Inhibition of thyroid secretion by sodium fluoride in vitro

BIOCHIMICA ET BIOPI-I~'SICAACTA

197

BBA 26 788 I N H I B I T I O N OF T H Y R O I D S E C R E T I O N BY SODIUM F L U O R I D E IN VITRO

C. WILLEMS, J. BERBEROF-VAN SANDE AND J. E. DUMONT Laboratory of Nuclear Medicine, School of Medicine, University of Brussels and Biology Department *, Euratom, Brussels (Belgium)

(Received September 7th, 1971)

SUMMARY N a F mimicked the activation by thyrotropin of iodide binding to proteins and of glucose C-I oxidation but not the accumulation of intracellular colloid droplets or the stimulation of secretion in dog thyroid slices in vitro. On the contrary, N a F inhibited the two latter thyrotropin effects. The inhibitory action of F - was partially relieved by the addition of glucose to the medium; it was mimicked by sodium oxamate. These data suggest that N a F depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. NaF inhibited the activation of colloid droplet accumulation and secretion by N 6 , 0 V - d i b u t y r y l adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) and the accumulation of cyclic AMP in thyrotropin-stimulated slices. This suggests an inhibition at the level of both cyclic AMP accumulation and cyclic AMP action. The inhibition by N a F and sodium oxamate of colloid droplet formation and thyroid secretion but not of glucose C-I oxidation in stimulated slices further confirms our conclusion that the latter effect is not merely a consequence of the activation by thyrotropin of colloid endocytosis.

INTRODUCTION F - is the most potent activator of adenyl cyclase in acellular systems from eucaryotes. However, it has not been shown to activate this enzyme in intact cells 1,2. We have demonstrated 3 that N a F mimics two effects of thyrotropin on dog thyroid which are believed to be mediated b y adenosine 3',5'-monophosphate (cyclic AMP) (i.e. which are reproduced by cyclic AMP and N 6 , 0 V - d i b u t y r y l cyclic AMP, and are potentiated by caffeine) : the activation of glucose C-I oxidation and of I - binding to proteins. However, contrary to thyrotropin action, N a F does not induce the accumulation of intracellular colloid droplets in this material 3. We suggested that this discrepancy could be due to an inhibition by N a F of one of the metabolic steps Abbreviations: cyclic AMP: adenosine 3',5'-monophosphate; dibutyryl cyclic AMP: N6,O2'-dibutyryladenosine 3',5'-monophosphate. The expression glucose C-I oxidation is used to represent 14CO~formation from (I-14C~glucoseby the thyroid slices. * Contribution No. 736. Biochim. Biophys. Acta, 264 (1972) 197-2o4

198

c. WILLEMSel al.

required in the phagocytosis of colloid droplets. The purpose of this work was to investigate the mechanism of action of F - on thyroid metabolism and secretion. METHODS

The methods used for the measurement of El~C~glucose oxidation 4, 125Ibinding to proteins, intracellullar colloid droplet formation 3, and 131I secretion 5, have been previously described. In summary, dogs (~: 15 kg) were administered 15o/~C of carrier-free 13~I- by subcutaneous injection; then for 3 days they received 15o mg of thyroid powder (Thyranon, Organon, Oss, Nederland) per day in their food. On Day 4, thyroid lobes were resected and thyroid slices prepared. The slices were incubated at 37 ° in Parker 199 culture medium with io % calf serum in an atmosphere of carbogen. The incubation medium contained 8 mM glucose and was supplemented with (a) o.5/tC/ml [I-~4C~glucose for the measurement of glucose C-I oxidation and the evaluation of intracellular colloid droplet formation ; with (b) io tim 125I- (specific activity 5 ° mC/mmole) for the measurement of iodide binding to proteins; and with (c) 2 mM methimazole and I mM NaCI04 for the measurement of thyroid secretion. Butanol-extractable ~3~I secretion was measured directly from the butanol extract of the incubation medium and not as previously described 5 from the difference between total radioactivity and non-butanol-extracted radioactivity of the medium. This new procedure gave lower and more reproducible basal butanol extractable 13'I release values. The incubations were carried out for 45 min for the measurement of I - binding to proteins, for I or 2 h for the study of glucose oxidation and colloid droplet formation and for 4 h for the evaluation of secretion. In the latter case, the slices were preincubated in the absence of a stimulatory or inhibitory agent, for I tl. For the measurement of cyclic AMP accumulation, the slices were incubated for 80 min in Krebs-Ringer bicarbonate buffer supplemented with i nile{ caffeine and [3Hjadenine (15o #C/ml). F was added after 6o min and thyrotropin after 7o min of incubation. After the incubation, the slices were boiled for 3 min in I ml water containing 2.5 tmaoles cyclic AMP, homogenized, and centrifuged at 2oooo × g. The supernatant of this homogenate was used for the determination of ATP by the luciferase method s. The measurement of L3HIATP after separation was determined by two-dimensional thin-layer chromatography (PEI-cellulose, 1.6 M LiC1 and 2 M formic acid (I:I, v/v) and o.5 M then 0. 7 M (NH~)2SO4) 7. The measurement of cyclic !3HJAMP was determined after precipitation with ZnSO4-Ba(OH )2 followed by chromatography on Dowex 5o(H+)8, and two-dimensional thin-layer chromatograph 3, (cellulose, methanol-water (60:40, v/v); isopropanol-saturated (NH4)2SO4-water (2:79 : 19, by vol.)) ~, 10. The recoveries of cyclic [3H]AMP and [3H~ATP were measured using cyclic [HC~AMP as tracer and non-radioactive ATP. Cyclic AMP accumulation was estimated from cyclic AMP = (ATP × cyclic [3H!AMP)/[3HJATP after correction for cyclic [3HIAMP and I3H!ATP recovery. The rationale and details of this method will be described elsewhere. Because of the wide variation of response to thyrotropin and of basal secretion from one thyroid to another, results of experiments carried out on different thyroids have not been pooled. The results of individual experiments are therefore presented, each one representing the means of two or three closely agreeing duplicates. [3HJAdenine (specific activity 21 C/mmole) was obtained from the Radiochemical Centre (Amersham, Great Britain), cyclic E14C]AMP (specific activity > Biochim. Biophys. Acta, 264 (1972) 197-2o4

NaF

199

INHIBITION OF THYROID SECRETION

4o mC/mmole) from NEN Chemicals (Frankfurt/Main, Germany) or from the Radiochemical Centre (specific activity > 25 ° mC/mmole). ATP and dibutyryl cyclic AMP were from Boehringer (Mannheim, Germany). The thyrotropin used was either Thytropar (Armour, Kankakee, U.S.A.) or N I H thyrotropin (NIH-TSH-B5 Bovine) (gift of Dr. P. G. Condliffe). RESULTS

As reported previously, low concentrations of thyrotropin (from o.i mU/ml upwards) stimulated in vitro the oxidation of glucose C-I, the binding of I - to proteins, the formation of intracellular colloid droplets, and the secretion of butanol-extractable ~axI by dog thyroid slices in our system. These effects were mimicked by rather low concentrations of dibutyryl cyclic AMp3, 5. F - at concentrations of 4-1o mM (ref. 3), but not at 3 mM, considerably enhanced glucose C-I oxidation (from 25o to 7oo % of the controls (Fig. I) 11-13. This effect was observed after 3o min, 6o min, and 12o min of incubation. The combined effects of thyrotropin and 5 mM NaF were higher than those of either agent alone although no true additivity has been demonstrated.

I

DROPLETS BE TM I release

% of

control

%

C- 1

PB 1311

of control

SECRETION

,

~00

10

300 200

6

100 2

0

-g

UV-- Z

_~

Ez

o

7"

Fig. I. Effect of F - a n d t h y r o t r o p i n in vitro on glucose C-I o x i d a t i o n , I - b i n d i n g to p r o t e i n s a n d secretion in dog t h y r o i d slices. R e s u l t s of a r e p r e s e n t a t i v e e x p e r i m e n t carried o u t on t h e t h y r o i d of one dog. M e a n s ~ r a n g e of duplicates. A b b r e v i a t i o n s : C-I, glucose C-I o x i d a t i o n ; PB131I, l a l I b i n d i n g to p r o t e i n s ; secretion, b u t a n o l - e x t r a c t a b l e 1311 release in t h e m e d i u m in p e r c e n t of t h e t o t a l 1~1I of t h e slices; colloid droplets, intracellular colloid d r o p l e t f o r m a t i o n ; T S H , t h y r o t r o p i n .

BURKE12has reported additive effects of NaF and thyrotropin on glucose C-I oxidation in sheep thyroid slices. F - also enhanced the binding of 1251- to proteins3; this effect already very marked at I raM, increased up to I0 mM (from 200 to 800 %). A similar effect of I0 mM NaF accompanied by enhanced iodothyronine formation has been observed by AHN AND ROSENBERG14. Caffeine did not potentiate the effects of NaF at any concentration. The effects of thyrotropin and NaF were not additive. At no Biochim. Biophys. Acta, 264 (1972) 197-2o4

C. WILLEMS et al.

200

concentration between I and Io mM did N a F elicit the formation of intracellular colloid droplets. At 5 mM, N a F completely inhibited the appearance of droplets in slices stimulated by o.25 or I mU/ml thyrotropin but not by 5o or ioo mU/ml thyrotropin. At no concentration between I and IO mM did NaF enhance butanol-extractable lali release by the slices. Similarly, AHN AND I{OSENBERG15 did not observe any increase in iodoprotein proteolysis in the presence of IO mM NaF in dog thyroid slices. On the contrary, 5 mM N a F decreased the butanol-extractable 1311 release. In the presence of glucose, the thyrotropin stimulated secretion was inhibited slightly at i mM, greatly (60-80 % inhibition) at 5 lnM, and almost completely at io mM. This effect was more pronounced for low concentrations of thyrotropin. Slices preincubated for 2 h in the presence of 5 mM N a F were, after washing, fully responsive to thyrotropin in a subsequent incubation. 5 mM N a F also inhibited the dibutyryl cyclic AMP stimulated secretion, although less than the thyrotropin action. I - I O mM N a F had no significant effect on the non butanol-extractable 131I release from the slices.

BE 131I release % of total 1311

TSH 1m U / r n [ NaF • 5 mM

with glucose

% of control

Lactate

without glucose

3.

2_

2OO

LL

u. z

~-rm o~-Z

Fig. 2. Inhibition of t h y r o i d secretion by F - in vitro in the presence and absence of glucose in the mediunl. I n the absence of glucose, no lactate was detected in the medium. Lactate formation is therefore only indicated for the flasks containing glucose (basal value 12. 5 # m o l e s / l o o mg per 4 h). I n c u b a t i o n m e d i u m : K r e b s - R i n g e r bicarbonate buffer. Means ± range of duplicates (one dog thyroid). Abbreviations: see Fig. i.

Thyrotropin-induced thyroid secretion was generally higher in the presence than in the absence of glucose (Fig. 2). The inhibition of this secretion by N a F was much stronger in the absence of glucose in the medium (Fig. 2). As previously observed, aerobic glycolysis was very active in dog thyroid slices incubated in the presence of glucose in vitro; it was activated by thyrotropin and no lactate was formed in the absence of glucose 16. 5 mM F - markedly inhibited basal and stimulated aerobic glycolysis (Fig. 2). These experiments suggested that glycolysis might have an important supporting role in secretion and that the F - effect might be secondary to the inhibition of this pathway. The action of oxamate, another inhibitor of aerobic Biochim. Biophys. Acta, 264 (1972) 197-2o4

N a F INHIBITION OF THYROID SECRETION

201

glycolysis ~7, on butanol-extractable as11 release was therefore investigated. 25 and 50 mM Oxamate partially inhibited spontaneous lactate formation (2o-5o% inhibition) but abolished its increase in the presence of thyrotropin. 25 and 5 ° mM NaC1 only slightly inhibited spontaneous lactate formation and stimulated thyroid aerobic glycolysis. 25 and 5o mM Oxamate consistently inhibited the thyrotropin-induced release of butanol extractable 1~1I from the thyroid slices. 25 and 5o mM NaC1 also inhibited this release, but to a much lesser extent (Fig. 3). Neither oxamate nor NaC1 inhibited the stimulation of glucose C-I oxidation in the thyroid slices at concentrations of thyrotropin which elicit the secretory effect (Fig. 3). As previously reported in other systemsL thyrotropin greatly enhanced cyclic AMP accumulation in our dog thyroid slices. Despite its well known action on partiOXIDATION 8-1 OF GLUCOSE

200

SECRETION

%OF CONTROL

+

T

i00

BE 1311 RELEASE % OF TOIAL 131[

I

i-

I

I +

p+

+

~

+

÷

+

o~

Fig. 3. Action of sodium o x a m a t e (oxa) and NaC1 on the stimulation b y thyrotropin of glucose C-I oxidation and on secretion. Means i range of duplicates (one dog thyroid). Abbreviations: see Fig. i.

TABLE I EFFECT OF THYROTROPIN AND F - ON CYCLIC AMP FORMATION ON DOG THYROID SLICES

Means ~ range of duplicates (all results pertain to the same dog thyroid). Cyclic A 3 f P formation (corrected disint. ~rain X -lO 4 per mg wet wt.)

Estimated cyclic A M P CO•Cn,

A T P conch. (pmoles/mg wet wt.)

(pmoles/mg wet wt.) 0.80 ~ 0.06

320 ~ 20

--

0.46

~ 0.04

Thyrotropin (5 mU/ml)

6.72

=L 0.6

13-1o i

1.2o

320 :~= 4 °

N a F (5 mM)

0-45

=L o.o8

0.66 i

o.12

230 ~2 15

N a F (IO mM)

0.445 =tz o.i

0.79 ± o.15

195 _-L io

N a F (5 raM) + thyrotropin (5 m U / m l )

2.61

4.20 4- o.o6

260 !

:t= o.07

80

Bioehim. Biophys. Acta, 264 (1972) 197-2o4

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C. WILLEMS e[ ctl.

culate adenyl cyclase, N a F had no such effect on tile slices (Table I), which confirms previous observations of ZOR el al. ~3 and AHN AND ROSENBERG 14. In contrast, NaF strongly inhibited the thyrotropin-induced cyclic AMP accmnulation. N a F also decreased the ATP content of the control and thyrotropin-stimulated slices which is consistent with its inhibitory action on glycolysis. DISCUSSION

On the basis of the facts that NaF mimicked the action of thyrotropin on glucose oxidation and I - binding to proteins but not the induction by the hormone of intracellular colloid droplet formation in dog thyroid slices, we have suggested that NaF may inhibit one of the metabolic steps required in the endocytosis of colloid by the thyroid follicular cell. However, PASTAN et al. 11 observed no inhibition by 3.5 mM N a F of the thyrotropin (5o mU/ml) induced intracellular colloid droplet formation, and therefore suggested that NaF action on thyroid metabolism was not related to the cyclic AMP system. In this work, it is clearly demonstrated that NaF blocks the endocytosis of colloid droplets by follicular cells and the consequent thyroid secretion. This effect of N a F was, however, partially overcome when pharmacological, largely supramaximal concentrations of thyrotropin (5o mU/ml) were used. As PASTAN st al. H have used such concentrations of thyrotropin (50 mU/ml), their failure to observe an inhibitory effect of N a F on intracellular colloid droplet formation is explained. Confirming our previous suggestion, NaF inhibits one of the metabolic steps required in colloid endocytosis. One could therefore not expect N a F to activate this process even if it mimicked in some way the action of thyrotropin or cyclic AMP. F - inhibits m a n y enzymes, including enolase, in the Embden-Meyerhof pathway 18. Aerobic glycolysis is very active in the thyroid cell and endocytosis requires energy. Inhibition by NaF of aerobic glycolysis in the thyroid cell could therefore account for the inhibition of the secretory process. At the concentration used in these experiments (5 mM), N a F inhibited lactate formation in the control and stimulated dog thyroid slices. In the absence of glucose in the medium, when lactate formation was not detectable, N a F completely blocked the accumulation of intracellular colloid droplets and thyroid secretion. On the other hand, in the presence of glucose, when lactate formation was inhibited, but not abolished, NaF only partially inhibited thyroid secretion. A more specific inhibitor of aerobic glycolysis, sodium oxamate iv also inhibited thyroid secretion i n vitro. Although hyperosmolarity per se depressed secretion to some extent, the action of oxamate was much stronger than the action of similar concentrations of NaC1. These data therefore support the hypothesis that N a F depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. Although aerobic glycolysis is very active in dog slices i n vilro, it can be calculated that this pathway only accounts for I5-2o % of the ATP formed in the cells ''°. Moreover, N a F only very partially depleted the cellular ATP stores. The importance of the inhibition of secretion when aerobic glycolysis is depressed is therefore striking. We have previously observed that glucose and aerobic glycolysis seemed to play a more important role in another ATP-requiring metabolism of the thyroid, i.e. the transport of I , than their participation in the overall ATP formation would suggest 2°. I - transport and endocytosis of colloid are plasma-membrane functions. Perhaps for spatial reasons, glycolytic ATP may have a Biochim. ]?,iophys. Acla, 264 (I972) 197-2o4

NaF

INHIBITION OF THYROID SECRETION

203

special role in the supply of ATP for membrane function. A close dependence of phagocytosis on aerobic glycolysis has also been demonstrated in leucocytes 21. The inhibitory action of NaF on thyroid secretion might affect cyclic AMP accunmlation or cyclic AMP action. NaF markedly inhibited dibutyryl cyclic AMP as well as thyrotropin-induced secretion in vitro, which shows an effect on cyclic AMP action. However, the inhibition was always more marked on thyrotropin than on dibutyryl cyclic AMP-induced secretion which also suggests an action on cyclic AMP accumulation. N a F per se did not enhance cyclic AMP accumulation in dog thyroid slices; rather it strongly inhibited the thyrotropin-induced increase. Therefore, the inhibitory action of N a F on thyrotropin-induced secretion is exerted at the two levels of cyclic AMP accumulation and cyclic AMP action. N a F mimicked the activation of thyrotropin on glucose C-I oxidation and I binding to proteins in dog thyroid slices without enhancing cyclic AMP accumulation. This suggests that the N a F mimicry of thyrotropin action, reflects an effect at a step further than cyclic AMP formation in the chain of cause-effect relationships, or as suggested by some authors n-13 that N a F acts on an entirely different site in thyroid metabolism. Evidence to be reported elsewhere suggests that the thyrotropin-like effects of N a F might be secondary to an inhibition of protein phosphatase, and therefore to an enhancement of the phosphorylation level of specific proteins which are phosphorylated by cyclic AMP-dependent protein kinase(s). The fact that NaF inhibits the thyrotropin-induced secretion but not the stimulation by the hormone of glucose C-I oxidation and I - binding to proteins, further suggests that these two latter thyrotropin effects are not secondary to the activation of endocytosis 3. This conclusion is confirmed by the fact that sodium oxamate inhibits the activation of thyroid secretion but not of glucose C-I oxidation in dog thyroid slices. ACKNOWLEDGEMENTS

The authors wish to thank Mr. A. Melis for his technical collaboration and Miss Ch. Borrey for the preparation of the manuscript. This work has been carried out under Contract of the Minist~re de la Politique Scientifique within the framework of the Association Euratom, University of Brussels, University of Pisa, and partially thanks to grant No. IO~I of the Fonds de la Recherche Scientifique M~dicale.

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Biochim. Biophys. dcta, 264 {I972 ) i 9 7 - 2 o 4