MICROTUBULES AND THYROID HORMONE MOBILIZATION J. Wolff and B. Bhattacharyya National Institute of Arthritis, Metabolism and Digestive Diseases National Institutes of Health Bethesda, Maryland 20014
Thyroid tissue contains soluble tubulin that can, after special care has been taken to remove thyroglobulin, be purified to homogeneity.’ It closely resembles tubulin from brain in that it has a dimeric molecular weight of 114,000 daltons, consists of two unequal subunits of 55,000 & 2000 daltons, contains 0.8 mole of covalently bound phosphate, and binds 1 mole of colchicine per dimer with an affinity constant of 7 X 1 0 M l . Although the amino acid composition shows a greater proportion of polar amino acids (TABLE l ) , the thyroid protein promotes fluorescence in colchicine in a manner identical to brain tubdin.‘ No histone kinase was associated with these preparations, of thyroid tubulin.’ Unlike cilia, neurites, etc., thyroid tubulin is assembled into cytoplasmic microtubules with little tendency to form more organized or complicated structures. Microtubules are distributed throughout the cytoplasm of the thyroid cell, although they appear to be more abundant near the lateral cell membranes.3-G They are also seen perpendicular and parallel to the apical cell membrane, and may be near nuclei, mitochondria or lysosomes, but there are no obvious terminations in any of these structures. Unlike most other hormones, the thyroid hormones are stored extracellularly, i.e. as the thyroglobulin of the follicular lumen. They are transported in this form into the cell by endocytosis of the luminal contents in the form of colloid droplets. These subsequently fuse with lysosomes where the thyroglobulin is hydrolized to release triiodothyronine and thyroxine (T,and T,) , which eventually find their way into the circulation by a process probably not involving exocytosis.gr Such a complicated system offers many opportunities for the intervention of a “mechanical” control mechanism, and, following the work of Lacy et al.,x we investigated the effect of colchicine on thyroid secretion.!’ This effect can be studied in vitro in two general ways. In the first, the endocytosis that follow stimulation of the gland is assessed semiquantitatively by counting the colloid droplets that have accumulated in the cell. In the second, the overall process of secretion is measured by measuring the release of 1 3 1 1 from prelabeled mouse thyroids into the incubation l o The two methods respond to secretory stimuli and inhibitors in a parallel fashion.” As far as is known at present, most of the responses of the thyroid gland are mediated vin cyclic adenosine-3,5-monophosphate(CAMP) or at least are mimicked by CAMP. The normal thyroid shows very little secretory activity unless it is stimulated by thyrotropin (TSH) or CAMP. When mouse thyroid glands are incubated with colchicine or vinblastine, the stimulatory effect of TSH on secretion is markedly attenuated or is even abolished (FIGURE1 ) . Sensitivity to colchicine can be markedly increased by preincubation of the glands before they are stimulated. Thus, the concentration of the drug required to yield a certain degree of inhibition of TSH-stimulated secretion can be
763
Annals New York Academy of Sciences
764
PROPERTIES OF
TABLE1 THYROID AND BRAIN TUBULIN
Molecular weight Subunits Colchicine Bound PO,
Thyroid
Brain
114,000 55,000 l/dimer O.l/dimer
115,000 5 5,000 1/dimer
7 x WM-' KA 42.2 Mole % (Lys & Arg & Asp & Glu) Fluorescence (uncorrected) 350-353 Xmax (excitation) 430 Xmax (Emission)
0.8/dimer 3 x lo" M-' 33.7
353 430
reduced by nearly two orders of magnitude by 4 hours of preincubation (FIGURE 2). Without preincubation complete inhibition of secretion is not obtained even M colchicine.lO With both colchicine and vinblastine, the microat 1 X tubules disappear from the cytoplasm and with vinblastine the characteristic paracrystalline aggregates are found in the cytoplasm of the thyroid cell.6*';, l2 Since TSH stimulation of thyroid tissue leads to the accumulation of CAMP,
5kf2-
! Bo Y
d
ID.?
10% 10% COCCHlClNE (MI
to*
16'
FIGURE1. Effect of colchicine or vinblastine on the release of I3lI from mouse thyroids prelabeled for 2 hours in vivo with 1311. Excised tissues were preincubated for 2 hours and then incubated for 4 hours with (upper curve in each panel) or without 2 mU/ml of TSH. From Williams and Wolff." By permission of the National Academy of Sciences.
Wolff & Bhattacharyya: Thyroid Hormone Mobilization
765
10.
5
-w
i
FIGURE2. The relation between
preincubation time and colchicine concentration required to inhibit TSHstimulated ""1 secretion from prel,abeled mouse thyroid glands by 40%. Data derived from Reference 9.
z0
1.0
I
V
dV 0.1 1 2 3 4 PREINCUBATION (hours)
5
we tested the effect of colchicine o n c A M P or N6,02'-dibutyryl cyclic adenosine3',5'-monophosphate (DBC) -stimulated secretion.6*l o The mouse is particularly responsive to c A M P without nonpolar substituents. Secretion induced by both of these nucleotides was blocked to roughly the same extent by the same concentrations of colchicine ( FIGURE 3 ) . This suggests strongly, that these drugs act as a stage after the generation of CAMP. It has been shown separately that neither colchicine nor vinblastine inhibit adenylate cyclase or the cyclic nucleotide phosphodiesterase.'; Of great interest is the recent finding that large concentrations of TSH can overcome the inhibitory effects of vinblastine o n secretion (FIGURE 4 ) . This reversal is accompanied by the disappearance of the paracrystalline structures and the reappearance of microtubules.12 Similarly, secretion stimulated by large concentrations of c A M P is less sensitive t o colchicine than that stimulated by smaller levels.'O Such data lend strong support to the notion that microtubular integrity is important in secretion and the secretory stimuli may have an effect on microtubules.
n FIGURE 3. Comparison of the antisecretory effect of
colchicine on the secretion of '."I from prelabeled mouse thyroids stimulated with TSH, cAMP or DBC. I r i i * i wlabeling and in vitro preincubation were each for 2 hours and incubation with stimulators for 4 hours. Data redrawn from References 9 & 10.
% 20
2
15 H
5
5
10
o 5 a
0
0.1
1.0
COLCHlClNE (ptvl)
1 0
766
Annals New York Academy of Sciences
In order to localize the point at which the antimitotic agents interfere with thyroid hormone secretion, we investigated the effect of colchicine on TSHstimulated colloid endocytosis. Any signs of stimulation are completely removed M colchicine and no intracellular droplets can be found." The by 2 X dose-response curve, latent period, and slow recovery are similar to that measured by secretion of 1311,10 and it seems reasonable to conclude the colchicine interferes with a very early step in secretion. Similar results have been obtained
m b o n a p n
H
FIGURE4. Reversal of vinblastine-induced inhibition of thyroid secretion iiz vivo by increasing doses of TSH. BRI is the blood radioiodine measured at 2 hours after TSH injection into rats previously injected with InI; it is expressed as the log 2hrva1ue x 100. From Endocrinology." By permission of the publisher and the 0 hr value authors.
with vinblastine 6 , l 2 and podophyllotoxin (unpublished results). Further localization has been obtained with electron microscopy, which reveals that pseudopod protrusion into the follicular lumen is abolished by these l ? Thus, the very first step of thyroid secretion recognized at present fails to occur in the presence of colchicine or vinblastine. It is of interest that two other antimitotic agents, griseofulvin and isopropylN-( 3-chlorophenyl) carbonate (chloroIPC) , which are believed to act by inter-
Wolff & Bhattacharyya: Thyroid Hormone Mobilization
.
767
fering with the nucleating or microtubule organizing centers (MTOC) , d o not inhibit thyroid secretion.s* This ineffectiveness contrasts to another endocrine secretory process and a variety of more obviously mobile systems such as mitosis or axostyle movement. We should, therefore, like to suggest that secretory processes may or may not require a MTOC and can be differentiated from those that require microtubular participation alone by use of such drugs. Since much of the case for microtubule involvement in secretion rests upon the use of inhibitors such as colchicine and vinblastine, it is surprising that so few investigators have concerned themselves with the specificity of these inhibitors, nor have they concerned themselves very often with the concentrations of colchicine or vinblastine that have to be used. In part this lack of concern is due to the impossibility of ever proving that an inhibitor is specific. In part it is also due to the fact that the secretory response provokes numerous metabolic events that must needs be disturbed when secretion is disturbed; hence, cause and effect may be difficult to establish. However, it is of crucial importance to establish specificity as far as is possible. We have already mentioned that all antimicrotubular agents are effective at low concentrations against secretion despite their widely differing chemical properties. Antimitotic agents that do not bind to tubulin, such as griseofulvin and IPC, are ineffective. Moreover, colchicine analogues inhibit secretion in proportion to their ability to bind to tubulin as judged either by displacement of tritiated colchicine or by fluorescence.2 The time course of colchicine binding to intact thyroid tissue parallels its antisecretory effect.1° Other parameters of thyroid function are not influenced in our hands by colchicine concentrations that markedly depress secretion. These include : Iodine transport; G, Organic iodine formation; Glucose and pyruvate oxidation; l5 Adenylate cyclase; cAMP levels; and cAMP diesterase.9 M colchicine In a recent study, however, it was reported that 1 X inhibited TSH stimulated organic iodine formation. This inhibitory effect was believed to be the result of H,O, deficiency caused by inhibition of [l-14C]glucose oxidation.lL Since neither the Vincn alkaloids nor D,O inhibit the oxidation of [l-lLC]glucose by thyroid tissue,6 whereas they do block secretion,5-9 - lo it seems unclear at present whether or not these oxidative processes are coupled obligatorily to the secretory process. In addition to these thyroid findings, the following metabolic parameters have been studied vis Ci vis the action of colchicine (references listed in Ref. 6) : No Inhibition Protein synthesis (most but not all reports) RNA synthesis QO, Nucleoside phosphorylation
ATP levels li Glucose production l i Urea synthesis li Fatty acid uptake and oxidation li
Questionable Oxidative phosphorylation Inhibit ion Nucleoside transport Aldose reductase (however, methyl glutarates, which inhibit this enzyme, have no effect on thyroid secretion ".
768
Annals New York Academy of Sciences
Finally, we easily get effects in the micromolar range or even less, whereas in a fair portion of other organs much higher concentrations have been used. We feel, therefore, that a fairly strong case for the specificity of the colchicine effect for tubulin has been made in the case of its effect on thyroid secretion. If we assume then that colchicine exhibits a rather high degree of specificity for tubulin, it seems appropriate to formulate a mechanism by which TSH or cAMP stimulate secretion that requires tubules and that can be blocked by antimicrotubular agents. This has proved to be difficult. The possibility exists that TSH or cAMP stimulate the polymerization of tubulin to microtubules. This mechanism has been proposed for stimulation in some other systems 1R-20 but is very sensitive to the selection of the appropriate electronmicrograph. Using all the cells of different mouse thyroid follicles incubated in vitro for 7W,we found that the average number of microtubules increased from 3.1 k 0.1/p2 to 4.5 f 0.8/p2 after exposure to TSH.G If these differences are not the result of fixation artifacts or other problems involving microtubule preservation, then this increase probably occurred before any increase in the tubulin content since in the guinea pig thyroid, at least, such increases commence only after 6-10 hours whereas secretion is maximally stimulated in less than 1 hour.'! It seems highly probable, then, that if the above microtubule count is indeed meaningful, it represents aggregation from preexisting tubulin dimers. It is not clear whether microtubules are supposed to participate as rigid structures,21,22 or must be allowed to remodel. D,O, which stabilizes the microtubule, permitted us to decide between these two possibilities. D,O and hexylene glycol both inhibited thyroid secretion as measured by lslI release or by colloid droplet formation.1° This is in conformity with the D,O effect on most secretory systems and suggest that remodeling is an important part of microtubule participation. Whether or not assembly and disassembly of microtubules in the thyroid involves Catt cannot be said at present. Because of the role of Ca++in stimulussecretion coupling in some endocrine systems, Williams 2 3 studied the effect of external Catt on 1311 secretion. No effect of Cat+ deletion could be shown. However, citrate decreased the TSH response. Furthermore, the Cat' ionophore A23187, does not influence droplet formation in our hands. Because the thyroid cell and follicular lumen are rich in Ca++,2.* the possibility that redistribution of Ca++resulting from a stimulus could influence the state of aggregation of tubulin should be studied. Several laboratories are engaged in this, but the results will have to be separated from the marked effects of Cat+ on TSH binding and adenylate c y c l a ~ e . ~ ~ An increase in the number of microtubules does not, however, tell us anything about how they might act. Firstly, although no major relocation of thyroid microtubules has so far been found after TSH administration, it is not clear which, if any, of the cytoplasmic microtubules are involved in secretion. Secondly, the initial endocytotic events involve the apical membrane, yet microtubules have not been found to be in contact with this membrane, although microfilaments come very close. Because of these anatomical problems we turned to examine the possibility of a role for colchicine-binding protein associated with organelles and especially membranes. It was known that brain contained up to 50% particulate colchicine-binding activity, present especially in the microsomal and synaptosomal 2i Beef thyroid homogenates contained from 15-25% of their
Wolff & Bhattacharyya: Thyroid Hormone Mobilization
769
in vitru colchicine-binding activity in the crude particulate fraction. Particle fractionation revealed some localization of colchicine-binding activity in the various denser particles without clear-cut separation into any one particle (see TABLE 2). That this was not trapped soluble tubulin was decided by sonication of these fractions, which caused only trivial solubilization of the particle-bound activity. A fresh plasma membrane preparation, purified on the basis of adenylate cyclase enrichment,Zx showed the highest colchicine/protein ratio in the membranes although the contribution to the total binding made by the membranes could not be specified because of losses during purification. AS far as we have been able to examine this material, it shows the same properties as does the soluble tubulin from thyroid tissue. Thus, the K A for colchicine is TABLE2 DISTRIBUTION OF COLCHICINE-BINDING ACTIVITY IN BEEF THYROID TISSUE Experiment
Fraction
Percent of Total Binding
I*
Soluble ( 100,000x g supernatant) Particulate
78 22
I1 t
Nuclei “Lysosomes, etc.” Mitochondria Microsomes 1 0 0 , 0 0 0 ~ supernatant g
4.1 5.2 4.1 2.6 83.9
’!‘ Experiment I: Beef thyroid glands were homogenized in PMG buffer (pH 7.0) containing 0.25 M sucrose and centrifuged at 100,000 x g for 1 hour. Washed 100,000 x g pellets were resuspended in PMG buffer with a glass homogenizer and incubated with 2.5 x M [“H]colchicine for 1 hour at 37” C; the bound colchicine was determined by the gradient isolation procedure of Moore and Wolff.” t Experiment 11: Subcellular fractionation by a slight modification of the procedure of Gray and Whittaker.” The crude mitochondria1 fraction was briefly sonicated and washed three times with 0.32 M sucrose to ensure removal of trapped soluble protein.
7 X lo5 M-’ and the Ki for podophyllotoxin is 3 X lo-“ M in both cases. It thus seems possible that the inhibitory effects of colchicine and vinblastine on secretion may result from an interaction of the alkaloid with a tubulin-like component of the membrane rather than cytoplasmic rnicrotubules. If this proves to be the case, it may become somewhat easier to account for the colchicine effects on secretion. References 1. BHATTACHARYYA, B. & J. WOLFF. 1974. Biochemistry. 13: 2364. 2. BHATTACHARYYA, B. & J. WOLFF. 1974. Proc. Nat. Acad. Sci. U.S.A. 72: 2627. 3. SELJELID, R. 1967. J. Ultrastruct. Res. 17: 401.
770
Annals New York Academy of Sciences
1971. Anat. Rec. 171: 81. 4. NBvE, P. & S. H. WOLLMAN. 5. NBvE, P., P. KETELBANT-BALASSE, C. WILLEMS& J. E. DUMONT.1972. Exp. Cell Res. 74: 227. 6. WOLFF,J. & 1. A. WILLIAMS.1973. Rec. Prog. Horm. Res. 29: 229-285. S. H. 1969. I n Lysosomes in Biology and Pathology. J. T. Dingle 7. WOLLMAN, & H. B. Fell, Eds. Vol. 1: 483-512. North Holland Publishing Co. Amster-
dam, The Netherlands. LACY,P. E., S.L. HOWELL,D. A. YOUNG& C. J. FINK.1968. Nature 219: 1177. WILLIAMS, J. A. & J. WOLFF. 1970. Proc. Nat. Acad. Sci. U S A . 67: 1901. WILLIAMS, J. A. & J. WOLFF. 1972. J. Cell Biol. 54: 157. WILLIAMS, J. A., S. C. BERENS& J. WOLFF. 1971. Endocrinology 88: 1385. EKHOLM,R., L. E. ERICSON,J. 0. JOSEFSSON & A. MELANDER. 1974. Endocrinology 94: 64 1. 13. SCHOFIELD, J. G . & E. N. COLE. 1971. Mem. SOC.Endocrinol. 19: 185. P. & I. N. ROSENBERG. 1974. Endocrinology 94: 1086. 14. CHIRASEVEENUPRAPUND, & J. WOLFF. 1971. Biochim. Biophys. Acta 252: 15. BERENS,S. C., J. A. WILLIAMS
8. 9. 10. 11. 12.
3 14. 16. WILLIAMS, J. A. 1972. Endocrinology 91: 1141. Y., A. SINGH, F. ASSIMACOPOULOS-JEANNET, L. ORCI, G. 17. LE MARCHAND, ROUILLER & B. JEANRENAUD. 1973. J. Biol. Chem. 248: 6862. 18. SOIFER,D., T. BRAUN& 0. HECHTER.1971. Science 172: 269. I., S. HOFFSTEIN,J. GALLIN& G. WEISSMAN.1973. Proc. Nat. 19. GOLDSTEIN, Acad. Sci. U S A . 70: 2916. 20. YAMADA, K. M. & N. K. WESSELS.1971. Exp. Cell Res. 66: 346. 1968. J. Pharmacol. Exp. 21. GILLESPIE,E., R. J. LEVINE& S. E. MALAWISTA. Therap. 164: 158. 22. POISNER, A. M. & J. BERNSTEIN.1971. J. Pharmacol. Exp. Therap. 177: 102. J. A. 1972. Endocrinology 90: 1459. 23. WILLIAMS, W. L., J. VANMIDDLESWORTH & D. DAVIS. 1971. J. Clin. Endocrinol. 24. ROBISON, Metab. 32: 786. 25. MOORE,W. V. & J. WOLFF. 1974. J. Biol. Chem. 249: 6255. 26. FEIT,H. & S. H. BARONDES. 1971. J. Neurochem. 17: 1355. J. & W. W. FRANKE. 1974. J. Cell Biol. 60: 297. 27. STADLER, 28. WOLFF,J. & A. B. JONES. 1971. J. Biol. Chem. 246: 3939. 29. MOORE,W. V. & J. WOLFF. 1973. J. Biol. Chem. 248: 5705. 1962. J. Anat. 96: 79. 30. GRAY,E. G. & V. P. WHITAKER.
Thyroid tissue contains soluble tubulin that can, after special care has been taken to remove thyroglobulin, be purified to homogeneity.’ It closely resembles tubulin from brain in that it has a dimeric molecular weight of 114,000 daltons, consists of two unequal subunits of 55,000 & 2000 daltons, contains 0.8 mole of covalently bound phosphate, and binds 1 mole of colchicine per dimer with an affinity constant of 7 X 1 0 M l . Although the amino acid composition shows a greater proportion of polar amino acids (TABLE l ) , the thyroid protein promotes fluorescence in colchicine in a manner identical to brain tubdin.‘ No histone kinase was associated with these preparations, of thyroid tubulin.’ Unlike cilia, neurites, etc., thyroid tubulin is assembled into cytoplasmic microtubules with little tendency to form more organized or complicated structures. Microtubules are distributed throughout the cytoplasm of the thyroid cell, although they appear to be more abundant near the lateral cell membranes.3-G They are also seen perpendicular and parallel to the apical cell membrane, and may be near nuclei, mitochondria or lysosomes, but there are no obvious terminations in any of these structures. Unlike most other hormones, the thyroid hormones are stored extracellularly, i.e. as the thyroglobulin of the follicular lumen. They are transported in this form into the cell by endocytosis of the luminal contents in the form of colloid droplets. These subsequently fuse with lysosomes where the thyroglobulin is hydrolized to release triiodothyronine and thyroxine (T,and T,) , which eventually find their way into the circulation by a process probably not involving exocytosis.gr Such a complicated system offers many opportunities for the intervention of a “mechanical” control mechanism, and, following the work of Lacy et al.,x we investigated the effect of colchicine on thyroid secretion.!’ This effect can be studied in vitro in two general ways. In the first, the endocytosis that follow stimulation of the gland is assessed semiquantitatively by counting the colloid droplets that have accumulated in the cell. In the second, the overall process of secretion is measured by measuring the release of 1 3 1 1 from prelabeled mouse thyroids into the incubation l o The two methods respond to secretory stimuli and inhibitors in a parallel fashion.” As far as is known at present, most of the responses of the thyroid gland are mediated vin cyclic adenosine-3,5-monophosphate(CAMP) or at least are mimicked by CAMP. The normal thyroid shows very little secretory activity unless it is stimulated by thyrotropin (TSH) or CAMP. When mouse thyroid glands are incubated with colchicine or vinblastine, the stimulatory effect of TSH on secretion is markedly attenuated or is even abolished (FIGURE1 ) . Sensitivity to colchicine can be markedly increased by preincubation of the glands before they are stimulated. Thus, the concentration of the drug required to yield a certain degree of inhibition of TSH-stimulated secretion can be
763
Annals New York Academy of Sciences
764
PROPERTIES OF
TABLE1 THYROID AND BRAIN TUBULIN
Molecular weight Subunits Colchicine Bound PO,
Thyroid
Brain
114,000 55,000 l/dimer O.l/dimer
115,000 5 5,000 1/dimer
7 x WM-' KA 42.2 Mole % (Lys & Arg & Asp & Glu) Fluorescence (uncorrected) 350-353 Xmax (excitation) 430 Xmax (Emission)
0.8/dimer 3 x lo" M-' 33.7
353 430
reduced by nearly two orders of magnitude by 4 hours of preincubation (FIGURE 2). Without preincubation complete inhibition of secretion is not obtained even M colchicine.lO With both colchicine and vinblastine, the microat 1 X tubules disappear from the cytoplasm and with vinblastine the characteristic paracrystalline aggregates are found in the cytoplasm of the thyroid cell.6*';, l2 Since TSH stimulation of thyroid tissue leads to the accumulation of CAMP,
5kf2-
! Bo Y
d
ID.?
10% 10% COCCHlClNE (MI
to*
16'
FIGURE1. Effect of colchicine or vinblastine on the release of I3lI from mouse thyroids prelabeled for 2 hours in vivo with 1311. Excised tissues were preincubated for 2 hours and then incubated for 4 hours with (upper curve in each panel) or without 2 mU/ml of TSH. From Williams and Wolff." By permission of the National Academy of Sciences.
Wolff & Bhattacharyya: Thyroid Hormone Mobilization
765
10.
5
-w
i
FIGURE2. The relation between
preincubation time and colchicine concentration required to inhibit TSHstimulated ""1 secretion from prel,abeled mouse thyroid glands by 40%. Data derived from Reference 9.
z0
1.0
I
V
dV 0.1 1 2 3 4 PREINCUBATION (hours)
5
we tested the effect of colchicine o n c A M P or N6,02'-dibutyryl cyclic adenosine3',5'-monophosphate (DBC) -stimulated secretion.6*l o The mouse is particularly responsive to c A M P without nonpolar substituents. Secretion induced by both of these nucleotides was blocked to roughly the same extent by the same concentrations of colchicine ( FIGURE 3 ) . This suggests strongly, that these drugs act as a stage after the generation of CAMP. It has been shown separately that neither colchicine nor vinblastine inhibit adenylate cyclase or the cyclic nucleotide phosphodiesterase.'; Of great interest is the recent finding that large concentrations of TSH can overcome the inhibitory effects of vinblastine o n secretion (FIGURE 4 ) . This reversal is accompanied by the disappearance of the paracrystalline structures and the reappearance of microtubules.12 Similarly, secretion stimulated by large concentrations of c A M P is less sensitive t o colchicine than that stimulated by smaller levels.'O Such data lend strong support to the notion that microtubular integrity is important in secretion and the secretory stimuli may have an effect on microtubules.
n FIGURE 3. Comparison of the antisecretory effect of
colchicine on the secretion of '."I from prelabeled mouse thyroids stimulated with TSH, cAMP or DBC. I r i i * i wlabeling and in vitro preincubation were each for 2 hours and incubation with stimulators for 4 hours. Data redrawn from References 9 & 10.
% 20
2
15 H
5
5
10
o 5 a
0
0.1
1.0
COLCHlClNE (ptvl)
1 0
766
Annals New York Academy of Sciences
In order to localize the point at which the antimitotic agents interfere with thyroid hormone secretion, we investigated the effect of colchicine on TSHstimulated colloid endocytosis. Any signs of stimulation are completely removed M colchicine and no intracellular droplets can be found." The by 2 X dose-response curve, latent period, and slow recovery are similar to that measured by secretion of 1311,10 and it seems reasonable to conclude the colchicine interferes with a very early step in secretion. Similar results have been obtained
m b o n a p n
H
FIGURE4. Reversal of vinblastine-induced inhibition of thyroid secretion iiz vivo by increasing doses of TSH. BRI is the blood radioiodine measured at 2 hours after TSH injection into rats previously injected with InI; it is expressed as the log 2hrva1ue x 100. From Endocrinology." By permission of the publisher and the 0 hr value authors.
with vinblastine 6 , l 2 and podophyllotoxin (unpublished results). Further localization has been obtained with electron microscopy, which reveals that pseudopod protrusion into the follicular lumen is abolished by these l ? Thus, the very first step of thyroid secretion recognized at present fails to occur in the presence of colchicine or vinblastine. It is of interest that two other antimitotic agents, griseofulvin and isopropylN-( 3-chlorophenyl) carbonate (chloroIPC) , which are believed to act by inter-
Wolff & Bhattacharyya: Thyroid Hormone Mobilization
.
767
fering with the nucleating or microtubule organizing centers (MTOC) , d o not inhibit thyroid secretion.s* This ineffectiveness contrasts to another endocrine secretory process and a variety of more obviously mobile systems such as mitosis or axostyle movement. We should, therefore, like to suggest that secretory processes may or may not require a MTOC and can be differentiated from those that require microtubular participation alone by use of such drugs. Since much of the case for microtubule involvement in secretion rests upon the use of inhibitors such as colchicine and vinblastine, it is surprising that so few investigators have concerned themselves with the specificity of these inhibitors, nor have they concerned themselves very often with the concentrations of colchicine or vinblastine that have to be used. In part this lack of concern is due to the impossibility of ever proving that an inhibitor is specific. In part it is also due to the fact that the secretory response provokes numerous metabolic events that must needs be disturbed when secretion is disturbed; hence, cause and effect may be difficult to establish. However, it is of crucial importance to establish specificity as far as is possible. We have already mentioned that all antimicrotubular agents are effective at low concentrations against secretion despite their widely differing chemical properties. Antimitotic agents that do not bind to tubulin, such as griseofulvin and IPC, are ineffective. Moreover, colchicine analogues inhibit secretion in proportion to their ability to bind to tubulin as judged either by displacement of tritiated colchicine or by fluorescence.2 The time course of colchicine binding to intact thyroid tissue parallels its antisecretory effect.1° Other parameters of thyroid function are not influenced in our hands by colchicine concentrations that markedly depress secretion. These include : Iodine transport; G, Organic iodine formation; Glucose and pyruvate oxidation; l5 Adenylate cyclase; cAMP levels; and cAMP diesterase.9 M colchicine In a recent study, however, it was reported that 1 X inhibited TSH stimulated organic iodine formation. This inhibitory effect was believed to be the result of H,O, deficiency caused by inhibition of [l-14C]glucose oxidation.lL Since neither the Vincn alkaloids nor D,O inhibit the oxidation of [l-lLC]glucose by thyroid tissue,6 whereas they do block secretion,5-9 - lo it seems unclear at present whether or not these oxidative processes are coupled obligatorily to the secretory process. In addition to these thyroid findings, the following metabolic parameters have been studied vis Ci vis the action of colchicine (references listed in Ref. 6) : No Inhibition Protein synthesis (most but not all reports) RNA synthesis QO, Nucleoside phosphorylation
ATP levels li Glucose production l i Urea synthesis li Fatty acid uptake and oxidation li
Questionable Oxidative phosphorylation Inhibit ion Nucleoside transport Aldose reductase (however, methyl glutarates, which inhibit this enzyme, have no effect on thyroid secretion ".
768
Annals New York Academy of Sciences
Finally, we easily get effects in the micromolar range or even less, whereas in a fair portion of other organs much higher concentrations have been used. We feel, therefore, that a fairly strong case for the specificity of the colchicine effect for tubulin has been made in the case of its effect on thyroid secretion. If we assume then that colchicine exhibits a rather high degree of specificity for tubulin, it seems appropriate to formulate a mechanism by which TSH or cAMP stimulate secretion that requires tubules and that can be blocked by antimicrotubular agents. This has proved to be difficult. The possibility exists that TSH or cAMP stimulate the polymerization of tubulin to microtubules. This mechanism has been proposed for stimulation in some other systems 1R-20 but is very sensitive to the selection of the appropriate electronmicrograph. Using all the cells of different mouse thyroid follicles incubated in vitro for 7W,we found that the average number of microtubules increased from 3.1 k 0.1/p2 to 4.5 f 0.8/p2 after exposure to TSH.G If these differences are not the result of fixation artifacts or other problems involving microtubule preservation, then this increase probably occurred before any increase in the tubulin content since in the guinea pig thyroid, at least, such increases commence only after 6-10 hours whereas secretion is maximally stimulated in less than 1 hour.'! It seems highly probable, then, that if the above microtubule count is indeed meaningful, it represents aggregation from preexisting tubulin dimers. It is not clear whether microtubules are supposed to participate as rigid structures,21,22 or must be allowed to remodel. D,O, which stabilizes the microtubule, permitted us to decide between these two possibilities. D,O and hexylene glycol both inhibited thyroid secretion as measured by lslI release or by colloid droplet formation.1° This is in conformity with the D,O effect on most secretory systems and suggest that remodeling is an important part of microtubule participation. Whether or not assembly and disassembly of microtubules in the thyroid involves Catt cannot be said at present. Because of the role of Ca++in stimulussecretion coupling in some endocrine systems, Williams 2 3 studied the effect of external Catt on 1311 secretion. No effect of Cat+ deletion could be shown. However, citrate decreased the TSH response. Furthermore, the Cat' ionophore A23187, does not influence droplet formation in our hands. Because the thyroid cell and follicular lumen are rich in Ca++,2.* the possibility that redistribution of Ca++resulting from a stimulus could influence the state of aggregation of tubulin should be studied. Several laboratories are engaged in this, but the results will have to be separated from the marked effects of Cat+ on TSH binding and adenylate c y c l a ~ e . ~ ~ An increase in the number of microtubules does not, however, tell us anything about how they might act. Firstly, although no major relocation of thyroid microtubules has so far been found after TSH administration, it is not clear which, if any, of the cytoplasmic microtubules are involved in secretion. Secondly, the initial endocytotic events involve the apical membrane, yet microtubules have not been found to be in contact with this membrane, although microfilaments come very close. Because of these anatomical problems we turned to examine the possibility of a role for colchicine-binding protein associated with organelles and especially membranes. It was known that brain contained up to 50% particulate colchicine-binding activity, present especially in the microsomal and synaptosomal 2i Beef thyroid homogenates contained from 15-25% of their
Wolff & Bhattacharyya: Thyroid Hormone Mobilization
769
in vitru colchicine-binding activity in the crude particulate fraction. Particle fractionation revealed some localization of colchicine-binding activity in the various denser particles without clear-cut separation into any one particle (see TABLE 2). That this was not trapped soluble tubulin was decided by sonication of these fractions, which caused only trivial solubilization of the particle-bound activity. A fresh plasma membrane preparation, purified on the basis of adenylate cyclase enrichment,Zx showed the highest colchicine/protein ratio in the membranes although the contribution to the total binding made by the membranes could not be specified because of losses during purification. AS far as we have been able to examine this material, it shows the same properties as does the soluble tubulin from thyroid tissue. Thus, the K A for colchicine is TABLE2 DISTRIBUTION OF COLCHICINE-BINDING ACTIVITY IN BEEF THYROID TISSUE Experiment
Fraction
Percent of Total Binding
I*
Soluble ( 100,000x g supernatant) Particulate
78 22
I1 t
Nuclei “Lysosomes, etc.” Mitochondria Microsomes 1 0 0 , 0 0 0 ~ supernatant g
4.1 5.2 4.1 2.6 83.9
’!‘ Experiment I: Beef thyroid glands were homogenized in PMG buffer (pH 7.0) containing 0.25 M sucrose and centrifuged at 100,000 x g for 1 hour. Washed 100,000 x g pellets were resuspended in PMG buffer with a glass homogenizer and incubated with 2.5 x M [“H]colchicine for 1 hour at 37” C; the bound colchicine was determined by the gradient isolation procedure of Moore and Wolff.” t Experiment 11: Subcellular fractionation by a slight modification of the procedure of Gray and Whittaker.” The crude mitochondria1 fraction was briefly sonicated and washed three times with 0.32 M sucrose to ensure removal of trapped soluble protein.
7 X lo5 M-’ and the Ki for podophyllotoxin is 3 X lo-“ M in both cases. It thus seems possible that the inhibitory effects of colchicine and vinblastine on secretion may result from an interaction of the alkaloid with a tubulin-like component of the membrane rather than cytoplasmic rnicrotubules. If this proves to be the case, it may become somewhat easier to account for the colchicine effects on secretion. References 1. BHATTACHARYYA, B. & J. WOLFF. 1974. Biochemistry. 13: 2364. 2. BHATTACHARYYA, B. & J. WOLFF. 1974. Proc. Nat. Acad. Sci. U.S.A. 72: 2627. 3. SELJELID, R. 1967. J. Ultrastruct. Res. 17: 401.
770
Annals New York Academy of Sciences
1971. Anat. Rec. 171: 81. 4. NBvE, P. & S. H. WOLLMAN. 5. NBvE, P., P. KETELBANT-BALASSE, C. WILLEMS& J. E. DUMONT.1972. Exp. Cell Res. 74: 227. 6. WOLFF,J. & 1. A. WILLIAMS.1973. Rec. Prog. Horm. Res. 29: 229-285. S. H. 1969. I n Lysosomes in Biology and Pathology. J. T. Dingle 7. WOLLMAN, & H. B. Fell, Eds. Vol. 1: 483-512. North Holland Publishing Co. Amster-
dam, The Netherlands. LACY,P. E., S.L. HOWELL,D. A. YOUNG& C. J. FINK.1968. Nature 219: 1177. WILLIAMS, J. A. & J. WOLFF. 1970. Proc. Nat. Acad. Sci. U S A . 67: 1901. WILLIAMS, J. A. & J. WOLFF. 1972. J. Cell Biol. 54: 157. WILLIAMS, J. A., S. C. BERENS& J. WOLFF. 1971. Endocrinology 88: 1385. EKHOLM,R., L. E. ERICSON,J. 0. JOSEFSSON & A. MELANDER. 1974. Endocrinology 94: 64 1. 13. SCHOFIELD, J. G . & E. N. COLE. 1971. Mem. SOC.Endocrinol. 19: 185. P. & I. N. ROSENBERG. 1974. Endocrinology 94: 1086. 14. CHIRASEVEENUPRAPUND, & J. WOLFF. 1971. Biochim. Biophys. Acta 252: 15. BERENS,S. C., J. A. WILLIAMS
8. 9. 10. 11. 12.
3 14. 16. WILLIAMS, J. A. 1972. Endocrinology 91: 1141. Y., A. SINGH, F. ASSIMACOPOULOS-JEANNET, L. ORCI, G. 17. LE MARCHAND, ROUILLER & B. JEANRENAUD. 1973. J. Biol. Chem. 248: 6862. 18. SOIFER,D., T. BRAUN& 0. HECHTER.1971. Science 172: 269. I., S. HOFFSTEIN,J. GALLIN& G. WEISSMAN.1973. Proc. Nat. 19. GOLDSTEIN, Acad. Sci. U S A . 70: 2916. 20. YAMADA, K. M. & N. K. WESSELS.1971. Exp. Cell Res. 66: 346. 1968. J. Pharmacol. Exp. 21. GILLESPIE,E., R. J. LEVINE& S. E. MALAWISTA. Therap. 164: 158. 22. POISNER, A. M. & J. BERNSTEIN.1971. J. Pharmacol. Exp. Therap. 177: 102. J. A. 1972. Endocrinology 90: 1459. 23. WILLIAMS, W. L., J. VANMIDDLESWORTH & D. DAVIS. 1971. J. Clin. Endocrinol. 24. ROBISON, Metab. 32: 786. 25. MOORE,W. V. & J. WOLFF. 1974. J. Biol. Chem. 249: 6255. 26. FEIT,H. & S. H. BARONDES. 1971. J. Neurochem. 17: 1355. J. & W. W. FRANKE. 1974. J. Cell Biol. 60: 297. 27. STADLER, 28. WOLFF,J. & A. B. JONES. 1971. J. Biol. Chem. 246: 3939. 29. MOORE,W. V. & J. WOLFF. 1973. J. Biol. Chem. 248: 5705. 1962. J. Anat. 96: 79. 30. GRAY,E. G. & V. P. WHITAKER.
