard conditions. In five experiments, cyclic AMP accumulation caused by 5 m U / m l TSH was reduced by 75% ( P < 0 . 0 0 1 ) by arachidonic acid, by 4% (P < 0.05) by linoleic ~cid and not by linolenic acid, all at a concentration of 160 #M. This effect has been compared to the prostaglandin Fl,~ inhibitory action. As can be seen in Fig. 3, the effect of arachidonic acid was immediate and was sustained for at least 2 h. Indeed in the experiment presented (Fig. 3 panel A), the cyclic AMP levels of arachidonic acid-treated slices was 10% of control slices after 2 h incubation as well as after 5 min. When arachidonic acid was added after 30 min TSH action (Fig. 3 panel B), the inhibitory effect was always smaller, which was not the case with prostaglandin Fl,,. The inhibitory effect of carbamylcholine on cyclic AMP accumulation in TSH-stimulated slices disappears in Ca2+-free incubation medium [1617]. As shown in Table III the effect of prostaglandin FI,~, but not of arachidonic acid, was clearly Ca 2+-dependent. The omission of Ca 2÷ in

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PGFIc~(~M ) Fig. 2. Effect of prostaglandin Fia on protein iodination and TSH induced thyroid secretion. (Q O) inhibitory effect of increasing prostaglandin Fi,, concentration on the secretory action of 1 m U / m l TSH. 3 days before the experiment, 250/~C of KI31I was injected intramuscularly to the dog. After I h preincubation, the thyroid slices were transferred to a fresh medium for a 4 h incubation with or without prostaglandin FI,, and 1 m U / m l TSH. After acidification, the incubation medium was extracted with butanol. Results represent the radioactivity present in the butanol extract as percent of the total radioactivity (medium + slices). Basal secretion was 0.16%---0.003. ( × - - X). Stimulatory effect of prostaglandin Fi,, on iodination. After I h preincubation and 45 min incubation with KI31I (40 ~M; spec. act. 12.5 Ci/mol) and prostaglandin Fla, the slices were homogenized and proteins precipitated with 10% trichloroacetic acid: results express the radioactivity present in this pellet as percent of the total radioactivity present in the slices. No effect on the active transport of iodide was observed.

~o of control

5

rain

~

rain

Fig. 3. Kinetics of cyclic AMP accumulation in 1 m U / m l TSHstimulated dog thyroid slices incubated in presence or absence of arachidonic acid (Ara. Ac. 160 #M). Panel A: TSH and arachidonic acid were added together: Panel B: Arachidonic acid was added 30 min after TSH. The arrows indicate the time of arachidonic acid addition to the medium. (O O) pmoles cyclic AMP per 100 mg wet weight tissue. ( × - - - × ) intracellular cyclic A M P levels in arachidonic acid treated slices expressed as percent of the values obtained in control slices. Phosphodiesterase inhibitor present in the medium was 0.1 mM Ro 20-1724.

57 TABLE III Effect of extracellular Ca 2+ on cyclic A M P levels in TSH-stimulated dog thyroid slices incubated with or without arachidonic acid 80 /~M or prostaglandin Fla 5.6/~M. The various Ca 2+ media (I,45 mM; no added Ca 2+ ; no added Ca 2+ + I mM EGTA) were present during the I h preincubation and the 35 rain test incubation. TSH and arachidonic acid were added at the beginning of the test incubation, prostaglandin Fla was added 5 min before its end. Results are expressed as pmol cyclic AMP per 100 mg wet wt. tissue. The basal value was 79-+ 9 pmol cyclic AMP per 100 mg wet wt. tissue. Variation in extracellular Ca 2+ was without effect on the basal value. Agents

Ca 2+ 0.45 mM)

No added Ca 2+

EGTA (I raM)

TSH (1 m U / m l ) T S H ( I m U / m l ) + a r a c h i d o n i c acid (80 #M) TSH (I m U / m l ) + p r o s t a g l a n d i n FI,, (5.6 #M)

2.962-*- 188 1.051-+ 77 1.739--- 64

2.309-+ 59 254 -ll0 2.182±217

1.715-+201 161 -+ 35 1.852 -+ 145

the incubation medium, increased the inhibition by arachidonic acid. Contrary to arachidonic acid, which is specific for the action of TSH, prostaglandin F~a-inhibited cyclic AMP accumulation independent of the nature of the stimulator (Table IV). Champion et al. [7] have reported that indomethacin prevents the inhibitory action of acetylcholine on cyclic AMP accumulation in pig thyroid slices. Indomethacin, a non-steroid antiinflammatory drug, blocks a major pathway of arachidonate metabolism i.e., the first enzyme (cyclooxygenase) of the synthesis of prostaglandins, thromboxanes and prostacyclin [18]. 28 pM

indomethacin, which completely blocked prostaglandin synthesis in our thyroid preparations [19], did not modify the effect of arachidonate on cyclic AMP accumulation. In one representative experiment cyclic AMP accumulation expressed in pmol per 100 mg wet wt. tissue were: control 63-+10; TSH ( l m U / m l ) 479-50; TSH and arachidonic acid (160 #M) 8 5 - 2; TSH and indomethacin (28 /~M) 514-+45, TSH plus arachidonic acid and indomethacin 88-4-4. TSH and arachidonic acid were added at the beginning of the 30 min test incubation, indomethacin was present during the 1 h preincubation and in the test incubation.

TABLE IV Effect of 160 # M arachidonic acid and 28 # M prostaglandin Fi,, on cylic AMP accumulation induced by TSH, prostagl~indin E I of cholera toxin. The test incubation lasted 30 min for TSH and prostaglandin El, and "60 rain for cholera toxin. Arachidonic acid was added at the beginning of the test incubation and prostaglandin Fl~ was added 5 min before the end of the incubation. 0,1 mM g o 20-1724 was present in the test incubation medium as phosphodiesterase inhibitor. Results are expressed as pmot cyclic AMP per 100 mg wet wt. tissue. The basal values were 2 6 ~ 5 in the upper experiment and 65---+20 in the lower experiment. Inhibitor

TSH (1 m U / m l )

Prostaglandin El a

None Arachidonic acid (160/~M)

1.532--- 88 214 -4- 73

418 458

None Prostaglandin FIo (28/~M)

3.524-+256 1.322± 177

Cholera toxin ( I ~ g/ml)

± 14 ± 16

1.353+ 131 1.226-+ 107

1.017-+47 578 -+92

1.205 -+ 138 573 -+ 36

a Prostaglandin E l was added at 2.8 # M concentration for the arachidonate experiment and control; the experiment with prostaglandin F m and control was done with 14 p M prostaglandin E I.

58 TABLE V E F F E C T OF P R O S T A G L A N D I N F2,~ ON CYCLIC G M P LEVELS IN DOG THYROID SLICES Results are expressed in pmol cyclic GMP-+S.E. per g wet wt. tissue. After 40 min preincubation, the slices were transfered to fresh medium containing-+-25 m U / m l TSH and 1 mM theophylline. After 15 min, 28 ~ M prostaglandin F2,~ or 10/t M carbamylcholine was added. The incubation was ended after I, 2, 3 or 5 min. These incubation conditions were chosen according to Jacquemin and coworkers [24]. Addition

Prostaglandin F2,~ (28 #M) 0 min

1 min

Carbamylcholine (10/~M) 2 min

3 min

5 min 5 min

None TSH (25 m U / m l )

34--+7 35-+4

29--+0.4 33+4

29--+7 36-+4

Adenosine inhibits adenylate cyclase at the P site in many system [20]. At high concentration (0.1-1 mM) it inhibits cyclic AMP accumulation in dog thyroid slices, an effect which is relieved by adenosine deaminase (3 U/ml) (Van Sande, J., unpublished data). Such a concentration of the enzyme did not suppress the inhibitory effect of prostaglandin Fl~ (data not shown). Cyclic GMP concentration has been reported to increase under prostaglandin F~ stimulation in some systems, including the dog thyroid [21-24]. Cyclic GMP activates the hydrolysis of cyclic AMP by one of the three thyroid phosphodiesterases, an effect which is blocked by methylxanthines [25]. An increased cyclic GMP level could therefore explain the prostaglandin F~ effect on cyclic AMP. We tried the effect of prostaglandin F~ and prostaglandin F2,, (0.28, 2.8, 28 and 70 #M) in a 30 min incubation. No increase in cyclic GMP was observed. We explored in six experiments the short time scale from 1, 2, 3, 5, 7, 10 to 15 min and the longer time scale from 15 to 60 min without any success. Each point was performed in triplicate. The values obtained were in the good range of the standard curve, approx. 30 pmol/g wet wt. tissue. Jacquemin et al. have reported that 28 #M prost a g l a n d i n F2a increase cyclic GMP concentration in dog thyroid slices, using a protocol in which the slices were prestimulated for 15 min with 25 m U / m l TSH, then exposed to 28 #M prostaglandin F2~ for 1, 2, 3 or 5 min. Reproducing this protocol and incubation conditions, we did not observe any significant effect on the cyclic GMP content of the slices. In the same experiment,

32-+7 35-+6

25 ~- 2 33± 10

218± 19

carbamylcholine induced a clear cyclic GMP increase (Table V). The effect of prostaglandin F2a on cyclic AMP levels was also measured in the same experiments. No inhibitory effect of the prostaglandin F could be observed in the presence of 1 mM theophylline (data not shown). Discussion

We have shown that, whereas high concentrations of prostaglandin F2~ (28 /~M) slightly increase the concentration of cyclic AMP in the resting dog thyroid, lower concentration.~ of prostaglandin FI~ or prostaglandin F2~ (2.8 #M) inhibit the accumulation of cyclic AMP in the stimulated dog thyroid. These observations can thus explain why in some studies prostaglandins F were reported to stimulate thyroid secretion, like TSH [3,6], whereas in other studies they antagonized TSH action [9]. It is well-known that prostaglandin E~ activates thyroid adenylate cyclase and reproduces most of the effects of TSH. The action of prostaglandins F on the thyroid was not so well defined: our study shows clearly that they are mainly antagonists of TSH action. That prostaglandins E and F exert opposite actions is a general phenomenon. Distinct receptors have been identified for prostaglandins E and F [28]. The inhibitory action of prostaglandin Fa on the accumulation of cyclic AMP in the stimulated dog thyroid is characterized by the following properties: rapid onset and short duration; suppression by methylxanthine inhibitors of phosphodiesterases, but not by a non-methyl-

59¸ xanthine inhibitor (Ro 20-1724); lack of selectivity for a particular stimulus of cyclic AMP accumulation; calcium dependency; accompanied by an activation of iodide binding to proteins and an inhibition of secretion. The latter characteristics show that the effect bears on the thyroid follicular cell. Champion and Jacquemin [24] did not observe an inhibitory effect of prostaglandin F:~ on cyclic AMP accumulation in the dog thyroid stimulated by TSH: this contradiction with our data is only apparent, since their experiments were performed in the presence of 1 mM theophylline, which, as we have shown, blocks the inhibitory effect of prostaglandin F~. It is remarkable that the same prostaglandins F inhibit cyclic AMP accumulation in parathyroid cells by an entirely different mechanism: this effect is not suppressed by methylxanthines inhibitors of phosphodiesterases and it is not Ca2+-dependent [29]. These findings raise three main questions: (1) Is the inhibitory action of prostaglandin F~ secondary to the release of a known negative regulator of the system? (2) Does the effect of prostaglandin Fa explain the previously reported arachidonic acid action [1517 (3) What is the biochemical mechanism of action of prostaglandin F,? Experiments with specific inhibitors have ruled out that the inhibitory effect of prostaglandins F, could be due to the release of endogenous epinephrine, acetylcholine or adenosine, three compounds known to inhibit cyclic AMP formation in the thyroid. Since arachidonic acid is converted into prostaglandin F2a by the dog thyroid the question could be asked whether the inhibitory effect of arachidonic acid is mediated by prostaglandin F2~. This is obviously not the case since the properties of the arachidonic acid effect are completely different from those of the prostaglandin F~ effect. Inhibition of cyclic AMP accumulation by arachidonic acid is sustained, maintained in the presence of methylxanthine, selective for the stimulation by TSH and increased rather than inhibited, in a calcium-free medium. Furthermore, the inhibitory effect of arachidonic acid was not suppressed by indomethacin, at concentrations

known tO completely block prostaglandin F2~ synthesis in the same experimental conditions [19]. The selective inhibition of TSH action by arachidonic acid suggests an effect at the level of adenylate cyclase TSH receptor. It might be tempting to speculate that the effect of arachidonic acid on the TSH stimulation of thyroid adenylate cyclase is somehow similar to the inhibition by arachidonic acid and other cis-polyunsaturated fatty acids, of capping in lymphocytes [30]. This effect, which appeared to be related to changes in membrane fluidity, was also independent of the cyclooxygenase and potentiated in calcium-free medium. This would be compatible with the hypothesis that microclustering of the receptor is an essential step in hormone action [31]. The lack of specificity of the prostaglandin F~ inhibition of cyclic AMP accumulation seems to exclude an action at the level of the TSH receptor or of its coupling with adenylate cyclase; it is compatible with an inhibition of the cyclase itself or an activation of cyclic AMP disposal. Prostaglandin F~ enhanced protein iodination which is completely dependent on free intracellular calcium levels [32]. Moreover, the inhibitory effects of prostaglandin F~ on cyclic AMP accumulation and thyroid secretion were abolished in calcium-depleted slices. These characteristics are similar to those of carbamylcholine action which has been demonstrated to be due to an increase calcium influx in the thyroid cell [32]. The action of carbamylcholine on cyclic AMP accumulation are also unspecific for the activator of cyclase and relieved by methylxanthines. The action of carbamylcholine has been related in part to the Ca 2+ dependent accumulation of cyclic GMP which enhances the hydrolysis of cyclic AMP by an activation of a thyroid phosphodiesterases [29], an effect suppressed by methylxanthines [25]. Champion and Jacquemin [24] have reported that prostaglandin F2~ increased cyclic GMP levels in dog thyroid slices. However in our system prostaglandin F~ was unable to raise cyclic GMP concentrations. The reason for the discrepancy is unclear. In the experiment presented, the effect of prostaglandin F2a (28 #M) on cyclic GMP levels was weak and barely significant [24]. Thus, an increase in cyclic GMP accumulation with its consequent activation of cyclic AMP hydrolysis can-

60

not explain prostaglandin F~ action. Therefore, although prostaglandin F~ action seems to be mediated by intracellular calcium, direct evidence of an activation of calcium translocation as well as an explanation of the methylxanthine effet are missing. Acknowledgments The authors would like to thank Mr W. Wasteels and Mr C. Massart for their technical collaboration, Mrs G. Wilmes for the drawing of the figures and Mrs D. Leemans for the typing of the manuscript. This work was supported by the contract of the Minist~re de la Politique Scientifique (Actions Concertb.es). References 1 Wolff, J. and Cook, G.H. (1973) J. Biol. Chem. 248, 350-355 2 Zor, U., Kaneko, T., Lowe, I.P., Bloom, G. and Field, J.B. (1969) J. Biol. Chem. 244, 5189-5195 3 Burke, G. (1970) Am. J. Physiol. 218, 1445-1452 4 Rodesch, F., Neve, P., Willems, C. and Dumont, J.E. (1969) Eur. J. Biochem. 8, 26-32 5 Dumont, J.E., Willems, C., Van Sande, J. and Neve, P. (1971) Ann. N.Y. Acad. Sci. 185, 291-316 6 Shenkman, L., Imai, Y., Kataoka, K., Hollander, C.S., Wan, L., Tang, S.C. and Avruskin, T. (1974) Science 184, 81-82 7 Champion, S., Haye, B. and Jacquemin, C. (1974) FEBS Lett. 46, 289-292 8 Yamamoto, M., Herman, E.A. and Rapoport, B. (1979) J. Biol. Chem. 254, 4046-4051 9 Mockel, J., Decaux, G., Unger, J. and Dumont, J.E. (1980) Endocrinology 107, 2069-2075 10 Willems, C., Rocmans, P.A. and Dumont, J.E. (1970) Biochim. Biophys. Acta 222, 474-481 11 Gilman, A.G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312 12 Van Sande, J. and Dumont, J.E. (1973) Biochim. Biophys. Acta 313,320-328

13 Ca.illa, H.L., Vannier, C.J. and Delaage, M.A. (1976) Anal. Biochem. 70, 195-202 14 Van Sande, J., Erneux, C. and Dumont, J.E. (1977) J. Cyclic Nucl. Res. 3, 335-345 15 Boeynaems, J.M., Van Sande, J., Decoster, C. and Dumont, J.E. (1980) Prostaglandins 19, 537-550 16 Van Sande, J., Decoster, C. and Dumont, J.E. (1975) Biochem. Biophys. Res. Commun. 62, 168-175 !7 Van Sande, J., Decoster, C. and Dumont, J.E. (1979) Mol. Cell. Endocr. 14, 45-57 18 Flower, R.J. (1974) Pharmacol. Rev. 26, 33-67 19 Boeynaems, J.M., Waelbroeck, M. and Dumont, J.E. (1979) Endocrinol. 105, 988-995 20 Londos, C., Wolff, J. and Cooper, D.M.F. (1979) in Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides (Baer, H.P. and Drummond, G.I., eds.), pp. 271-281, Raven Press, New York 21 Kuehl, F.A. Jr, Cirillo, V.J., Ham, E.A. and Humes, J.L. (1973) in Advances in Biosciences, International Conference on Prostaglandins (S. Bergstr6m and Bernhard, S., eds.), pp. 155-172, Pergamon Press, Vieweg, Braunschweig 22 De Asua, L.J., Clingan, D. and Rudland, P.S. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2724-2728 23 Dunham, E.W., Haddox, M.K. and Goldberg, N.D. (1974) Proc. Natl. Acad. Sci. U.S.A. 71,815-819 24 Champion, S. and Jacquemin, C. (1978) FEBS Lett. 92, 156-158 25 Erneux, C., Van Sande, J., Dumont, J.E. and Boeynaems, J.M. (1977) Eur. J. Biochem. 72, 137-147 28 Yamashita, K., Yamashita, S. and Ogata, E. (1979) Life Sci. 24, 563-570 27 Sherwin, J.R. and Mills, I. (1980) Endocrinology 106, 28-34 28 Rao, Ch.V. and Harker, C.W. (1978) Biochem. Biophys. Res. Commun. 85, 1054-1060 29 Gardner, D.G., Brown, E.M., Windeck, R. and Aurbach, G.D. (1979) Endocrinology 104, 1-7 30 Klausner, R.D., Bhalla, D.K., Dragsten, P., Hoover, R.L. and Karnovsky, M.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 437-441 31 Schlessinger, J., Yarden, Y., Barak, L., Lax, I., Gabbay, M. and Geiger, B. (1981) Horm. Cell Regul. 5, 197-208 32 Decoster, C., Mockel, J., Van Sande, J., Unger, J. and Dumont, J.E. (1980) Eur. J. Biochem. 104, 199-208