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Stimulation in vitro by thyrotropin, cyclic 3′,5′-AMP, dibutyryl cyclic 3′,5′-AMP and prostaglandin E1, of secretion by dog thyroid slices
474BBA
BIOCHIMICA ET BIOPHYS*ICA ACTA
26454
STIMULATION I N V I T R O BY THYROTROPIN, CYCLIC 3',5'-AMP, D I B U T Y R Y L CYLCLIC 3',5'-AMP AND PROSTAGLANDIN E 1, OF SECRETION BY D O G T H Y R O I D SLICES C. W I L L E M S , P. A. R O C M A N S AND J. E. D U M O N T
Laboratory of Nuclear ,TVIedicine, School of Medicine, University of Brussels, and Biology Department, Euratom*, iooo-Brussels (Belgium) ( R e c e i v e d J u l y 6th, 197 o)
SUMMARY
The release of 1811 radioactivity extracted by butanol (thyroid hormones and iodide) in vitro from slices of prelabeled thyroids removed from pretreated dogs is measured. Results are compared with measurements of intracellular colloid droplet formation. Several arguments support the hypothesis that the in vitro release corresponds to secretion. Secretion in vitro was apparently delayed. Secretion in vitro as well as intracellular colloid droplet formation was enhanced by thyroid stimulating hormone (TSH), cyclic AMP (cAMP), N6-2-0-dibutyryl cyclic AMP (b2cAMP), and prostaglandin El, but not by caffeine, AMP or ATP. The kinetics of these effects were similar for the 4 stimulating agents. Concentration effect relationships for the 4 compounds are presented. Orders of magnitude of equieffective concentrations were TSH 0.25 mU/ml, cAMP 3.5-1o mM, b2cAMP o.I mM. The concentration effect curve of prostaglandin E 1 was biphasic. The results support the hypothesis that the induction by TSH of thyroid secretion is mediated by cAMP. For low concentrations of TSH intracellular colloid droplet formation varied greatly from cell to cell, thus suggesting a marked intercellular functional heterogeneity. Several facts suggest that in the in vitro system thyroglobulinolysis may become the limiting step of secretion at high levels of thyroid stimulation.
INTRODUCTION
During the early phase of thyroid stimulating hormone (TSH) action on the thyroid, the main physiological effect of the hormone is the stimulation of secretion 1-~. To study this early effect and its biochemical correlates, it would be of great interest to use an in vitro system. Unfortunately, up to the present time, no truly satisfactory system has been proposed. Thyroid perfusion with heterologous plasma allows the testing of one variable per experiment 5. Intracellular colloid droplet formation, observed by light microscopy~,6-10 in incubated thyroid slices, only measures an A b b r e v i a t i o n s : c A M P , cyclic A M P ; b2cAMP, N ° - 2 - O ' - d i b u t y r y l cyclic A M P (i oo/~g/ml -- o.20 raM); BEa31I, 1311 r a d i o a c t i v i t y e x t r a c t e d b y butano115; NBE131I, 1311 r a d i o a c t i v i t y r e m a i n i n g in t h e p r o t e i n p r e c i p i t a t e a f t e r b u t a n o l i c extractionl~; T S H , t h y r o i d s t i m u l a t i n g h o r m o n e . * C o n t r i b u t i o n No. 61I of t h e Biology D e p a r t m e n t , E u r a t o m .
Biochim. Biophys. Acta, 222 (197 o) 474 481
DOG THYROID SECRETION
in vitro
475
equilibrium state between endocytosis and digestion of the colloid. The number of colloid droplets per cell m a y not even represent a good measurement of the amount of colloid in the follicular cells, as the size and density of these droplets m a y vary, and as small droplets m a y escape detection. Iodine release by mouse thyroid in culture is a good system 11, but mouse thyroids, small and attached to the tracheal block, are unsuitable for metabolic studies not involving iodine. The release of 1811- or 1271- from beef or dog thyroid slices is increased in the presence of TSHT,12,18, but because of the large error in such measurements, this method has not been systematically exploited. We propose a new method for the study of secretion by dog thyroid slices in vitro. This system has been used to study the effect on thyroid secretion, of TSH, of other hormones, of neurohumors and of TSHmimicking agents cyclic AMP (cAMP), N~-2-O'-dibutyryl cyclic AMP (b2cAMP) and prostaglandin E 1 (ref. IO). METHODS
Dogs (15-3o kg) were administered 15o #C of carrier-free181I - by subcutaneous injection, then for 3 days received 300 mg of thyroid powder (Thyranon, Organon Oss, Nederland), in their food. On day 4, thyroid lobes were resected and thyroid slices prepared~,l°, 14. Slices were incubated in Parker 199 culture medium (Burroughs Wellcome, London, England): this medium was reinforced with insulin o.I unit/ml, freshly prepared 5 mM ascorbate, 15 mM glutathione and io mg/ml bovine serum albumin, all neutralized to p H 7-4, and with 2 mM methimazole and I mM NaC104. Calf serum (Burroughs Wellcome, London, England) was added to the medium (I vol./io vol.), which was incubated at 37 ° in an atmosphere of carbogen. After a first preincubation of i h, the slices were blotted on filter paper, weighed and incubated individually for another 4 h in flasks containing 2 ml of incubation medium. The slices were then fixed in Bouin solution and their radioactivity was counted in a well type ~ scintillation counter (Philips, Eindhoven, The Netherlands). The incubation medium was also counted before and after butanolic extraction 15. The butanolextractable 1~1I (BE131I) was calculated from the difference of total radioactivity of the medium and radioactivity of the extracted medium (NBE131I). BE131I and NBE131I releases from the slices were expressed as percentages of the total radioactivity of the incubation flask (slices and medium 181I). Colloid droplet formation was estimated either qualitativelyg, 1° or as number of droplets per nucleus counted on 20 randomly chosen follicles. Means, standard deviations of the mean and Student's t values were calculated from the data of individual follicles. For the release experiments, means of the duplicates of individual experiments were compared by paired t tests. Bovine T S H (IO units/mg) was purified in our laboratory 16. Bovine growth hormone (NIH-6H-BI4, i unit/mg) and prolactin (NIH-P-B2, 20 units/mg) were kindly given by Endocrinology Study Section of the National Institutes of Health (Bethesda, U.S.A.). Prostaglandin E 1 was a gift of S. Bergstr6m (Karolinska Institutet Stockholm, Sweden), and of J. Pike (Upjohn Cy, Kalamazoo, U.S.A.). b2cAMP, cAMP, 5'-AMP, ATP and epinephrine bitartrate were obtained from Calbiochem (Los Angeles, U.S.A.). Synthetic ACTH, serotonin creatine sulfate, carbamylcholine chloride, and commercial bovine T S H were obtained from Sandoz (Basle, SwitzerBiochim. Biophys. Acta, 222 (197 o) 474-481
47 6
c. WILLEMS et al.
land), Calbiochem (Los Angeles, U.S.A.) and Armour (Kankakee, U.S.A.), respectively. RESULTS
Previous experiments with prelabeled dog thyroid slices had demonstrated an increase of 131I- formation in the presence of TSH:. 131I- measured as the difference between total 131I of the homogenate of the slices in the incubation medium and TCA-precipitable 131I of this homogenate, was small. The error in such measurements was important. Several changes in the methodology have therefore been introduced. Dogs were pretreated with thyroid extract '°. Slices were preincubated to remove colloid from the follicles disrupted during the preparation. They were incubated in a very enriched medium and, to discharge all the iodide formed, in the presence of NaC10~ and methimazole 11. BE131I was measured in the incubation medium only. The release was expressed as a fraction of the lalI of the slices at the beginning of the incubation and not per tissue weight; thus, only follicles containing colloid were taken into account. Using this technique, a low BE131I release from the slices was observed in 23 experiments, mean i . i % (range o-2 %), which was, except in one experiment, greatly enhanced in the presence of TSH (1.25 munits/ml), mean 4.5 % (range 1.3-9.9 %). The basal release m a y be overestimated, as any loss of 131I-labeled protein during extraction would contribute to this value. BElalI in the slices was negligible. Spontaneous NBElZlI release from the slices varied greatly from one experiment to the other, mean 24 % (range lO.2-43.8 %). Absence of ascorbate, GSH 17, insulin, the biochemical components of Parker medium or of albumin and calf serum in the medium, did not significantly modify spontaneous or TSH (1.25 munits/ml) induced release of BE131I. Ascorbate, GSH and insulin were not added in later experiments. No colloid droplet was observed in the cells of control slices. In stimulated slices, large numbers of colloid droplets were evidenced. The number of droplets per cell varied greatly from one cell to another in the same follicles. Heterogeneity between follicles was not evident. The size of colloid droplets varied greatly even in one cell. In follicles where the colloid appeared retracted, pseudopods protruding from the apex of the stimulated follicular cells were observed. Paper chromatography of the incubation medium of the slices in butanol acetic water and 2 M NH4OH saturated isoamyl alcohoP demonstrated the presence of origin material, presumably '311- labeled proteins corresponding to NBE131I, and small quantities of 131I- and thyroxine. Stimulation of the slices with TSH greatly increased 131I- and thyroxine formation. No F131Iliodotyrosines were demonstrated. BE131I appearing in the incubation medium might result from unspecific hydrolysis in the medium of l~lI-labeled proteins released from the slices, or from intracellular hydrolysis of medium 131I-labeled proteins taken up by follicular cells of the slices. To investigate these possibilities, dog thyroid slices labeled in, vivo with 125I were incubated in a medium containing 181I-labeled proteins released from lalI-prelabeled slices. Whereas TSH greatly increased BE125I in the incubation medium, BE131I remained negligible. BE131I release was always greater in the presence rather than in the absence Biochim. Biophys. Acta, 222 (197 o) 474-481
DOG THYROID SECRETION i n VitrO
477
of 0.25 munit/ml T S H (2.4 v s . 0.5 %, P < 0.005, N* ---=9), in the presence of 1.25 munit/ml T S H rather than in the presence of 0.25 m unit/ml T S H (3.6 v s . 2.4 %, P < o . o o I , N ---- 9); IOO munits/ml TSH did not further enhance the release (6.0 % v s . 5.6% for 1.25 munits/ml TSH, 0. 4 < P < o.5, N = 7) (Fig. I). In 4 out of 5 experiments, a stimulation was obtained at o.I munit/ml TSH. The effect of TSH was decreased at p H 6.8 and 8.0 (46 % and 68 % of the controls, respectively). Because of the high background release of NBE~81I from the control slices, the effect of T S H on this release was more difficult to ascertain. Nevertheless, a distinct stimulation was observed in 3 out of 9 experiments for 0.25 munit/ml TSH, in i i out of 23 experiments for 1.25 munits/ml T S H and in 6 out of 7 experiments for ioo munits/ml TSH. In the positive experiments, the release of N B E I ~ I was greater for IOO munits/ml TSH than for 1.25 munits/ml T S H (33.1% v s . 24.2 %, P < o.ooi, .,\" 6).
BE131Irelease % of tot(at 1131
5 3 2 .
Number of droplets ~ per nucleus
~
/
/
1 ~
f B
"
10
~
BEI
-
E
I
o 0
5
control
0-~ " ~
0
0.05
0.25
1.25
100
[TSH]~U~ml
Fig. I. Effect of various TSH concentrations in vitro on BE131I release and intracellular colloid droplet formation in dog thyroid slices. Results of a representative experiment. G, BE181I release; bars, droplets.
At concentrations of o.oi and o.o5 munit/ml, T S H induced the formation of colloid droplets in some cells while having no effect at all on the vast majority of cells. The stimulated cells, scattered individually in m a n y different follicles, contained as m a n y droplets as the cells in maximally stimulated slices. Colloid droplet formation greatly increased between 0.25 and 1.25 munits/ml but further increased at higher concentrations of hormone (Fig. I). It was already very marked after i h but further increased during the second hour of incubation (Fig. 2). The size of the droplets seemed to increase from the first to the 4th h of incubation. The effect of commercial T S H was reproduced b y purified TSH. Insulin (o.I munit/ml), prolactin (50 #g/ml), growth hormone (IO #g/ml) and ACTH (I /~g/ml) did not increase the release of BE131I or NBElS~I from the slices. Likewise, epinephrine (50 /zM), serotonin (50 #M) and carbamylcholine (0.2 mM) did not enhance these releases. The polypeptide hormones as well as the neurohumors did not elicit the formation of colloid droplets in the follicular cells of the slices. In the presence of caffeine (i raM), cAMP stimulated BE~3~I release from dog thyroid slices. This effect was already detected at I mM and was maximal at * Number of experiments. Biochim. Biophys. Acta ,222 (197 o) 474-481
478
c. WILLEMSet al.
Io mM (Fig. 3) at the latter concentration, the effect was not potentiated by caffeine. cAMP (3.5-1o raM) and TSH (0.25 munit/ml) were approximately equally effective. No increase of BE181I release was observed in the presence of caffeine (I mM and 5 raM), ATP (5 mM) and caffeine (i raM), or AMP (5 mM) and caffeine (I raM). b2cAMP also enhanced BE TM I release. This effect was detected at 0.05 mM and was maximal at 0.20 mM. The maximal effect of b2cAMP was higher than the maximal effect of TSH (Fig. 3). b2cAMP (o.I mM) and TSH (0.25 munit/ml) were approximately equally effective, b,cAMP and cAMP elicited the formation of a great number of colloid droplets in follicular cellsS,1°. Prostaglandin E 1 enhanced BElSlI secretion in 6 out of 7 experiments in which the effect of TSH was clearly demonstrated. Generally, the effect was detected at o.I #g/ml and decreased for higher concentrations (Fig. 3). The occurrence of Release Number of droplets per nucleus
pB125I /
-2O i
"15 10 5
I
2
3
/,
o
hours
_
Fig. 2. Kinetics of the effect of TSH (I.25 munits/ml) in vitro on BElSlI release and intracellular colloid droplet formation in dog thyroid slices. Results of a representative experiment. A kinetic curve of cumulative pB125I release from a stimulated (3o units of TSH b y intravenous injection) dog thyroid in vivo is shown for comparison.
BE131T release % of tota1131I
BE1:31! release /, ] % of total t31I,
,/ /~--t/-'--~
6
• d25
i t
COntrot 1.'25 // 160 [TSH]mU/ml
[ TSH
~control
1
3.S
10
['cAMPJrnM
BE131Irelease
BEI311 release % of total 131I
% of total lmI t TSH
' ~ c o
I TSH
control
ntrot
i
0.1
1 3.5 10 [PGE1]'~t
Fig. 3. Relationships between concentrations of TSH, bicAMP, cAMP and prostaglandin E 1 and effects on B E 131I release from dog thyroid slices in vitro. Results of representative experiments. Biochirn. Biophys. Acta, 222 (197 o) 474-481
DOG THYROID SECRETION
in vitro
479
a m a x i m u m in the concentration effect was constant but not the situation of this m a x i m u m on the concentration scale. In one experiment, the effect of prostaglandin E x was not detectable below io #g/ml. In 3 out of 6 experiments, no effect was observed at io /,g/ml. The maximal effects of prostaglandin E 1 and TSH (1.25 munits/ml) were similar. Prostaglandin E 1 elicited the formation of intracellular colloid droplets: sometimes at o.i #g/ml, most of the times at i and 3.5 /,g/ml, in 3 out of 6 experiments not at IO #g/ml and never at 25 /,g/ml. This observation m a y explain why no effect of prostaglandin E~ was demonstrated previously 1°. With cAMP, b2cAMP and prostaglandin E~ as with T S H (1.25 and IOO munits/ml) the enhancement of BE131I release was observed after 2 and 4 h, but not after I h of incubation (Fig. 2). Enhancement of NBEXalI release from the thyroid slices was only observed at the highest levels of stimulation b y TSH-mimicking agents. DISCUSSION
Under conditions in which iodide is neither bound to proteins nor retained in the tissue, TSH enhanced the release of thyroid hormone and iodide from dog thyroid slices in vitro as in vivo 3,4. Experimental evidence supports the hypothesis t h a t the TSH-induced release in vitro is the counterpart of the in vivo secretion. Only thyroid hormone and iodide but not iodotyrosines were released. The release was enhanced b y TSH and TSH-mimicking agents (cAMP, b2cAMP ) but not b y other hormonesZ, 4,1s. The release in vitro was preceded by the formation of colloid droplets in the follicular cells. Electron micrographs of similarly treated thyroid slices further showed, as in vivo stimulated dog thyroids xg, pseudopod formation with phagocytic engulfment of colloid in the follicular cells 2°. Secretion in vitro appeared to be delayed. This delay was of the order of i h, whereas in vivo thyroid secretion begins lO-15 min after intravenous T S H administration3, 4 (Fig. 2). This apparent delay m a y be partly due to the experimental conditions. The background radioactivity in the venous outflow of the thyroid in vivo is very low, while the background activity of the medium in vitro is not. However, several facts suggest that secretion is really delayed in vitro. No stimulation was detected after I h of incubation, whether the stimulating agent was TSH, cAMP, b2cAMP or prostaglandin E 1. Extrapolation of the kinetics of induced secretion also suggests that the secretion began after I h of incubation with the stimulating agent. Electron micrographs of similarly treated dog thyroid slices after i h of incubation showed pseudopods and colloid droplet formation but little evidence of fusion of lysosomes with droplets, digestion of the droplets or progress of the droplets towards the basis of the cells ~°. The cause of the delay of in vitro secretion is unknown. Other effects of T S H on similar slices, enhancement of glucose carbon i oxidation or of iodide organification, began very soon after T S H addition ~°. Secretory effects of cAMP, b2cAMP, and prostaglandin E 1 were also delayed. These data suggest that the delay in secretion is not due to a delay in T S H access to the cells or in its primary action on the cell but rather to a delay in the secretory process itself. Thyroid slices of pretreated dogs are extremely sensitive to T S H : droplets could be demonstrated for a concentration of o.oi munit/ml of TSH, i.e. at T S H levels for which no effect on the metabolism of the whole slice was detected. There Biochim. Biophys. Acta, 222 (197o) 474-481
480
C. WlLLEMS et al.
is striking heterogeneity of response to TSH in vitro from one follicular cell to another; for low concentrations of TSH, scarce cells in various follicles appeared filled up with colloid droplets, while in the great majority of the other cells, no colloid droplet at all was demonstrated. This finding confirms the importance ill the action of T S H or~ thyroid tissue of the individual reactivity of the cells TM. It suggests an all or none response of the cells to TSH. The enhancement of intracellular colloid droplet formation, of iodide organification, and sometimes of glucose carbon I oxidation can be detected at a concentration of 0.o5 munit/ml 1°. BE131I release was a less sensitive index of T S H stimulation. Iodide organification, and BE~8~I release seemed to approach an asymptotic value at 1.25 munits/ml, while glucose carbon I oxidation and colloid droplet formation further increased above this concentration. Enhancement of glucose I oxidation at high T S H concentrations m a y therefore result from the stimulation of endocytosis but not from increased secretion. Several data suggest that thyroglobulinolysis m a y become the limiting step of secretion in our system. Colloid uptake, estimated by the number and size of intracellular colloid droplets, further increased during the incubation while the secretory rate remained constant. At high T S H concentrations, colloid uptake, but not secretion, further increased. TSH and T S H mimicking agents, at concentrations higher than those required to initiate secretion, enhanced the release of NBE~81I. However, it is not known if this NBE~SlI is released from the droplets or directly from the follicular lumen of loosened follicles. It is of interest that in vivo thyroglobulin is released in the lymphatic outflow of the stimulated thyroid zm. cAMP, and b,cAMP, but not AMP or ATP, mimicked the effect of TSH on thyroid secretion in vitro, cAMP had been shown previously to enhance secretion by mouse thyroid in vitro 2~, but this finding had been questioned 28. In our dog thyroid slices, these agents acted in the same range of concentrations at which they mimicked other T S H effects 1°. These data therefore further support the hypothesis that the effects of T S H on secretion, iodide binding to proteins and for low T S H concentrations, glucose carbon I oxidation, are mediated b y cAMP. There is now growing evidence relating T S H action on the thyroid to its activation of thyroid adenylcyclase ~4,~5. The fact that b2cAMP, but not cAMP, consistently gave a higher maximal stimulation of thyroid secretion than TSH, is not due to a difference in the kinetics of their actions. It is compatible with the hypothesis that intracellular cAMP, but not b2cAMP or its monobutyryl derivative, would give rise in the thyroid cell to an inhibitor of cAMP action such as has been described in the brain 26. The TSH-mimicking action of prostaglandin E~ on secretion further supports the hypothesis that it m a y in thyroid as in other tissues activate adenyl cyclase and reproduce by this mechanism the effects of T S H 1°. It is of interest in this regard that prostaglandin E 2 activates adenylcyclase in dog thyroid homogenates 2v.
ACKNOWLEDGEMENTS
This work was realized under Contract of the MinistSre de la Programmation Scientifique within the framework of the Association E u r a t o m - U n i v e r s i t y of BrusselsUniversity of Pisa (No. 026-63-4 BIAC), and thanks to grant. IOli of the Fonds Biochim. Biophys. Acta, 222 (197o) 474-481
DOG THYI~OID SECRETION
in vitro
de la Recherche Scientifique M~dicale National de la Recherche Scientifique to The authors would like to thank Ch. Borrey and Miss J. Hennaux for the
481 and Cr6dit aux Chercheurs of the Fonds Dr. P. A. Rocmans. Mr. A. MELIS for his technical help, Miss typing of the manuscript.
REFERENCES i V. SODERBERG, Physiol. Rev., 39 (I959), 777. 2 J. E. DUMONT AND P. ROCMANS, Proc. 2nd Intern. Congr. Endocrinol., E x c e r p t a Medica, A m s t e r d a m , 1964, p. 81. 3 P. ROCMANS AND J. E. DUMONT, J. Physiol., 174 (1964) 26. 4 I. N. ROSENEERG, J. C. ATHANS AND G. H. ISAACS, Recent Progr. Hormone Res., 21 (I965),335 p. A. ROCMANS AND W. G. AUSTEN, in J. C. NORMAN, Organ Perfusion and Preservation, A p p l e t o n - C e n t u r y - C r a f t Publ., N e w York, 1968, p. 767 . 6 L. C. JUNQUEIRA, Endocrinology, 4 ° (1947) 286. 7 J. E. DUMONT, Bull. Soc. Chim. Biol., 48 (1966) 419 • 8 I. PASTAN AND S. H. WOLLMAN, J. Cell Biol., 35 (1967) 262. 9 T. ONAYA, D. H. SOLOMON AND W. D. DAVlDSON, Endocrinology, 85 (1969) 15o. i o F. R. RODESCla, P. NEVE, C. WILLEMS AND J. E. DUMONT, European J. Biochem., 8 (1969) 26. i i J. BROWN AND D. S. MUNRO, J. Endocrinol., 38 (1967) 439. 12 P. L. POFFENBARGER, R. C. POWELL AND W. P. DEISS, J. Clin. Invest., 42 (1963) 239. 13 R. C. POWELL, M. A. RAHMAN AND W. P. DEISS, Endocrinology, 74 (1964) 39514 J. E. DUMONT AND J. VAN SANDE, Bull. Soc. Chim. Biol., 47 (1965) 321. 15 L. J. DEGROOT AND J. B. STANBURY, Am. J. Med., 27 (1969) 586. 16 P. G. CONDLIFFE, R. W. BATES, AND R. M. FRAPS, Biochim. Biophys. Acta, 34 /I959) 43 °. 17 W. P. DEISS AND R. L. PEAKE, Ann. Intern. Med., 69 (1968) 881. 18 C. S. AHN, J. C. ATHANS AND I. INT.ROSENBERG, Endocrinology, 85 (1969) 22419 P. NEVE AND J. E. DUMONT, Zellforsch. Mikroshop. Anat., lO 3 (197 o) 61. 20 P. •EVE AND J. E. DUMONT, Exptl. Cell Res., in the press. 21 P. M. DANIEL, L. G. PLASKETT AND O. E. PRATT, J. Physiol., 138 (1967) 25. 22 J. M. ENSOR AND D. S. MUNRO, J. Endocrinol., 43, (1969) 477. 23 Y. OCHI AND L. J. DEGROOT, Metabolism, 18 (1969) 331. 24 J. E. DUMONT, Bull. Soc. Chim. Biol., 5 ° (1968) 24Ol. 25 J. B. FIELD, Metabolism, 17 (1968), 226. 26 F. MURAD, T. W. RALL AND M. VAUGHAN, Biochim. Biophys. Acta, 192 (1969) 43 o. 27 V. ZOR, T. KANEKO, I. P. LOWE, C-. BLOOM AND J. B. FIELD, J. Biol., Chem. 244 (1969) 5189 •
Biochim. Biophys. Acta, 222 (197 o) ¢74-481
BIOCHIMICA ET BIOPHYS*ICA ACTA
26454
STIMULATION I N V I T R O BY THYROTROPIN, CYCLIC 3',5'-AMP, D I B U T Y R Y L CYLCLIC 3',5'-AMP AND PROSTAGLANDIN E 1, OF SECRETION BY D O G T H Y R O I D SLICES C. W I L L E M S , P. A. R O C M A N S AND J. E. D U M O N T
Laboratory of Nuclear ,TVIedicine, School of Medicine, University of Brussels, and Biology Department, Euratom*, iooo-Brussels (Belgium) ( R e c e i v e d J u l y 6th, 197 o)
SUMMARY
The release of 1811 radioactivity extracted by butanol (thyroid hormones and iodide) in vitro from slices of prelabeled thyroids removed from pretreated dogs is measured. Results are compared with measurements of intracellular colloid droplet formation. Several arguments support the hypothesis that the in vitro release corresponds to secretion. Secretion in vitro was apparently delayed. Secretion in vitro as well as intracellular colloid droplet formation was enhanced by thyroid stimulating hormone (TSH), cyclic AMP (cAMP), N6-2-0-dibutyryl cyclic AMP (b2cAMP), and prostaglandin El, but not by caffeine, AMP or ATP. The kinetics of these effects were similar for the 4 stimulating agents. Concentration effect relationships for the 4 compounds are presented. Orders of magnitude of equieffective concentrations were TSH 0.25 mU/ml, cAMP 3.5-1o mM, b2cAMP o.I mM. The concentration effect curve of prostaglandin E 1 was biphasic. The results support the hypothesis that the induction by TSH of thyroid secretion is mediated by cAMP. For low concentrations of TSH intracellular colloid droplet formation varied greatly from cell to cell, thus suggesting a marked intercellular functional heterogeneity. Several facts suggest that in the in vitro system thyroglobulinolysis may become the limiting step of secretion at high levels of thyroid stimulation.
INTRODUCTION
During the early phase of thyroid stimulating hormone (TSH) action on the thyroid, the main physiological effect of the hormone is the stimulation of secretion 1-~. To study this early effect and its biochemical correlates, it would be of great interest to use an in vitro system. Unfortunately, up to the present time, no truly satisfactory system has been proposed. Thyroid perfusion with heterologous plasma allows the testing of one variable per experiment 5. Intracellular colloid droplet formation, observed by light microscopy~,6-10 in incubated thyroid slices, only measures an A b b r e v i a t i o n s : c A M P , cyclic A M P ; b2cAMP, N ° - 2 - O ' - d i b u t y r y l cyclic A M P (i oo/~g/ml -- o.20 raM); BEa31I, 1311 r a d i o a c t i v i t y e x t r a c t e d b y butano115; NBE131I, 1311 r a d i o a c t i v i t y r e m a i n i n g in t h e p r o t e i n p r e c i p i t a t e a f t e r b u t a n o l i c extractionl~; T S H , t h y r o i d s t i m u l a t i n g h o r m o n e . * C o n t r i b u t i o n No. 61I of t h e Biology D e p a r t m e n t , E u r a t o m .
Biochim. Biophys. Acta, 222 (197 o) 474 481
DOG THYROID SECRETION
in vitro
475
equilibrium state between endocytosis and digestion of the colloid. The number of colloid droplets per cell m a y not even represent a good measurement of the amount of colloid in the follicular cells, as the size and density of these droplets m a y vary, and as small droplets m a y escape detection. Iodine release by mouse thyroid in culture is a good system 11, but mouse thyroids, small and attached to the tracheal block, are unsuitable for metabolic studies not involving iodine. The release of 1811- or 1271- from beef or dog thyroid slices is increased in the presence of TSHT,12,18, but because of the large error in such measurements, this method has not been systematically exploited. We propose a new method for the study of secretion by dog thyroid slices in vitro. This system has been used to study the effect on thyroid secretion, of TSH, of other hormones, of neurohumors and of TSHmimicking agents cyclic AMP (cAMP), N~-2-O'-dibutyryl cyclic AMP (b2cAMP) and prostaglandin E 1 (ref. IO). METHODS
Dogs (15-3o kg) were administered 15o #C of carrier-free181I - by subcutaneous injection, then for 3 days received 300 mg of thyroid powder (Thyranon, Organon Oss, Nederland), in their food. On day 4, thyroid lobes were resected and thyroid slices prepared~,l°, 14. Slices were incubated in Parker 199 culture medium (Burroughs Wellcome, London, England): this medium was reinforced with insulin o.I unit/ml, freshly prepared 5 mM ascorbate, 15 mM glutathione and io mg/ml bovine serum albumin, all neutralized to p H 7-4, and with 2 mM methimazole and I mM NaC104. Calf serum (Burroughs Wellcome, London, England) was added to the medium (I vol./io vol.), which was incubated at 37 ° in an atmosphere of carbogen. After a first preincubation of i h, the slices were blotted on filter paper, weighed and incubated individually for another 4 h in flasks containing 2 ml of incubation medium. The slices were then fixed in Bouin solution and their radioactivity was counted in a well type ~ scintillation counter (Philips, Eindhoven, The Netherlands). The incubation medium was also counted before and after butanolic extraction 15. The butanolextractable 1~1I (BE131I) was calculated from the difference of total radioactivity of the medium and radioactivity of the extracted medium (NBE131I). BE131I and NBE131I releases from the slices were expressed as percentages of the total radioactivity of the incubation flask (slices and medium 181I). Colloid droplet formation was estimated either qualitativelyg, 1° or as number of droplets per nucleus counted on 20 randomly chosen follicles. Means, standard deviations of the mean and Student's t values were calculated from the data of individual follicles. For the release experiments, means of the duplicates of individual experiments were compared by paired t tests. Bovine T S H (IO units/mg) was purified in our laboratory 16. Bovine growth hormone (NIH-6H-BI4, i unit/mg) and prolactin (NIH-P-B2, 20 units/mg) were kindly given by Endocrinology Study Section of the National Institutes of Health (Bethesda, U.S.A.). Prostaglandin E 1 was a gift of S. Bergstr6m (Karolinska Institutet Stockholm, Sweden), and of J. Pike (Upjohn Cy, Kalamazoo, U.S.A.). b2cAMP, cAMP, 5'-AMP, ATP and epinephrine bitartrate were obtained from Calbiochem (Los Angeles, U.S.A.). Synthetic ACTH, serotonin creatine sulfate, carbamylcholine chloride, and commercial bovine T S H were obtained from Sandoz (Basle, SwitzerBiochim. Biophys. Acta, 222 (197 o) 474-481
47 6
c. WILLEMS et al.
land), Calbiochem (Los Angeles, U.S.A.) and Armour (Kankakee, U.S.A.), respectively. RESULTS
Previous experiments with prelabeled dog thyroid slices had demonstrated an increase of 131I- formation in the presence of TSH:. 131I- measured as the difference between total 131I of the homogenate of the slices in the incubation medium and TCA-precipitable 131I of this homogenate, was small. The error in such measurements was important. Several changes in the methodology have therefore been introduced. Dogs were pretreated with thyroid extract '°. Slices were preincubated to remove colloid from the follicles disrupted during the preparation. They were incubated in a very enriched medium and, to discharge all the iodide formed, in the presence of NaC10~ and methimazole 11. BE131I was measured in the incubation medium only. The release was expressed as a fraction of the lalI of the slices at the beginning of the incubation and not per tissue weight; thus, only follicles containing colloid were taken into account. Using this technique, a low BE131I release from the slices was observed in 23 experiments, mean i . i % (range o-2 %), which was, except in one experiment, greatly enhanced in the presence of TSH (1.25 munits/ml), mean 4.5 % (range 1.3-9.9 %). The basal release m a y be overestimated, as any loss of 131I-labeled protein during extraction would contribute to this value. BElalI in the slices was negligible. Spontaneous NBElZlI release from the slices varied greatly from one experiment to the other, mean 24 % (range lO.2-43.8 %). Absence of ascorbate, GSH 17, insulin, the biochemical components of Parker medium or of albumin and calf serum in the medium, did not significantly modify spontaneous or TSH (1.25 munits/ml) induced release of BE131I. Ascorbate, GSH and insulin were not added in later experiments. No colloid droplet was observed in the cells of control slices. In stimulated slices, large numbers of colloid droplets were evidenced. The number of droplets per cell varied greatly from one cell to another in the same follicles. Heterogeneity between follicles was not evident. The size of colloid droplets varied greatly even in one cell. In follicles where the colloid appeared retracted, pseudopods protruding from the apex of the stimulated follicular cells were observed. Paper chromatography of the incubation medium of the slices in butanol acetic water and 2 M NH4OH saturated isoamyl alcohoP demonstrated the presence of origin material, presumably '311- labeled proteins corresponding to NBE131I, and small quantities of 131I- and thyroxine. Stimulation of the slices with TSH greatly increased 131I- and thyroxine formation. No F131Iliodotyrosines were demonstrated. BE131I appearing in the incubation medium might result from unspecific hydrolysis in the medium of l~lI-labeled proteins released from the slices, or from intracellular hydrolysis of medium 131I-labeled proteins taken up by follicular cells of the slices. To investigate these possibilities, dog thyroid slices labeled in, vivo with 125I were incubated in a medium containing 181I-labeled proteins released from lalI-prelabeled slices. Whereas TSH greatly increased BE125I in the incubation medium, BE131I remained negligible. BE131I release was always greater in the presence rather than in the absence Biochim. Biophys. Acta, 222 (197 o) 474-481
DOG THYROID SECRETION i n VitrO
477
of 0.25 munit/ml T S H (2.4 v s . 0.5 %, P < 0.005, N* ---=9), in the presence of 1.25 munit/ml T S H rather than in the presence of 0.25 m unit/ml T S H (3.6 v s . 2.4 %, P < o . o o I , N ---- 9); IOO munits/ml TSH did not further enhance the release (6.0 % v s . 5.6% for 1.25 munits/ml TSH, 0. 4 < P < o.5, N = 7) (Fig. I). In 4 out of 5 experiments, a stimulation was obtained at o.I munit/ml TSH. The effect of TSH was decreased at p H 6.8 and 8.0 (46 % and 68 % of the controls, respectively). Because of the high background release of NBE~81I from the control slices, the effect of T S H on this release was more difficult to ascertain. Nevertheless, a distinct stimulation was observed in 3 out of 9 experiments for 0.25 munit/ml TSH, in i i out of 23 experiments for 1.25 munits/ml T S H and in 6 out of 7 experiments for ioo munits/ml TSH. In the positive experiments, the release of N B E I ~ I was greater for IOO munits/ml TSH than for 1.25 munits/ml T S H (33.1% v s . 24.2 %, P < o.ooi, .,\" 6).
BE131Irelease % of tot(at 1131
5 3 2 .
Number of droplets ~ per nucleus
~
/
/
1 ~
f B
"
10
~
BEI
-
E
I
o 0
5
control
0-~ " ~
0
0.05
0.25
1.25
100
[TSH]~U~ml
Fig. I. Effect of various TSH concentrations in vitro on BE131I release and intracellular colloid droplet formation in dog thyroid slices. Results of a representative experiment. G, BE181I release; bars, droplets.
At concentrations of o.oi and o.o5 munit/ml, T S H induced the formation of colloid droplets in some cells while having no effect at all on the vast majority of cells. The stimulated cells, scattered individually in m a n y different follicles, contained as m a n y droplets as the cells in maximally stimulated slices. Colloid droplet formation greatly increased between 0.25 and 1.25 munits/ml but further increased at higher concentrations of hormone (Fig. I). It was already very marked after i h but further increased during the second hour of incubation (Fig. 2). The size of the droplets seemed to increase from the first to the 4th h of incubation. The effect of commercial T S H was reproduced b y purified TSH. Insulin (o.I munit/ml), prolactin (50 #g/ml), growth hormone (IO #g/ml) and ACTH (I /~g/ml) did not increase the release of BE131I or NBElS~I from the slices. Likewise, epinephrine (50 /zM), serotonin (50 #M) and carbamylcholine (0.2 mM) did not enhance these releases. The polypeptide hormones as well as the neurohumors did not elicit the formation of colloid droplets in the follicular cells of the slices. In the presence of caffeine (i raM), cAMP stimulated BE~3~I release from dog thyroid slices. This effect was already detected at I mM and was maximal at * Number of experiments. Biochim. Biophys. Acta ,222 (197 o) 474-481
478
c. WILLEMSet al.
Io mM (Fig. 3) at the latter concentration, the effect was not potentiated by caffeine. cAMP (3.5-1o raM) and TSH (0.25 munit/ml) were approximately equally effective. No increase of BE181I release was observed in the presence of caffeine (I mM and 5 raM), ATP (5 mM) and caffeine (i raM), or AMP (5 mM) and caffeine (I raM). b2cAMP also enhanced BE TM I release. This effect was detected at 0.05 mM and was maximal at 0.20 mM. The maximal effect of b2cAMP was higher than the maximal effect of TSH (Fig. 3). b2cAMP (o.I mM) and TSH (0.25 munit/ml) were approximately equally effective, b,cAMP and cAMP elicited the formation of a great number of colloid droplets in follicular cellsS,1°. Prostaglandin E 1 enhanced BElSlI secretion in 6 out of 7 experiments in which the effect of TSH was clearly demonstrated. Generally, the effect was detected at o.I #g/ml and decreased for higher concentrations (Fig. 3). The occurrence of Release Number of droplets per nucleus
pB125I /
-2O i
"15 10 5
I
2
3
/,
o
hours
_
Fig. 2. Kinetics of the effect of TSH (I.25 munits/ml) in vitro on BElSlI release and intracellular colloid droplet formation in dog thyroid slices. Results of a representative experiment. A kinetic curve of cumulative pB125I release from a stimulated (3o units of TSH b y intravenous injection) dog thyroid in vivo is shown for comparison.
BE131T release % of tota1131I
BE1:31! release /, ] % of total t31I,
,/ /~--t/-'--~
6
• d25
i t
COntrot 1.'25 // 160 [TSH]mU/ml
[ TSH
~control
1
3.S
10
['cAMPJrnM
BE131Irelease
BEI311 release % of total 131I
% of total lmI t TSH
' ~ c o
I TSH
control
ntrot
i
0.1
1 3.5 10 [PGE1]'~t
Fig. 3. Relationships between concentrations of TSH, bicAMP, cAMP and prostaglandin E 1 and effects on B E 131I release from dog thyroid slices in vitro. Results of representative experiments. Biochirn. Biophys. Acta, 222 (197 o) 474-481
DOG THYROID SECRETION
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479
a m a x i m u m in the concentration effect was constant but not the situation of this m a x i m u m on the concentration scale. In one experiment, the effect of prostaglandin E x was not detectable below io #g/ml. In 3 out of 6 experiments, no effect was observed at io /,g/ml. The maximal effects of prostaglandin E 1 and TSH (1.25 munits/ml) were similar. Prostaglandin E 1 elicited the formation of intracellular colloid droplets: sometimes at o.i #g/ml, most of the times at i and 3.5 /,g/ml, in 3 out of 6 experiments not at IO #g/ml and never at 25 /,g/ml. This observation m a y explain why no effect of prostaglandin E~ was demonstrated previously 1°. With cAMP, b2cAMP and prostaglandin E~ as with T S H (1.25 and IOO munits/ml) the enhancement of BE131I release was observed after 2 and 4 h, but not after I h of incubation (Fig. 2). Enhancement of NBEXalI release from the thyroid slices was only observed at the highest levels of stimulation b y TSH-mimicking agents. DISCUSSION
Under conditions in which iodide is neither bound to proteins nor retained in the tissue, TSH enhanced the release of thyroid hormone and iodide from dog thyroid slices in vitro as in vivo 3,4. Experimental evidence supports the hypothesis t h a t the TSH-induced release in vitro is the counterpart of the in vivo secretion. Only thyroid hormone and iodide but not iodotyrosines were released. The release was enhanced b y TSH and TSH-mimicking agents (cAMP, b2cAMP ) but not b y other hormonesZ, 4,1s. The release in vitro was preceded by the formation of colloid droplets in the follicular cells. Electron micrographs of similarly treated thyroid slices further showed, as in vivo stimulated dog thyroids xg, pseudopod formation with phagocytic engulfment of colloid in the follicular cells 2°. Secretion in vitro appeared to be delayed. This delay was of the order of i h, whereas in vivo thyroid secretion begins lO-15 min after intravenous T S H administration3, 4 (Fig. 2). This apparent delay m a y be partly due to the experimental conditions. The background radioactivity in the venous outflow of the thyroid in vivo is very low, while the background activity of the medium in vitro is not. However, several facts suggest that secretion is really delayed in vitro. No stimulation was detected after I h of incubation, whether the stimulating agent was TSH, cAMP, b2cAMP or prostaglandin E 1. Extrapolation of the kinetics of induced secretion also suggests that the secretion began after I h of incubation with the stimulating agent. Electron micrographs of similarly treated dog thyroid slices after i h of incubation showed pseudopods and colloid droplet formation but little evidence of fusion of lysosomes with droplets, digestion of the droplets or progress of the droplets towards the basis of the cells ~°. The cause of the delay of in vitro secretion is unknown. Other effects of T S H on similar slices, enhancement of glucose carbon i oxidation or of iodide organification, began very soon after T S H addition ~°. Secretory effects of cAMP, b2cAMP, and prostaglandin E 1 were also delayed. These data suggest that the delay in secretion is not due to a delay in T S H access to the cells or in its primary action on the cell but rather to a delay in the secretory process itself. Thyroid slices of pretreated dogs are extremely sensitive to T S H : droplets could be demonstrated for a concentration of o.oi munit/ml of TSH, i.e. at T S H levels for which no effect on the metabolism of the whole slice was detected. There Biochim. Biophys. Acta, 222 (197o) 474-481
480
C. WlLLEMS et al.
is striking heterogeneity of response to TSH in vitro from one follicular cell to another; for low concentrations of TSH, scarce cells in various follicles appeared filled up with colloid droplets, while in the great majority of the other cells, no colloid droplet at all was demonstrated. This finding confirms the importance ill the action of T S H or~ thyroid tissue of the individual reactivity of the cells TM. It suggests an all or none response of the cells to TSH. The enhancement of intracellular colloid droplet formation, of iodide organification, and sometimes of glucose carbon I oxidation can be detected at a concentration of 0.o5 munit/ml 1°. BE131I release was a less sensitive index of T S H stimulation. Iodide organification, and BE~8~I release seemed to approach an asymptotic value at 1.25 munits/ml, while glucose carbon I oxidation and colloid droplet formation further increased above this concentration. Enhancement of glucose I oxidation at high T S H concentrations m a y therefore result from the stimulation of endocytosis but not from increased secretion. Several data suggest that thyroglobulinolysis m a y become the limiting step of secretion in our system. Colloid uptake, estimated by the number and size of intracellular colloid droplets, further increased during the incubation while the secretory rate remained constant. At high T S H concentrations, colloid uptake, but not secretion, further increased. TSH and T S H mimicking agents, at concentrations higher than those required to initiate secretion, enhanced the release of NBE~81I. However, it is not known if this NBE~SlI is released from the droplets or directly from the follicular lumen of loosened follicles. It is of interest that in vivo thyroglobulin is released in the lymphatic outflow of the stimulated thyroid zm. cAMP, and b,cAMP, but not AMP or ATP, mimicked the effect of TSH on thyroid secretion in vitro, cAMP had been shown previously to enhance secretion by mouse thyroid in vitro 2~, but this finding had been questioned 28. In our dog thyroid slices, these agents acted in the same range of concentrations at which they mimicked other T S H effects 1°. These data therefore further support the hypothesis that the effects of T S H on secretion, iodide binding to proteins and for low T S H concentrations, glucose carbon I oxidation, are mediated b y cAMP. There is now growing evidence relating T S H action on the thyroid to its activation of thyroid adenylcyclase ~4,~5. The fact that b2cAMP, but not cAMP, consistently gave a higher maximal stimulation of thyroid secretion than TSH, is not due to a difference in the kinetics of their actions. It is compatible with the hypothesis that intracellular cAMP, but not b2cAMP or its monobutyryl derivative, would give rise in the thyroid cell to an inhibitor of cAMP action such as has been described in the brain 26. The TSH-mimicking action of prostaglandin E~ on secretion further supports the hypothesis that it m a y in thyroid as in other tissues activate adenyl cyclase and reproduce by this mechanism the effects of T S H 1°. It is of interest in this regard that prostaglandin E 2 activates adenylcyclase in dog thyroid homogenates 2v.
ACKNOWLEDGEMENTS
This work was realized under Contract of the MinistSre de la Programmation Scientifique within the framework of the Association E u r a t o m - U n i v e r s i t y of BrusselsUniversity of Pisa (No. 026-63-4 BIAC), and thanks to grant. IOli of the Fonds Biochim. Biophys. Acta, 222 (197o) 474-481
DOG THYI~OID SECRETION
in vitro
de la Recherche Scientifique M~dicale National de la Recherche Scientifique to The authors would like to thank Ch. Borrey and Miss J. Hennaux for the
481 and Cr6dit aux Chercheurs of the Fonds Dr. P. A. Rocmans. Mr. A. MELIS for his technical help, Miss typing of the manuscript.
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