Stimulation of Thyroid Metabolism by Thyrotropin, Cyclic 3 : 5 -AMP, Dibutyryl Cyclic 3 : 5 -AMP and Prostaglandin E,

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1 European J. Biochem. 8 (1969) Stimulation of Thyroid Metabolism by Thyrotropin, Cyclic 3 : 5 -AMP, Dibutyryl Cyclic 3 : 5 -AMP and Prostaglandin E, F. RODESCH, P. NEVE, C. WILLEMS, and J. E. DUMONT Laboratoires de Mkdecine Nuclkaire, de Gynkcologie et Obstktrique, de Pathologie, et de Mkdecine Exp&irnentale, Facnltk de MBdecine, Universitk de Bruxelles, et Dkpartement de Biologie, Euratom, Bnixelles (Received August 16/November 13, 1968).Under appropriate conditions, thyrotropin stimulated the binding of [1311]iodide to proteins in sheep, calf and dog thyroid slices. In dog thyroid slices, thyrotropin, dibutyl cyclic 3 :5 - adenosine monophosphate, and cyclic 3 : 5 -AMP in the presence of caffeine, stimulated glucose oxidation at C-1, [1311]iodide binding to proteins and intracellular colloid droplet formation; no such effect was obtained with butyrate, 5 -AMP, ADP, ATP or caffeine. The stimulation of [131I]iodide binding to proteins and of glucose oxidation at C-1 by low concentrations of thyrotropin (0.6 mu/ml) was enhanced in the presence of caffeine. These data support the hypothesis that many effects of thyrotropin on thyroid tissue are, to a large extent if perhaps not exclusively, secondary to the activation of thyroid adenyl cyclase by this hormone. Fluoride, which activates adenyl cyclase in acellular systems, mimicked the effects of thyrotropin on glucose C-1 oxidation and on [lsii]iodide binding to proteins. Prostaglandin E,, which in some tissues increases the intracellular levels of cyclic 3 : B -AMP, also stimulated these metabolic steps. No effect of fluoride and prostaglandin El on intracellular colloid droplet formation was observed. Four criteria have to be fulfilled for ascribing a hormone effect on a target cell to an increased formation of cyclic 3 :5 -AMP in the cell through activation of adenyl cyclase by the hormone [ 1, 21 : the hormone should activate adenyl cyclase in tissue homogenate or in a fraction of this homogenate ; the hormone should induce an increase in the level of cyclic 3 : 5 -AMP in the intact cell, this increase preceding other effects of the hormone; cyclic 3 : B -AMP itself should specifically reproduce the effects of the hormone; inhibitors of cyclic 3 :5 - AMP phosphodiesterase such as caffeine or theophylline should potentiate the effects of the hormone. Some of these criteria have been met for different actions of thyrotropin on thyroid tissue. Thyrotropin activates the formation of cyclic3 : 5 -AMP by thyroid particulate preparation [3] and homogenates [4]. It has been reported that thyrotropin increases the levels of cyclic 3 : 5 -AMP in thyroid slices [5]. Cyclic 3 : 5 -AMP, as thyrotropin, stimulates the stereospecific transport of sugars in thyroid slices [6] and thyroid secretion in vivo [7]; it does not however, stimulate glucose oxidation, 3aP incorporation in phospholipids, or colloid droplet formation in thyroid slices [8-121.The latter thyrotropin effects have been reproduced with dibutyl cyclic 3 :5 -AMP [lo-1.21, a derivative of cyclic 3 :$-AMP which enters cells more readily than the parent compound and is resistant to enzymatic hydrolysis [13]. In the experiments carried out in vivo, for which controls with other nucleotides were reported, 5 -AMP, ADP, and ATP mimicked the secretory effect of thyrotropin and of cyclic 3 : B -AMP, which shows that the action of the latter nucleotide was not specific, Potentiation of thyrotropin action by theophylline has been observed for the secretory effect only [7]. Neither the third, nor the fourth criteria of Sutherland have therefore been completely fulfilled for the investigated effects of thyrotropin on thyroid tissue. The purpose of this work was to investigate the effects of cyclic 3 :5 -AMP, its analogue dibutyl cyclic 3 :5 -AMP, and of compounds which are known to influence the metabolism of cyclic 3 :5 - AMP, such as caffeine, fluoride and prostaglandin El, on three thyrotropin sensitive metabolic events in thyroid tissue : glucose oxidation, iodide organification and colloid droplet formation. Criteria 3 and 4 of Sutherland have been substantiated for the first two effects. MATERIALS AND METHODS Dogs were pretreated with thyroid extract (300 mg/day) for 2 to 3 days before the emo oval of the thyroids. Dog thyroid slices were prepared at Unusuul Abbreviations. Bu,-3 : 5 -AMP, No-2-0-dibutyl temperature immediately and incubated less cyclic 3 : 5 -adenosine monophosphate ; protein bound l3ii, 1311 radioactivity precipitated by trichloroacetic acid from than 30 after the Of the lobes. The prepthe homogenate of the slices incubated with [lali]iodide. aration of sheep and calf thyroid slices, the incubation

2 ~ Vo1.S. Nu.1, 1969 F. RODESCH, P. NEVE, C. WILLEMS, and J. E. DUMONT 27 procedure, and the method for measuring the oxidation of [l -14C]glucose oxidation have been previously described [14,15]. The incubation medium was Krebs Ringer phosphate buffer; the incubation time, 1 hour. For the study of iodide organification, the incubation medium contained 0.1 mc/1 of [1311]iodide, the incubation time was 45 min. At the end of the incubation the slices incubated in the presence of [l.14c]glucose were fixed in Bouin, embedded in paraffin, sectioned at 7 p, and stained with periodic acid Schiff haemotoxylin, while the slices incubated with [1311]iodide were blotted on filter paper and frozen in 2 mm methimazole. Sections were observed by light microscopy in order to get a qualitative evaluation of the formation of intracellular periodic acid Schiff + droplets; sections in which most of the cells displayed droplets were classified + +, sections in which some of the follicles did not evidence many intracellular droplets were classified +, sections in which careful examination did not evidence periodic acid Schiff + inclusions were classified as negative. The preparation, incubation and collection after incubation, of isolated sheep thyroid cells, were carried out as previously reported [16]. The 1311-labeled slices and cells were homogenized in the methimazole solution. The radioactivity of the homogenate and of the twice washed I0 Ol0 trichloroacetic acid precipitate of the homogenates (protein bound [1311]iodine) were measured in a well type scintillation counter. Iodide organification was evaluated from the protein bound [131I]iodine, or, in order to avoid the possible interference of effects on iodide trapping, by the ratio of protein bound 1311 to total 1311 of the homogenate. For the same reason, isolated cells were incubated in the presence of 1 mm NaC10, [17]. When the results of different experiments were pooled, the mean, the standard deviation of the mean and Student s t values have been calculated from the logarithms of the data [14]. Results are expressed as antilogarithms of the mean and of the mean minus or plus the standard deviation of the mean or as arithmetic means of at least two closely agreeing duplicates. Thyrotropin (thytropar) was obtained from Armour (Kankakee, U.S.A.), N6-2 -O-dib~tyl-~y~li~ 3 :5 AMP (Bu,- 3 :5 -AMP), cyclic 3 :5 -AMP, 5 -AMP, ADP, and ATP from Calbiochem (Los Angeles, U.S.A.). Purified thyrotropin (15 Ujmg), purified Bu,-3 : 5 -AMP, and prostaglandin El, were graciously furnished by P. G. Condliffe (National Institute of Health, Bethesda, U.S.A.), T. Posternak (Ecole de Chimie, GenPve, Suisse), S. Bergstrom (Karolinska Institutet, Stockholm, Sweden) and J. Pike (Upjohn Cy, Kalamazoo, U.S.A.) respectively. RESULTS Thyrotropin stimulated the incorporation of L131IIiodide in the proteins of sheep thyroid slices. This effect was also observed in Krebs Ringer bicar- bonate buffer. Optimal conditions for obtaining the effect were: incubation time of 1 hour or less, iodide concentration 10 to 50 pm. The following incubations were therefore carried out for 45min with 10pM iodide. Under these conditions, thyrotropin stimulated the organification of iodide in calf and dog thyroid slices and in the slices of 8 out of 11 sheep thyroids (Table 1). This confirms the data of Kondo Table 1. Effect of thyrotropin on iodide organification in thyroid slices of sheep, calf and dog Iodide organification was measured by the ratio protein bound l3ii : total l3ii of the homogenate. Sheep control ratios varied from 0.09 to Calf control ratios varied from 0.07 to Dog control ratios varied from 0.09 to N = number of experiments [Thyrotropinl Iodide organiliration S control Sheep ( ) 5 <0.001 Calf ( ) 6 < Doe ( ) 8 < c - -3 g m-1-0- I o 0.01 a KX) Thyrotropin (rnulrnl) I 131, OOOOO Fig. 1. Ilelation between the concentration of thyrotropin and its effect on glucose C-1 oxidation (C-l), iodide organification (Ia1I), and colloid droplet formation in thyroid slim of one dog on pig thyroid slices [18]. Thyrotropin (1 mu to 100 mu/ml) markedly stimulated the organification of iodide in the slices of 16 out of 18 dog thyroids. The stimulation, by thyrotropin as well as by other agents, was slight in 2 thyroids, characterized by a high ratio protein bound 1311 : homogenate lali in the controls (0.57 and 0.68). Thyrotropin stimulated iodide organification, glucose oxidation at C-1 [14] and the formation of intracellular colloid droplets [ 11,191 in dog thyroid slices. A plot of effects on the 3 variables us. thyrotropin concentration for one typical thyroid is shown in Fig. I. The 3 cellular processes were well stimulated

3 ~ ~ ~.. ~~~ 28 Thyroid Stimulation by Thyrotropin, Cyclic 3 : 6 -AMP and Prostaglandin E, European J. Biorhem. at a concentration of 0.1 mu/ml, iodide organification being already increased at a thyrotropin concentration of 0.01 mu/ml. The action of thyrotropin on iodide organification and on colloid droplet formation, but not the stimulation of glucose C-I oxidation reached a plateau at a concentration of 10mU/ml. The data on glucose oxidation confirm previous results [14]. The effects of thyrotropin have been reproduced with purified thyrotropin. Dibutyl cyclic 3 :5 -AMP stimulated glucose C-I oxidation and induced the formation of intracellular colloid droplets in dog thyroid slices (Table 2, Fig.2). These effects were obtained with the crude as well as with the purified preparation in Krebs Ringer glucose C- I oxidation, iodide organification or colloid droplet formation in dog thyroid slices (Table 2). Cyclic 3 :5 -AMP in the presence of caffeine stimulated glucose C-1 oxidation, iodide organification, and colloid droplet formation in dog thyroid slices (Table 2, Fig.2). Other authors have failed to obtain thyrotropin like effects with this compound [8-121 ; our successful results are probably explained by the presence of caffeine and by the low level of activity of the thyroids due to the suppression of endogenous thyrotropin stimulation by the pretreatment with thyroid extract. Caffeine did not stimulate the oxidation of glucose, the organification of iodide or the formation of colloid droplets. Table 2. Effect of thyrotropin, dibutyl cyclic 3 :5 -AMP, and cyclic 3 :5 -AMP on glucose C-1 oxidation, iodide organification, and colloid phagocytosis in dog thyroid Control values for glucose C-1 oxidation ranged from 52 to 321 counts/min/mg barium carbonate (total 40 mg) per 100 mg of tissue wet weight, Iodide organification was measured by the protein bound ls1i of the slices homogenate; control values ranged from 1,570 to 9,450 counts/min/100 mg tissue wet weight. The number of experiments is given in parentheses Variiilrlc Glucose C oxidation 100 (,ontrol Thyrotropiri ~ (100 mt/ml) [1311]Iodide binding 100 to Droteins 742 (6) ( ) 502 (8) ( ) 664 (8) ( ) 767 (9) ( ) 521 (14) ( ) Dibutyl cyclic 3 : 5 -AMP 7 mm cyclic 1 nim cyclic ~ st: 5,-ARIP + 3 :5 -AMP niiu 0.75 nim 1 nibf caffeine 1 mm caffeinr 250 (6) ( ) 270a (8) ( ) pcrrentagc of control valur 280 (8) ( ) 217 (9) ( ) 313 (9) 273 (6) 400 (5) ( ) ( ) ( ) Intracellular colloid droplets 0 (12) ++ (12) ++a (12) ++ (8) ++ (4) 3 Purified dibutyl cyclic 3 :5 -AMP. ( affeinr ( ) 293 (4) 78.8 (8) ( ) ( ) (6) ( ) phosphate, as well as in Krebs Ringer bicarbonate buffer. The stimulation of glucose oxidation by Bu2-3 :5 -AMP as well as by thyrotropin was observed after 30min (but not after l0min) and was maximal after 1 hour of incubation. Dibutyl cyclic 3 : 5 -AMP also stimulated the organification of iodide. Preliminary experiments had shown an inhibition of this metabolic step by this compound [20]. These results were explained when it was discovered that the preparation of Bu8-3 :5 -AMP used was heavily contamined with iodide (3.6O/,); in this first preparation barium iodide had been used to precipitate the nucleotide as a barium salt [20a]. B%-3 : 5 - AMP (0.5 mm) also stimulated the organification of iodide by isolated sheep thyroid cells incubated in the presence of I mm perchlorate (137 Ole, P < 0.01). 0.6 mm butyrate, 0.6 mm isobutyrate, 7 mm S-AMP, ADP and ATP with 1 mm caffeine did not stimulate At low concentrations of thyrotropin (0.5 and 1 mu/ml), caffeine substantially increased the effects of the hormone on glucose C-1 oxidation and iodide organification (Fig. 3). Fluoride stimulated iodide organification, and, as previously observed [ 191, the oxidation of glucose C-1 (Table 3). As thyrotropin, fluoride stimulated iodide organification at concentrations which did not influence glucose C-I oxidation (1 mm). At all the concentrations used (1 mm, 3 mm, IOmM), and in the presence as in the absence of caffeine, fluoride failed to induce the formation of colloid droplets. Prostaglandin E, decreases the level of cyclic 3 : 5 -AMP in isolated fat cells while it increases this level in several other tissues [21]. In dog thyroid slices, prostaglandin El stimulated both glucose C- 1 oxidation and iodide organification (Table 3). These effects were observed at concentrations of 0.01 pg/ml

4 Vol.8, No. 1, 1869 F. RODESCH, P. NEVE, C. WILLEMS, and J. E. DUMONT 29 Fig. 2. Effect of thyrotropin, dibutyl cyclic 3 :5 -AMP, and cyclic 3 :5 -AMP on intracellular colloid droplet formation in dog thymid slices. (A) Control: no colloid droplets (magnification x 1,100). (B) Thyrotropin (100mU/ml) : periodic acid Schiff + droplets at the top of the cells (+) (magnification x 1,100). (C) 0.30pM dibutyl cyclic 3 :5 -AMP : numerous droplets (++) (magnification x 1,100). (D) 1 mm cyclic 3 :5 -AMP + 1 mm caffeine : colloid droplets (+) (magnification ~900)

5 Thyroid Stimulation by Thyrotropin, Cyclic 3' : 5'-AMP and Prostaglandin E, European J. Biochetn. 300 I I 0 C T T+C 0 C T T+C Fig. 3. Potentiation of thyrotropin stimulation of iodide organificution and glucose C-1 oxidation in dog thyroid slices by 1 mm caffeine mulml thyrotropin. C-1 : glucose carbon 1 oxidation; 1311: incorporation of [lsii]iodide in proteins; C : caffeine; T : thyrotropin. N = number of experiments; P = probability. Control values for glucose C-1 oxidation ranged from 182 to 410 counts/min/mg barium carbonate (total 40 mg) per 100 mg of tissue wet weight. Iodide organification was measured by the protein bound 1311 of the slices homogenate; control values ranged from 1,630 to 5,520 counts/min/100 mg tissue wet weight Table 3. Effect of fluoride and prostaglandin El on glucose C-1 oxidation, iodide organification and colloid droplet formation in dog thyroid slices Control values for glucose carbon 1 oxidation ranged from 42 to 117 counts/min/mg barium carbonate (total 40 mg) per 100 mg of tissue wet weight. Iodide organification was measured by the protein bound 1311 of the slices homogenate; control values ranged from 1,450 to 9,450 counts/min/ 100 mg tissue wet weight. The number of experiments is given in parentheses Adnitivc Concen- Glucoac C-1 [';:?$ Colloid tration oxidation to Droteinfi drop'cts percentage of control - NaF 3 mm 443 (5) 402 (6) 0 (6) ( ) ( ) Prost,aglan- 20 pg/ml 334 (10) 396 (10) 0 (6) din E, ( ) ( ) to 20 pglml, and were maximal at a concentration of 0.1 pg/ml. At no concentration did prostaglandin E, stimulate the formation of colloid droplets. Thyrotropin 100 mu/ml markedly increased the protein bound 1311 in dog thyroid slices while significantly decreasing their [1311]iodide content (74 "lo ; P < 0.001); the total uptake of iodide ([1311]iodide+ protein bound 1311) was therefore increased (1 18 O/o ; P < 0.001) in these slices. No such effects were observed in sheep thyroid slices. Dibutyl cyclic 3':5'- AMP (0.75 mm) also decreased the [1311]iodide content of' dog thyroid slices (74O/,; P < 0.01) and increased slightly but not significantly the total 1311 content. Thyrotropin (10 mu/ml), 0.3 mm Bu,-3':5'- AMP, 7 mm cyclic 3':5'-AMP (with 1 mm caffeine), 3 mm fluoride and prostaglandin El (20 pglml), all decreased the [1311]iodide content and increased the total 1311 content of the slices, but these effects were small and not statistically significant. These data exclude the possibility that the increase in the iodide incorporation in the proteins of stimulated thyroid slices is caused by an enhancement of iodide transport in these slices ; they do not indicate whether the decrased [l3'i]iodide content of the slices is a consequence of the activated iodide organification, of an inhibition of iodide transport or of both. 0.6 mm isobutyrate, 0.6 mm butyrate, 1 mm caffeine, 7 mm ATP, 7 mm ADP, and 7 mm 5'-AMP (with 1 mm caffeine) did not modify significantly the [l3l1]iodide or the total 1311 content of the dog thyroid slices. The effects of thyrotropin, Bu,-3' : 5'-AMP, 3' : 5'-AMP, fluoride and prostaglandin E, on glucose C-1 oxidation and iodide organification were obtained in all the experiments in which they were looked for. On the contrary in 4 out of 20 dog thyroids no colloid droplet formation was observed by optical microscopy and for one thyroid by electron microscopy, in the presence of thyrotropin or of other compounds. There was no obvious relation between the basal levels of glucose oxidation or of iodide organification and the inducibility of droplet formation. For the 16 other thyroids, occasional slices incubated in the presence of thyrotropin, 3':5'-AMP or Bu2-3':5'-AMP did not evidence any formation of colloid droplets. There was no relation between the importance of the stimulation of glucose oxidation and the formation of droplets. DISCUSSION Sutherland et al. [l-31 have proposed a general model of hormonal mechanism of action by activation by the hormones of tissue specific adenyl cyclases. As a thyrotropin preparation activated the formation of cyclic 3' :5'-AMP by a particulate preparation of sheep thyroid, they suggested that this model could be applied to thyrotropin and thyroid tissue [3]. Several seemingly unrelated experimental facts were consistent with this hypothesis : a) Thyrotropin stimulates lipolysis in rat adipose tissue [22], a process which seems to depend on cyclic 3' : 5'-AMP formation [23]. b) Fluoride, an unspecific activator of adenyl oyclase [24], mimicks the effects of thyrotropin on glucose oxidation [19]. c) The known intracellular effects of cyclic 3': 5'- AMP [25] could account for several of the thyrotropin effects on thyroid metabolism : inhibition of glucose incorporation in glycogen [9,26] and in the fatty acids of lipids [26], stimulat'ion of glycolysis [la, 261. d) Caffeine stimulates thyroid secretion in dogs ~271.

6 Vo1.8,zTo. 1, 1969 F. RODESCH, P. NEVE, C. WILLEMS, and J. E. DUMONT 31 The fact that cyclic 3':5'-AMP itself did not stimulate glucose catabolism in beef thyroid slices [8,9] or the hexose monophosphate pathway in sheep thyroid homogenates [28] cast some doubt about the applicability of the model of Sutherland to thyroid. The first finding could be explained by a poor penetration of cyclic 3' : 5'-AMP in intact cells, while the latter could merely indicate that stimulation of the hexose monophosphate pathway is not a direct effect of cyclic 3' : 5'-AMP ; both findings were negative and admit other explanations. Present evidence strongly supports the hypothesis that many effects of thyrotropin are secondary to the activation by this hormone of thyroid adenyl cyclase. Purified thyrotropin activates adenyl cyclase activity in beef and dog thyroid homogenates [4]. Thyrotropin in vitro increases the intracellular concentration of cyclic 3':5'-AMP in thyroid slices Dibutyl cyclic 3':5'- AMP qualitatively mimics several effects of thyrotropin on thyroid slices : the stimulation of glucose C-1 oxidation [lo], i. e. of the hexose monophosphate pathway [9,26], the stimulation of [32P]phosphate incorporation in phospholipids [lo], the formation of intracellular colloid droplets [ll], and as shown in this work, the enhancement of iodide organification. Neither butyrate nor adenine nucleotides stimulate glucose oxidation, iodide organification or droplet formation. Cyclic 3' : B'-AMP itself, as thyrotropin, stimulates the transport of sugar [6] in vitro, and the secretion of thyroid iodine in vivo [7] ; the latter effects were, however, not specific as they were also obtained with 5'-AMP, ADP, and ATP. In this work it has been shown that, when added with caffeine, cyclic 3' :5'-AMP stimulates the oxidation of glucose at C- 1, the organification of iodide and the formation of colloid droplets; these effects are not obtained with other adenine nucleotides. Theophylline potentiates the stimulation by thyrotropin of iodine secretion by the thyroid in vivo [7] ; caffeine potentiates the stimulation by thyrotropin of glucose oxidation and iodide organification in dog thyroid slices. The 4 criteria of applicability of the model of Sutherland have therefore been satisfied for thyroid tissue and thyrotropin in different systems and for different effects. All the effects of thyrotropin which have been tested so far can be explained by a primary activation of thyroid adenyl cyclase. The stimulatory effects of Bu,-3' : 5'-AMP and cyclic 3':5'-AMP on glucose oxidation and iodide organification have always been smaller than the corresponding thyrotropin effects. A similar conclusion may be drawn from the data of Pastan et al. on the stimulation of glucose oxidation and of [32P]Pi and [3H]inositol incorporation in phospholipids [10,12] and from the data of Tarui et al. on the stimulation of stereospecific sugar transport [6]. Several explanations could account for these discrepancies : a) The slow penetration of Bu2-3':5'-AMP and even more so of cyclic 3':5'-AMP in the thyroid cells may not allow a build up to the intracellular level of these nucleotides as great as in thyrotropin stimulated cells. As the concentrations of these compounds in the medium are already high, further increases of these concentrations may bring about new artefacts. b) Bu,-3' : 5'-AMP and its intracellular derivatives (i.e. monobutyl cyclic 3':5'-AMP) may be less efficient than cyclic 3' : 5'-AMP itself in the activation of intracellular enzymatic systems. c) The possibility that the effects of thyrotropin may not be mediated exclusively via adenyl cyclase is not excluded. Fluoride activates adenyl cyclase in the homogenates of many tissues [l,24]. However, an effect of fluoride on cyclic 3' : 5'-AMP formation in an intact tissue has never been demonstrated [l]. The fact that fluoride inhibits glycolysis in thyroid tissue shows that it penetrates the thyroid cell. It could therefore be assumed that this compound would activate adenyl cyclase in the intact cell. Fluoride indeed mimics two effects of thyrotropin, the stimulation of glucose oxidation and of iodide orgadcation, which supports this assumption. The lack of stimulation of colloid droplet formation does not bear against this hypothesis since fluoride, as a general inhibitor, may well inhibit some of the steps required for the initiation of phagocytosis. Prostaglandin El inhibits the effects of several hormones which act by activation of adenyl cyclase (catecholamines, glucagon, ACTH and thyrotropin on adipose tissue and vasopressin on the toad bladder) [21,29]. As prostaglandin decreases the 3': 5'- AMP levels in fat cells, and as it does not inhibit the stimulatory effects of Bu,-3' : 5'-AMP in adipose tissue and in the toad bladder, it is believed that this compound interferes with the activation by the hormones of adenyl-cyclase in the target cells [21]. On the other hand, prostaglandin stimulates the accumulation of 3':5'-AMP in slices of lung, spleen, diaphragm kidney and testes; it also increases 3':5'- AMP accumulation in broken cell preparations of human leukocytes and platelets [21]. In dog thyroid slices, prostaglandin mimics two etlects of thyrotropin and 3' : 5'-AMP, i. e. the stimulation of glucose oxidation and of iodide organification ; these data suggest that in dog thyroid slices, prostaglandin also enhances 3' : 5'-AMP accumulation. The striking similarity between the metabolic changes induced in the thyroid cell by thyrotropin and in phagocytosing cells by phagocytosis led us several years ago [15,19,28,30] to propose the hypothesis that many metabolic effects of thyrotropin, and notably the stimulation of the hexose monophosphate pathway, were the consequences of the induction by thyrotropin of colloid phagocytosis by the thyroid cells. This hypothesis has been later

7 32 F. RODESCH ct al.: Thyroid Stimulation by Thyrotropin, Cyclic 3 :5 -AMP and Prostaglandin E, European J. Biochem. emphasized by Pastan [31]. In these experiments the stimulation of phagocytosis by thyrotropin, 3 : 5 - AMP and Bu,-3 : 5 -AMP was not observed in some tissues and in some slices while the stimulation of the hexose monophosphate pathway was present. Colloid phagocytosis may have occurred without being evidenced in these slices. However, in one of these cases the absence of phagocytosing activity was checked by electron microscopy. Similarly prostaglandin E, and fluoride mimicked the effects of thyrotropin on iodide organification and glucose oxidation but did not induce phagocytosis. These discrepancies cast some doubt about the hypothesis that the stimulation of the hexose monophosphate pathway by thyrotropin and 3 :B -AMP is a consequence of the induction of phagocytosis in the thyroid cell. This work was realized under contract Euratom-Univcrsity of Brussels -University of Pis& ( BIAC) (Contribution no 419 of the Euratom Biology Department) and under contract no 1011 of the Fonds de la Recherche Scicntifique MAdicale. The authors thank Mr. A. Melis and Mr. W. Wasteels for their help and Miss C. Borrey for the preparation of the manuscript REFERENCES Sutherland, E. W., Robinson, G. A., and Butcher, R.W., Circulation, 37 (1968) 279. Sutherland, E. W., Oye, I., and Butcher, R. W., Recent Progr. Hormone Res. 21 (1965) 623. Klainer, L. M., Chi, Y. M., Freidberg, S. L., Rall, T. W., and Sutherland, E. W., J. Biol. Chem. 237 (1962) Pastan, I., and Katzen, R., Biochem. Biqhys. Res. Commun. 29 (1967) 792. Gilman, G. A,, and Rall, T. W., Federation Proc. 25 (1966) 617. Tarui, S., Nonaka, K., Ikura, Y., and Shima, K., Biochem. Biophys. Res. Comrnun. 13 (1963) 329. Bastomsky, C. H., and McKenzie, J. M., Am. J. Physiol. 213 (1967) Field, J. B., Pastan, I., Herring, B., and Johneon, P., Biochim. Biophys. Acta, 50 (1961) Merlevelde, W., Weaver, G., and Landau, B. R., J. Clin. Invest. 42 (1963) Pastan, I., Biochem. Biqphys. Res. Commun. 25 (1966) Pastan, I., and Wollman, S. H., J. Cell Biol. 35 (1967) Pastan, I., and Macchia, V., J. Biol. Chem. 242 (1967) Posternak, T., Sutherland, E. W., and Henion, W. F., Biochim. Biophys. A&, 65 (1962) Dumont, J. E., Bull. SOC. Chim. Biol. 46 (1964) Dumont. J. E.. and Van Sande., J.., Bull. SOC. Chim. Biol. 47 (1965) Rodesch. F.. and Dumont. J. E.. Exvtl. Cell Res. 47 I & (1967) Rodesch, F., Jortay, A., and Dumont, J. E., Experientia, 24 (1968) Kondo, Y., J. Biochem. (Tokyo), 50 (1961) Dumont, J. E., Bull. Soe. Chim. Biol. 48 (1966) Rodesch, F., and Dumont, J. E., Arch. Intern. Physiol. Biochim. 75 (1967) a. Posternak, T., personal communication. 21. Butcher, R. W., and Baird, C. E., J. Biol. Chem. 243 (1968) Freinkel, N., J. Clin. Invest. 40 (1961) Butcher, R. W., Ho, R. J., Meng, H. C., and Sutherland, E. W., J. Biol. Chem. 240 (1965) Sutherland, E. W., Rall, T. W., and Menon, T. S., J. Biol. Chem. 237 (1962) Sutherland, E. W., and Robison, G. A., Pharmacol. Rev. 18 (1966) Dumont, J. E., and Tondeur-Montenez, T., Biochim. Biwhvs. Acta. 111 (1965) AmvLgova, M. C., Byul. Eksperim. Biol. i Med. 54 (1962) Dumont, J. E., and Eloy, J., Bull. Soc. Chim. Biol. 48 (1966) Bergstrom, S., Science, 157 (1967) Dumont, J. E;, and Rocmark, P.; J. Physiol. (London), 174 (1964) Pastan, I., Ann. Rev. Biochem. 35 (1966) 369. F. Rodesch, P. Neve, C. Willems, and J. E. Dumont Laboratoire de MBdecine Nuclbaire de l U.L.B. 115 Boulevard de Waterloo, Bruxelles 1, Belgium