In Vitro Stimulation of Thyroid Ornithine Decarboxylase Activity and Polyamines by Thyrotropin STEVEN J. SCHEINMAN* AND GERARD N. BURROWf Department of Internal Medicine, Yale University Neic Haven, Connecticut 06510 ABSTRACT. Thyrotropin (TSH) stimulation of ornithine decarboxylase (ODC) activity and polyamine levels was studied in vitro in rat thyroids. The elevation in ODC activity was related to the concentration of TSH in the incubation medium with peak activity at a concentration of 25 mU/ml. ODC activity with 50 mU/ml of TSH was 3 to 5-fold higher than control activity at 5 h of incubation; this stimulation was enhanced by the addition of 0.5 mM 3-isobutyl-l-methyl xanthine (MIX), a phosphodiesterase inhibitor. Dibutyryl cyclic AMP (DbcAMP) also stimulated ODC activity with a dose response up to 2.0 mM. The increase in ODC activity with TSH and MIX was prevented by in-
T
HE POLYAMINES spermidine and spermine, and their biosynthetic precursor, putrescine, are widely distributed in biological systems and have been demonstrated to accumulate in rapidly growing tissue concomitantly with an increase in nucleic acids (1). Polyamines have a number of well-characterized effects on nucleic acid metabolism and function, protein synthesis, and cell membranes (1-3). The rate-limiting step in polyamine biosynthesis, is probably the formation of putrescine from ornithine which is catalyzed by the enzyme ornitliine decarboxylase (ODC) (EC 4.1.1.17) (4). ODC activity is increased in a variety of endocrine target organs under hormonal stimulation (1,5-8). TSH has also been demonstrated to increase ODC activity in the thyroid in vivo (9-12). TSH stimulation of ODC activity is mimicked by the administration of dibutyryl cAMP, but it is difficult to separate direct Received September 2, 1976. Supported by USPHS Grant #AM 17060. * Presented to the Faculty of the Yale University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Medicine. f Present address: Toronto General Hospital, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
School of Medicine, 333 Cedar
Street,
cubation with actinomycin D (10 fxg/m\) or puromycin (0.2 HIM). Putrescine concentrations in rat thyroids rose to three times basal levels after 6 h of incubation with TSH and MIX; no significant elevation in spermidine or spermine was observed after up to 7 h incubation. The increase in tissue putrescine preceded a rise in [3H]uridine incorporation into acid-soluble material that occurred at 7 h. The results suggest that stimulation of thyroid ODC activity by TSH is mediated by cyclic AMP; the data further are consistent with a role for polyamines in the control of RNA synthesis in the thyroid. (Endocrinology 101: 1088, 1977)
hormonal effects from more indirect ones (9). To obviate this problem, we have studied the factors involved in the regulation of thyroid ODC activity in vitro and have related the increase in ODC activity to the time course for synthesis of polyamines and RNA. Materials and Methods Tissue incubation Male Sprague Dawley rats weighing 200 g were obtained from Charles River Laboratories. The thyroid glands were removed and randomized with 4-12 lobes placed in a beaker. The lobes were preincubated in 5 ml Krebs-Ringerbicarbonate buffer (KRB) for 15 min and then incubated for 1-7 h in KRB in a Dubnoff metabolic shaker at 37 C in 95% O 2 -5% CO2. Quantitative bacterial determinations of samples of the media were made at intervals up to 7 h and no significant contamination occurred. After incubation, the thyroids were homogenized in appropriate media (specified below) with a mechanically driven Teflon pestle in a PotterElvehjem tube. Homogenates were then centrifuged at 4 C and the supernatants assayed for ODC activity or polyamines. For ODC assay, 4 lobes representing 40-60 ing of tissue were homogenized in 1.4 ml of buffer composed of 0.25M sucrose, 0.5 mM EDTA and 10 mM 2-
1088
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
THYROID ODC ACTIVITY IN VITRO mercaptoethanol, and centrifuged at 100,000 x g for 30 min. For polyamine determinations, 8-10 lobes were homogenized in 0.4 ml of 0.4N perchloric acid (PCA) and centrifuged at 1,000 x g for 10 min. Enzyme assay ODC activity was assayed by measuring 14CO2 produced from DL-[l-14C]ornithine as described by Russell and Snyder (13) and modified by Chen et al. (14). The assay mixture contained 0.6 niM DL-ornithine, 0.1 mM pyridoxal-5-phosphate, 25 mM Tris-HCl buffer (pH 7.88 at 37 C), 13 /Amoles mercaptoethanol, and 100 /xl of supernatant representing 0.06-0.2 mg protein, in a final volume of 215 fxl. Within this range of protein concentration, ODC activity was linear with thyroid protein concentration. Ornithine was prepared for assay by acidification to O.lN HC1, heating on a rotary evaporator under vacuum at 54 C for 30 min to remove a volatile; contaminant which otherwise gave high assay blanks, and then diluted to O.OIN HC1. Unlabelled ornithine was added to give a final specific activity of 5.8 mCi/mmole. Decarboxylation was carried out in #2057 Falcon tubes with a polyethylene well containing 0.2 ml of monoethanolamine: ethylene glycol monoethyl ether (1:2, vol/vol). Decarboxylation was linear with time for up to 2 h of incubation (Fig. .1). Assay tubes in the present study were incubated for 90 min at 37 C in a shaker bath. The reaction was stopped by injection of 0.4 ml 2N citric acid through the needle. After agitation for an additional 60 min at 37 C, each well was removed, placed in 15 ml Aquasol (New England Nuclear) and isotope content was determined and counted on a Packard Tri-Carb or a Searle Mark II scintillation counter. The amount of 14CO2 released from substrate in tubes with no enzyme was subtracted from the value for all other samples. Picomoles of 14 CO2 released from the L-isomer were calculated (15). ODC activity was expressed as picomoles 14CO2 released per milligram of protein per hour of assay incubation. ODC activity was determined in triplicate in all experiments. Measurements were made on supernatants from a single beaker. All experiments were repeated and confirmed at least once, more often several times. Protein determination was by the method of Lowry et al. (16) using a least squares calculation for a BSA standard curve.
1089
ODC activity was measured between pH 6.6 and 9.9 and found to be maximal from 7.7 to 8.1. Maximal ODC activity was seen at 48 C. Kinetic studies reproducibly demonstrated a Km for ornithine of 0.12 mM, closely comparable to the ODC Km observed in other mamalian systems though at variance with the only other Km reported for the thyroid enzyme (10). Our samples were assayed at pH 7.88, 37 C with 0.6 mM DL-ornithine. Polyamine assay Tissue levels of putrescine, spermidine and spermine were determined by dansylation according to the method of Dion and Herbst (17). PCA supernatant (0.2 ml) was added to 0.4 ml of dansyl chloride (30 mg/ml acetone) and 18.5 mg anhydrous Na2CO.(. After 16 h in the dark at room temperature, 0.1 ml of proline (100 mg/ml H2O) was added for 30 min to convert excess dansyl chloride to dansyl proline. Dansyl derivatives were extracted into 0.5 ml benzene and 10 fx\ of the benzene layer was spotted onto 250 (iini Silica gel TLC plates which had been heated to 110 C for 1 h prior to spotting. Standard samples of all three amines were spotted on each plate as well. Plates were run twice with 2:3 ethylacetate/cyclohexane (vol/vol) in the dark, and sprayed immediately with 1:4 triethanolamine/ethylene glycol (vol/vol). Plates were dried in the dark, in vacua for 16 h and allowed to equilibrate to atmospheric pressure for one hour before scanning on a Turner III fluorimeter with TLC scanner. Blanks were scanned between the putrescine and spermidine spots and were subtracted from values for each sample. The area under each peak was proportional to the amount of dansyl amine at that spot; the area was proportional between 0-5 nmoles for putrescine and 0-20 nmoles for spermidine and spermine. The assay had a reproducibility of ±11.7% for putrescine, 14.2% for spermidine and 13.7% for spermine. Tissue levels were expressed as nmoles/g of tissue weighed after incubation. Medium was lyophilized, solubilized in 0.4N PCA and assayed as above. RNA synthesis For those experiments in which RNA synthesis was measured, thyroids were incubated as above with [3H]uridine (10 /xCi/ml) added to the incubation medium. The pellet remaining after
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1977 Vol 101 • No 4
SCHEINMAN AND BURROW
1090 o 1400 -
2
4
5
6
7
TIME (hours)
FiG. 1. The time course of ODC activity in thyroids incubated with 50 mU/ml TSH and 0.5 mM MIX ( ) and in controls (: -r). A typical experiment is shown. Each point represents the mean ± SEM of three determinations.
centrifugation of the PCA homogenate was washed 3 times in 5% trichloroacetic acid and resuspended in 0.2 ml I N NaOH; 20 (JL\ aliquots were counted in Aquasol. [3H]Uridine incorporation into RNA was expressed as cpm per g of tissue.
(MIX), a potent phosphodiesterase inhibitor, led to marked enhancement of this stimulation, with peak ODC activity occurring usually at 6 h (Fig. 1); stimulation of ODC activity by MIX alone did not significantly differ from stimulation by 50 mU/ml of TSH at 5 h of incubation. The rise in control ODC activity with time is unexplained but a similar rise was observed by Cohen et al. in chick oviducts in vitro (18). The stimulation of ODC activity by TSH was dose-related; 5 mU/ml of TSH significantly elevated ODC activity over control and maximal stimulation was seen with 25 mU/ml (Fig. 2). All subsequent incubations with TSH were done at a concentration of 50 mU/ml. Incubation of thyroids with dibutyryl cyclic AMP (DbcAMP) resulted in doserelated stimulation of ODC activity up to a concentration of 2 mM. Incubation with butyrate alone had no stimulatory effect on ODC activity. There was no stimulation over control at 5 mM DbcAMP, as confirmed by several repetitions (Fig. 3). The stimulation of ODC activity by 1 mM
§ 600 -
Materials TSH was kindly supplied by the National Institutes of Health (NIH-TSH-B7). 3-Isobutyl-lmethyl xanthine was purchased from the Aldrich Chemical Company. N6,O2'-Dibutyryl 3',5' cyclic monophosphoric acid, actinomycin D, puromycin, putrescine, spermidine, spermine, dansyl chloride, and iproniazid phosphate were purchased from Sigma Chemicals. [6H3]Uridine (24.2 Ci/nmol lot #908-053) and DL[l-14C]ornithine monohydrochloride (lot #822-176) were obtained from New England Nuclear.
Z 500 -
Results Stimulation of thyroid ODC in vitro Incubation of thyroids with 50 mU/ml of TSH resulted in a 3 to 5-fold stimulation of ODC activity at 5 h (P < .001). The addition of 0.5 mM 3-isobutyl-l-methyl xanthine
25 [TSH] (mU/ml)
50
FIG. 2. Effect of varying concentrations of TSH on thyroid ODC activity at 5 h of incubation. Each point represents the mean ± SEM of three determinations.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
1091
THYROID ODC ACTIVITY IN VITRO DbcAMP was not enhanced by the addition of MIX.
Effects of inhibitors of RNA and protein synthesis on thyroid ODC activity The addition of 0.2 mM puromycin or 10 /u,g/ml actinomycin D to the preincubation and incubation medium inhibited stimulation of ODC activity in response to TSH and MIX (Fig. 4). Puromycin completely prevented the ten-fold rise in ODC activity in stimulated thyroids and even blocked basal ODC activity of control thyroids. Studies were not done with DbcAMP. A concentration of 1% ethanol was required to solubilize the actinomycin D; this concentration of ethanol was found to eliminate the ODC activity in the incubated control and to attenuate the ODC response to TSH and MIX. Thus, while the data show inhibition of ODC activity by actinomycin D, it is impossible to dis-
PUROMYCIN
1000
800 'c o o
-
600 -
3
Z 400 c
5 Z 200 _ a CK
E Control
ACTINOMYCIN D
TSH + Methyl Xanthine
T
1
200 -
150 -
100 -
50 -
T
T Control
900 -
Puromycin + TSH + Methyl Xanthine
TSH + Methyl Xonthine
Actinomycin D + TSH + Methyl Xonthine
800 -
FIG. 4. Upper: Effect of 0.2 mM puromycin on the stimulation of thyroid ODC activity in vitro at 5 h of incubation as compared with stimulated thyroids without inhibitor and controls. Lower: Effect of 10 /xg/ml actinomycin D on the stimulation of ODC activity as compared with stimulated thyroids and controls. Stimulation was with 50 mU/ml TSH and 0.5 mM MIX. Each point represents the mean ± SEM of three determinations.
700 -
tinguish complete from incomplete inhibition. Stimulation of thyroid amines in vitro
.t
.5
TSH
i [Ob CAMP] (mM)
FlC. 3. Effect of varying concentrations of DbcAMP on thyroid ODC activity at 5 h of incubation. Each point represents the mean ± SEM of three determinations. Bar graph represents stimulation with 50 mU/ml TSH alone.
Thyroid lobes contained basal levels of about 300 nmoles/g of spermidine and spermine and one-tenth as much putrescine. Tissue levels of all three amines did not rise before 5 h of incubation with TSH and MIX. At 6 h, a three-fold increase in tissue putrescine levels was observed (P < .05), together with marked leakage of putrescine into the medium, when thyroids were in-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
tion of tritiated uridine into acid-insoluble material, rose significantly at 7 h.
600
500
Endo • 1977 Vol 101 . N o 4
SCHEINMAN AND BURROW
1092
Discussion
I I Control VP7A TSH + Methyl Xonthine
o 400 E
5 300
200
100
TISSUE
MEDIUM
FIG. 5. Effect of TSH (50 mU/ml) and MIX (0.5 DIM) on putrescine levels in thyroid tissue and incubation medium at 6 h of incubation. All beakers included 5 mM iproniazid.
This is the first report of stimulation of thyroid ODC in vitro. Our results match the earlier in vivo observations that a single injection of TSH in rats yields peak activity of thyroid ODC 4 to 6 h later (9,10,12). The 3-5-fold rise in vitro is comparable to the degree of stimulation observed in vivo in hypophysectomized rats, but significantly less than that found with intact rats (9). Dibutyryl cyclic AMP has been shown to stimulate ODC activity in the adrenals of hypophysectomized rats (7), cultured mammary tissue (8), and when given with aminophylline in thyroids of normal rats (9). Enzyme activity in livers of intact rats was stimulated by DbcAMP (5,20), but not consistently in livers of hypophysectomized rats (21). As DbcAMP is known to cause release of pituitary hormones (22), in vivo
cubated in TSH and MIX (Fig. 5). Spermidine and spermine were not significantly elevated at 6 h. Both control and stimulated samples in this experiment were incubated with 5 mM iproniazid, an amine oxidase inhibitor, to prevent polyamine degradation (19).
1,000 eoo -
Time course of stimulation ofODC activity, putrescine content, and RNA synthesis Figure 6 illustrates the time course for ODC activity, putrescine levels, and [3H]uridine incorporation into RNA in a representative incubation with TSH and MIX. ODC activity rose at 4 h, peaked at 5 h, and declined thereafter. Although these samples were incubated without iproniazid, putrescine levels were significantly elevated over controls at 6 h (P < .05), following the ODC peak. Spermidine and spermine levels were again not significantly elevated during this time period. Putrescine leakage into the medium closely paralleled tissue levels. RNA synthesis, as measured by incorpora-
= 40,000 20.000 Z
3
o: Z 2
4
5
INCUBATION TIME (hours)
FIG. 6. The time course of ODC activity, tissue putrescine levels and RNA synthesis in thyroids incubated with 50 mU/ml TSH and 0.5 mM MIX.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
THYROID ODC ACTIVITY IN VITRO studies must be interpreted carefully. Further, there are systems in which ODC stimulation occurs without changes in tissue cAMP levels (23). Insulin, which lowers cAMP levels in target tissues, stimulates ODC activity in liver (21,24) and in mammary explants (29). Thus, in some systems induction of ODC activity appears to involve a mechanism independent of cAMP. In vivo results have varied regarding the role of cyclic AMP in thyroidal ODC stimulation. Earlier work from our laboratory (9) demonstrated 12-fold stimulation of rat thyroid ODC with a combination of DbcAMP and aminophylline, although either compound alone was ineffective. However, Richman et al., using identical concentrations, observed only slight stimulation (10). In the present study we have demonstrated a dose-related stimulation of up to 8-fold in thyroid ODC activity in vitro with 2 mM DbcAMP. We have further shown that the ODC response to TSH is enhanced by a phosphodiesterase inhibitor. Stimulation of ODC activity thus resembles other effects of TSH on thyroid metabolism which appear to be mediated by cAMP (25), perhaps through the action of cAMP-dependent protein kinase (26). The elevation of putrescine content with ODC activity implies that the increase in ODC activity is physiologic rather than an assay artifact. Polyamine levels found in the thyroid in the present study are comparable to those found in other rat organs (27,28) if a correction is made for the thyroglobulin content of the thyroid. The accumulation of putrescine alone during the 7 h incubation in the present study is similar to the finding in regenerating rat liver that putrescine levels rise by 2-3-fold beginning 4 h after a partial hepatectomy, while spermidine rises by much less and not until 8 h (28). Matsuzaki and Suzuki reported elevation of putrescine and spermidine levels in the thyroid beginning on the second day of methylthiouracil treatment (12). The degree of putrescine leakage into the medium was unexpected. In the pres-
1093
ence of 5 mM iproniazid it is unlikely that this represents putrescine produced by the breakdown of oxidized spermidine or spermine (19). Putrescine is less well bound to cellular constituents than are spermidine and spermine and this might explain the elevation of putrescine in the medium in the absence of any change in spermidine or spermine content of the medium. We have found that ODC activity and putrescine levels rise prior to an increase in [ 3 H]uridine incorporation into RNA. Stimulation of [ 3 H]uridine incorporation has been observed in vitro after 1 to 4 h of incubation of the thyroid with TSH by several (29-31) though not all (32) investigators. In none of these studies was incubation carried out for 7 h; it is possible that our methods were too insensitive to detect a small earlier change in uridine uptake into RNA. Studies using metabolic inhibitors indicated that stimulation of ODC activity requires new synthesis of RNA (9,10). It also has been proposed that polyamines participate in the regulation of RNA synthesis (1), which could be supported by our findings. Conceivably, an undetectable early increase in RNA production might lead to polyamine accumulation which would be followed by greater augmentation of nucleic acid synthesis. ; Regulation of ODC activity in the thyroid seems to depend on synthesis of new protein as well as of RNA (9,10); this is suggested not only by inhibitor studies, but also by the time lag in ODC stimulation by TSH as compared to other effects of TSH (25,33). However, it is not known whether there is an actual induction of enzyme protein or whether there is modulation of the activity of pre-existing ODC. Puromycin has other effects and might not be specific for TSH induction of ODC. In experiments not reported here, an antibody to partially purified hepatic ODC (34) inactivated about 90% of the ODC activity in TSH-stimulated thyroid supernatants. Further, radio-iodinated antibody
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
1094
SCHEINMAN AND BURROW
Endo • 1977 Vol 101 • No 4
11. Matsuzaki, S., and M. Suzuki, Endocrinol Jap 21: 529, 1974. 12. Matsuzaki, S., and M. Suzuki, Endocrinol Jap 22: 339, 1975. 13. Russell, D. H., and S. H. Synder, Proc Natl. Acad Sci USA 60: 1420, 1968. 14. Chen, K., J. Heller, and E. S. Canellakis, Biochem Biophys Res Commun 68: 401, 1976. 15. Raina, A., and J. Janne, Ada Chem Scand 22: 2375, 1968. 16. Lowry, O. H., A. L. Farr, N. J. Rosebrough, and R. J. RandallJ Biol Chem 193: 265, 1951. 17. Dion, A. S., and E. J. Herbst, Ann NY Acad Sci 171: 723, 1970. 18. Cohen, S., B. W. O'Malley, and M. Stasny, Science 170: 336, 1970. 19. Fillingame, R. H., and D. R. Morris, Biochemistry 12: 4479, 1973. 20. Beck, VV. T., R. A. Bellantone, and E. S. Cannellakis, Biochem Biophys Res Commun 48: 1649, 1972. Acknowledgments 21. Eloranta, T., and A. Raina, FEBS Lett 55: 22, 1975. We would like to thank Dr. John Heller for his kind 22. Hertelendy, F., H. Todd, G. T. Peake, L. J. Machlin, G. Johnston, and G. Pounds, Endocrinhelp in setting up the ODC assay. We are also indebted ology 89: 1256, 1971. to Dr. Zoe N. Canellakis and T. C. Theoharides for the use of their ODC antibody, to Dr. Stephen W. 23. Zor, U., Y. Koch, S. A. Lamprecht, J. Auslier, and H. R. Lindner,7 Endocrinol 58: 525, 1973. Spaulding for his helpful comments, and to Ms. 24. Levine, J. J., D. J. Jones, A. B. Learning, and P. Martha Jansen for excellent technical assistance. Raskin, Clin Res 24: 30A, 1976 (Abstract). 25. Dumont, J. E., Vitam Horm 29: 287, 1971. References 26. Spaulding, S. W., and G. N. Burrow, Endocrinology 96: 1018, 1975. 1. Raina, A., and J. Janne, Med Biol 53: 121, 1975. 27. Inoue, H., and A. Mizutani, Anal Biochem 56: 2. Janne, J., C. W. Bardin, and S. T. Jacob, Biochem408, 1973. istry 14: 3589, 1975. 28. Janne, J., Ada Physiol Scand Suppl 71: 300, 1967. 3. Fuchs, E., and C. M. Fuchs, FEBS Lett 19: 29. Adiga, P. R., P. V. N. Murthy, and J. M. McKenzie, 159, 1971. Biochemistry 10: 702, 1971. 4. Williams-Ashman, H. C , J. Janne, G. L. Coppoc, 30. Begg, D. J., and H. N. Munro, Nature 207: 483, M. E. Geroch, and A. Schenone, Adv Enzyme 1965. Regul 10: 225, 1971. 31. Lecocq, R. E., and J. E. Dumont, Biochem J 104: 13A, 1967. 5. Holtta, E., and A. Raina, Ada Endocrinol (Kbh) 73: 794, 1973. 32. El-Khatib, S. M., J. Haldar, and J. L. Starr, Proc 6. Levine, J. H., W. E. Nicholson, A. Peytremann, and Soc Exp Biol Med 143: 869, 1973. D. N. Orth, Endocrinology 97: 136, 1975. 33. Zor, U., T. Kaneko, I. P. Lowe, G. Bloom, and 7. Richman, R., C. Dobbins, S. Voina, L. UnderJ. B. FieldJ Biol Chem 244: 5189, 1969. wood, D. Mahafee, H. J. Gitelman, J. Van Wyk, 34. Theoharides, T. C , and Z. N. Canellakis,; Biol and R. L. N e y J Clin Invest 52: 2007, 1973. Chem 251: 1781, 1976. 8. Oka, T., and J. W. Perry, J Biol Chem 251: 1738, 35. Knopp, J., V. Stole, and W. Tong, ; Biol Chem 1976. 245: 4403, 1970. 9. Zusman, D. R., and G. N. Burrow, Endocrinology 36. Pavlovic-Hournac, M., L. Rappaport, and J. Nunes, 97: 1089, 1975. Endocrinology 89: 1477, 1971. 10. Richman, R., S. Park, M. Akbar, S. Yu, and G. Burke, 37. Sherwin, J. R., and W. Tong, Biochim Biophys Endocrinology 96: 1403, 1975. Ada 425: 502, 1976.
appeared to precipitate protein as it inactivated ODC, suggesting that TSH stimulation of ODC activity did in fact represent induced synthesis of enzyme protein. TSH has several immediate effects on the thyroid such as the stimulation of adenylate cyclase and the release of thyroid hormones (25,34). Continued TSH stimulation results in additional effects including enhanced uptake of iodide and thyroglobulin synthesis (35,36). These delayed effects appear to differ from the immediate ones in that they require synthesis of protein and RNA (37). Polyamines might play a role in modulating this latter phase of TSH stimulation in the thyroid.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
T
HE POLYAMINES spermidine and spermine, and their biosynthetic precursor, putrescine, are widely distributed in biological systems and have been demonstrated to accumulate in rapidly growing tissue concomitantly with an increase in nucleic acids (1). Polyamines have a number of well-characterized effects on nucleic acid metabolism and function, protein synthesis, and cell membranes (1-3). The rate-limiting step in polyamine biosynthesis, is probably the formation of putrescine from ornithine which is catalyzed by the enzyme ornitliine decarboxylase (ODC) (EC 4.1.1.17) (4). ODC activity is increased in a variety of endocrine target organs under hormonal stimulation (1,5-8). TSH has also been demonstrated to increase ODC activity in the thyroid in vivo (9-12). TSH stimulation of ODC activity is mimicked by the administration of dibutyryl cAMP, but it is difficult to separate direct Received September 2, 1976. Supported by USPHS Grant #AM 17060. * Presented to the Faculty of the Yale University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Medicine. f Present address: Toronto General Hospital, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
School of Medicine, 333 Cedar
Street,
cubation with actinomycin D (10 fxg/m\) or puromycin (0.2 HIM). Putrescine concentrations in rat thyroids rose to three times basal levels after 6 h of incubation with TSH and MIX; no significant elevation in spermidine or spermine was observed after up to 7 h incubation. The increase in tissue putrescine preceded a rise in [3H]uridine incorporation into acid-soluble material that occurred at 7 h. The results suggest that stimulation of thyroid ODC activity by TSH is mediated by cyclic AMP; the data further are consistent with a role for polyamines in the control of RNA synthesis in the thyroid. (Endocrinology 101: 1088, 1977)
hormonal effects from more indirect ones (9). To obviate this problem, we have studied the factors involved in the regulation of thyroid ODC activity in vitro and have related the increase in ODC activity to the time course for synthesis of polyamines and RNA. Materials and Methods Tissue incubation Male Sprague Dawley rats weighing 200 g were obtained from Charles River Laboratories. The thyroid glands were removed and randomized with 4-12 lobes placed in a beaker. The lobes were preincubated in 5 ml Krebs-Ringerbicarbonate buffer (KRB) for 15 min and then incubated for 1-7 h in KRB in a Dubnoff metabolic shaker at 37 C in 95% O 2 -5% CO2. Quantitative bacterial determinations of samples of the media were made at intervals up to 7 h and no significant contamination occurred. After incubation, the thyroids were homogenized in appropriate media (specified below) with a mechanically driven Teflon pestle in a PotterElvehjem tube. Homogenates were then centrifuged at 4 C and the supernatants assayed for ODC activity or polyamines. For ODC assay, 4 lobes representing 40-60 ing of tissue were homogenized in 1.4 ml of buffer composed of 0.25M sucrose, 0.5 mM EDTA and 10 mM 2-
1088
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
THYROID ODC ACTIVITY IN VITRO mercaptoethanol, and centrifuged at 100,000 x g for 30 min. For polyamine determinations, 8-10 lobes were homogenized in 0.4 ml of 0.4N perchloric acid (PCA) and centrifuged at 1,000 x g for 10 min. Enzyme assay ODC activity was assayed by measuring 14CO2 produced from DL-[l-14C]ornithine as described by Russell and Snyder (13) and modified by Chen et al. (14). The assay mixture contained 0.6 niM DL-ornithine, 0.1 mM pyridoxal-5-phosphate, 25 mM Tris-HCl buffer (pH 7.88 at 37 C), 13 /Amoles mercaptoethanol, and 100 /xl of supernatant representing 0.06-0.2 mg protein, in a final volume of 215 fxl. Within this range of protein concentration, ODC activity was linear with thyroid protein concentration. Ornithine was prepared for assay by acidification to O.lN HC1, heating on a rotary evaporator under vacuum at 54 C for 30 min to remove a volatile; contaminant which otherwise gave high assay blanks, and then diluted to O.OIN HC1. Unlabelled ornithine was added to give a final specific activity of 5.8 mCi/mmole. Decarboxylation was carried out in #2057 Falcon tubes with a polyethylene well containing 0.2 ml of monoethanolamine: ethylene glycol monoethyl ether (1:2, vol/vol). Decarboxylation was linear with time for up to 2 h of incubation (Fig. .1). Assay tubes in the present study were incubated for 90 min at 37 C in a shaker bath. The reaction was stopped by injection of 0.4 ml 2N citric acid through the needle. After agitation for an additional 60 min at 37 C, each well was removed, placed in 15 ml Aquasol (New England Nuclear) and isotope content was determined and counted on a Packard Tri-Carb or a Searle Mark II scintillation counter. The amount of 14CO2 released from substrate in tubes with no enzyme was subtracted from the value for all other samples. Picomoles of 14 CO2 released from the L-isomer were calculated (15). ODC activity was expressed as picomoles 14CO2 released per milligram of protein per hour of assay incubation. ODC activity was determined in triplicate in all experiments. Measurements were made on supernatants from a single beaker. All experiments were repeated and confirmed at least once, more often several times. Protein determination was by the method of Lowry et al. (16) using a least squares calculation for a BSA standard curve.
1089
ODC activity was measured between pH 6.6 and 9.9 and found to be maximal from 7.7 to 8.1. Maximal ODC activity was seen at 48 C. Kinetic studies reproducibly demonstrated a Km for ornithine of 0.12 mM, closely comparable to the ODC Km observed in other mamalian systems though at variance with the only other Km reported for the thyroid enzyme (10). Our samples were assayed at pH 7.88, 37 C with 0.6 mM DL-ornithine. Polyamine assay Tissue levels of putrescine, spermidine and spermine were determined by dansylation according to the method of Dion and Herbst (17). PCA supernatant (0.2 ml) was added to 0.4 ml of dansyl chloride (30 mg/ml acetone) and 18.5 mg anhydrous Na2CO.(. After 16 h in the dark at room temperature, 0.1 ml of proline (100 mg/ml H2O) was added for 30 min to convert excess dansyl chloride to dansyl proline. Dansyl derivatives were extracted into 0.5 ml benzene and 10 fx\ of the benzene layer was spotted onto 250 (iini Silica gel TLC plates which had been heated to 110 C for 1 h prior to spotting. Standard samples of all three amines were spotted on each plate as well. Plates were run twice with 2:3 ethylacetate/cyclohexane (vol/vol) in the dark, and sprayed immediately with 1:4 triethanolamine/ethylene glycol (vol/vol). Plates were dried in the dark, in vacua for 16 h and allowed to equilibrate to atmospheric pressure for one hour before scanning on a Turner III fluorimeter with TLC scanner. Blanks were scanned between the putrescine and spermidine spots and were subtracted from values for each sample. The area under each peak was proportional to the amount of dansyl amine at that spot; the area was proportional between 0-5 nmoles for putrescine and 0-20 nmoles for spermidine and spermine. The assay had a reproducibility of ±11.7% for putrescine, 14.2% for spermidine and 13.7% for spermine. Tissue levels were expressed as nmoles/g of tissue weighed after incubation. Medium was lyophilized, solubilized in 0.4N PCA and assayed as above. RNA synthesis For those experiments in which RNA synthesis was measured, thyroids were incubated as above with [3H]uridine (10 /xCi/ml) added to the incubation medium. The pellet remaining after
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1977 Vol 101 • No 4
SCHEINMAN AND BURROW
1090 o 1400 -
2
4
5
6
7
TIME (hours)
FiG. 1. The time course of ODC activity in thyroids incubated with 50 mU/ml TSH and 0.5 mM MIX ( ) and in controls (: -r). A typical experiment is shown. Each point represents the mean ± SEM of three determinations.
centrifugation of the PCA homogenate was washed 3 times in 5% trichloroacetic acid and resuspended in 0.2 ml I N NaOH; 20 (JL\ aliquots were counted in Aquasol. [3H]Uridine incorporation into RNA was expressed as cpm per g of tissue.
(MIX), a potent phosphodiesterase inhibitor, led to marked enhancement of this stimulation, with peak ODC activity occurring usually at 6 h (Fig. 1); stimulation of ODC activity by MIX alone did not significantly differ from stimulation by 50 mU/ml of TSH at 5 h of incubation. The rise in control ODC activity with time is unexplained but a similar rise was observed by Cohen et al. in chick oviducts in vitro (18). The stimulation of ODC activity by TSH was dose-related; 5 mU/ml of TSH significantly elevated ODC activity over control and maximal stimulation was seen with 25 mU/ml (Fig. 2). All subsequent incubations with TSH were done at a concentration of 50 mU/ml. Incubation of thyroids with dibutyryl cyclic AMP (DbcAMP) resulted in doserelated stimulation of ODC activity up to a concentration of 2 mM. Incubation with butyrate alone had no stimulatory effect on ODC activity. There was no stimulation over control at 5 mM DbcAMP, as confirmed by several repetitions (Fig. 3). The stimulation of ODC activity by 1 mM
§ 600 -
Materials TSH was kindly supplied by the National Institutes of Health (NIH-TSH-B7). 3-Isobutyl-lmethyl xanthine was purchased from the Aldrich Chemical Company. N6,O2'-Dibutyryl 3',5' cyclic monophosphoric acid, actinomycin D, puromycin, putrescine, spermidine, spermine, dansyl chloride, and iproniazid phosphate were purchased from Sigma Chemicals. [6H3]Uridine (24.2 Ci/nmol lot #908-053) and DL[l-14C]ornithine monohydrochloride (lot #822-176) were obtained from New England Nuclear.
Z 500 -
Results Stimulation of thyroid ODC in vitro Incubation of thyroids with 50 mU/ml of TSH resulted in a 3 to 5-fold stimulation of ODC activity at 5 h (P < .001). The addition of 0.5 mM 3-isobutyl-l-methyl xanthine
25 [TSH] (mU/ml)
50
FIG. 2. Effect of varying concentrations of TSH on thyroid ODC activity at 5 h of incubation. Each point represents the mean ± SEM of three determinations.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
1091
THYROID ODC ACTIVITY IN VITRO DbcAMP was not enhanced by the addition of MIX.
Effects of inhibitors of RNA and protein synthesis on thyroid ODC activity The addition of 0.2 mM puromycin or 10 /u,g/ml actinomycin D to the preincubation and incubation medium inhibited stimulation of ODC activity in response to TSH and MIX (Fig. 4). Puromycin completely prevented the ten-fold rise in ODC activity in stimulated thyroids and even blocked basal ODC activity of control thyroids. Studies were not done with DbcAMP. A concentration of 1% ethanol was required to solubilize the actinomycin D; this concentration of ethanol was found to eliminate the ODC activity in the incubated control and to attenuate the ODC response to TSH and MIX. Thus, while the data show inhibition of ODC activity by actinomycin D, it is impossible to dis-
PUROMYCIN
1000
800 'c o o
-
600 -
3
Z 400 c
5 Z 200 _ a CK
E Control
ACTINOMYCIN D
TSH + Methyl Xanthine
T
1
200 -
150 -
100 -
50 -
T
T Control
900 -
Puromycin + TSH + Methyl Xanthine
TSH + Methyl Xonthine
Actinomycin D + TSH + Methyl Xonthine
800 -
FIG. 4. Upper: Effect of 0.2 mM puromycin on the stimulation of thyroid ODC activity in vitro at 5 h of incubation as compared with stimulated thyroids without inhibitor and controls. Lower: Effect of 10 /xg/ml actinomycin D on the stimulation of ODC activity as compared with stimulated thyroids and controls. Stimulation was with 50 mU/ml TSH and 0.5 mM MIX. Each point represents the mean ± SEM of three determinations.
700 -
tinguish complete from incomplete inhibition. Stimulation of thyroid amines in vitro
.t
.5
TSH
i [Ob CAMP] (mM)
FlC. 3. Effect of varying concentrations of DbcAMP on thyroid ODC activity at 5 h of incubation. Each point represents the mean ± SEM of three determinations. Bar graph represents stimulation with 50 mU/ml TSH alone.
Thyroid lobes contained basal levels of about 300 nmoles/g of spermidine and spermine and one-tenth as much putrescine. Tissue levels of all three amines did not rise before 5 h of incubation with TSH and MIX. At 6 h, a three-fold increase in tissue putrescine levels was observed (P < .05), together with marked leakage of putrescine into the medium, when thyroids were in-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
tion of tritiated uridine into acid-insoluble material, rose significantly at 7 h.
600
500
Endo • 1977 Vol 101 . N o 4
SCHEINMAN AND BURROW
1092
Discussion
I I Control VP7A TSH + Methyl Xonthine
o 400 E
5 300
200
100
TISSUE
MEDIUM
FIG. 5. Effect of TSH (50 mU/ml) and MIX (0.5 DIM) on putrescine levels in thyroid tissue and incubation medium at 6 h of incubation. All beakers included 5 mM iproniazid.
This is the first report of stimulation of thyroid ODC in vitro. Our results match the earlier in vivo observations that a single injection of TSH in rats yields peak activity of thyroid ODC 4 to 6 h later (9,10,12). The 3-5-fold rise in vitro is comparable to the degree of stimulation observed in vivo in hypophysectomized rats, but significantly less than that found with intact rats (9). Dibutyryl cyclic AMP has been shown to stimulate ODC activity in the adrenals of hypophysectomized rats (7), cultured mammary tissue (8), and when given with aminophylline in thyroids of normal rats (9). Enzyme activity in livers of intact rats was stimulated by DbcAMP (5,20), but not consistently in livers of hypophysectomized rats (21). As DbcAMP is known to cause release of pituitary hormones (22), in vivo
cubated in TSH and MIX (Fig. 5). Spermidine and spermine were not significantly elevated at 6 h. Both control and stimulated samples in this experiment were incubated with 5 mM iproniazid, an amine oxidase inhibitor, to prevent polyamine degradation (19).
1,000 eoo -
Time course of stimulation ofODC activity, putrescine content, and RNA synthesis Figure 6 illustrates the time course for ODC activity, putrescine levels, and [3H]uridine incorporation into RNA in a representative incubation with TSH and MIX. ODC activity rose at 4 h, peaked at 5 h, and declined thereafter. Although these samples were incubated without iproniazid, putrescine levels were significantly elevated over controls at 6 h (P < .05), following the ODC peak. Spermidine and spermine levels were again not significantly elevated during this time period. Putrescine leakage into the medium closely paralleled tissue levels. RNA synthesis, as measured by incorpora-
= 40,000 20.000 Z
3
o: Z 2
4
5
INCUBATION TIME (hours)
FIG. 6. The time course of ODC activity, tissue putrescine levels and RNA synthesis in thyroids incubated with 50 mU/ml TSH and 0.5 mM MIX.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
THYROID ODC ACTIVITY IN VITRO studies must be interpreted carefully. Further, there are systems in which ODC stimulation occurs without changes in tissue cAMP levels (23). Insulin, which lowers cAMP levels in target tissues, stimulates ODC activity in liver (21,24) and in mammary explants (29). Thus, in some systems induction of ODC activity appears to involve a mechanism independent of cAMP. In vivo results have varied regarding the role of cyclic AMP in thyroidal ODC stimulation. Earlier work from our laboratory (9) demonstrated 12-fold stimulation of rat thyroid ODC with a combination of DbcAMP and aminophylline, although either compound alone was ineffective. However, Richman et al., using identical concentrations, observed only slight stimulation (10). In the present study we have demonstrated a dose-related stimulation of up to 8-fold in thyroid ODC activity in vitro with 2 mM DbcAMP. We have further shown that the ODC response to TSH is enhanced by a phosphodiesterase inhibitor. Stimulation of ODC activity thus resembles other effects of TSH on thyroid metabolism which appear to be mediated by cAMP (25), perhaps through the action of cAMP-dependent protein kinase (26). The elevation of putrescine content with ODC activity implies that the increase in ODC activity is physiologic rather than an assay artifact. Polyamine levels found in the thyroid in the present study are comparable to those found in other rat organs (27,28) if a correction is made for the thyroglobulin content of the thyroid. The accumulation of putrescine alone during the 7 h incubation in the present study is similar to the finding in regenerating rat liver that putrescine levels rise by 2-3-fold beginning 4 h after a partial hepatectomy, while spermidine rises by much less and not until 8 h (28). Matsuzaki and Suzuki reported elevation of putrescine and spermidine levels in the thyroid beginning on the second day of methylthiouracil treatment (12). The degree of putrescine leakage into the medium was unexpected. In the pres-
1093
ence of 5 mM iproniazid it is unlikely that this represents putrescine produced by the breakdown of oxidized spermidine or spermine (19). Putrescine is less well bound to cellular constituents than are spermidine and spermine and this might explain the elevation of putrescine in the medium in the absence of any change in spermidine or spermine content of the medium. We have found that ODC activity and putrescine levels rise prior to an increase in [ 3 H]uridine incorporation into RNA. Stimulation of [ 3 H]uridine incorporation has been observed in vitro after 1 to 4 h of incubation of the thyroid with TSH by several (29-31) though not all (32) investigators. In none of these studies was incubation carried out for 7 h; it is possible that our methods were too insensitive to detect a small earlier change in uridine uptake into RNA. Studies using metabolic inhibitors indicated that stimulation of ODC activity requires new synthesis of RNA (9,10). It also has been proposed that polyamines participate in the regulation of RNA synthesis (1), which could be supported by our findings. Conceivably, an undetectable early increase in RNA production might lead to polyamine accumulation which would be followed by greater augmentation of nucleic acid synthesis. ; Regulation of ODC activity in the thyroid seems to depend on synthesis of new protein as well as of RNA (9,10); this is suggested not only by inhibitor studies, but also by the time lag in ODC stimulation by TSH as compared to other effects of TSH (25,33). However, it is not known whether there is an actual induction of enzyme protein or whether there is modulation of the activity of pre-existing ODC. Puromycin has other effects and might not be specific for TSH induction of ODC. In experiments not reported here, an antibody to partially purified hepatic ODC (34) inactivated about 90% of the ODC activity in TSH-stimulated thyroid supernatants. Further, radio-iodinated antibody
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
1094
SCHEINMAN AND BURROW
Endo • 1977 Vol 101 • No 4
11. Matsuzaki, S., and M. Suzuki, Endocrinol Jap 21: 529, 1974. 12. Matsuzaki, S., and M. Suzuki, Endocrinol Jap 22: 339, 1975. 13. Russell, D. H., and S. H. Synder, Proc Natl. Acad Sci USA 60: 1420, 1968. 14. Chen, K., J. Heller, and E. S. Canellakis, Biochem Biophys Res Commun 68: 401, 1976. 15. Raina, A., and J. Janne, Ada Chem Scand 22: 2375, 1968. 16. Lowry, O. H., A. L. Farr, N. J. Rosebrough, and R. J. RandallJ Biol Chem 193: 265, 1951. 17. Dion, A. S., and E. J. Herbst, Ann NY Acad Sci 171: 723, 1970. 18. Cohen, S., B. W. O'Malley, and M. Stasny, Science 170: 336, 1970. 19. Fillingame, R. H., and D. R. Morris, Biochemistry 12: 4479, 1973. 20. Beck, VV. T., R. A. Bellantone, and E. S. Cannellakis, Biochem Biophys Res Commun 48: 1649, 1972. Acknowledgments 21. Eloranta, T., and A. Raina, FEBS Lett 55: 22, 1975. We would like to thank Dr. John Heller for his kind 22. Hertelendy, F., H. Todd, G. T. Peake, L. J. Machlin, G. Johnston, and G. Pounds, Endocrinhelp in setting up the ODC assay. We are also indebted ology 89: 1256, 1971. to Dr. Zoe N. Canellakis and T. C. Theoharides for the use of their ODC antibody, to Dr. Stephen W. 23. Zor, U., Y. Koch, S. A. Lamprecht, J. Auslier, and H. R. Lindner,7 Endocrinol 58: 525, 1973. Spaulding for his helpful comments, and to Ms. 24. Levine, J. J., D. J. Jones, A. B. Learning, and P. Martha Jansen for excellent technical assistance. Raskin, Clin Res 24: 30A, 1976 (Abstract). 25. Dumont, J. E., Vitam Horm 29: 287, 1971. References 26. Spaulding, S. W., and G. N. Burrow, Endocrinology 96: 1018, 1975. 1. Raina, A., and J. Janne, Med Biol 53: 121, 1975. 27. Inoue, H., and A. Mizutani, Anal Biochem 56: 2. Janne, J., C. W. Bardin, and S. T. Jacob, Biochem408, 1973. istry 14: 3589, 1975. 28. Janne, J., Ada Physiol Scand Suppl 71: 300, 1967. 3. Fuchs, E., and C. M. Fuchs, FEBS Lett 19: 29. Adiga, P. R., P. V. N. Murthy, and J. M. McKenzie, 159, 1971. Biochemistry 10: 702, 1971. 4. Williams-Ashman, H. C , J. Janne, G. L. Coppoc, 30. Begg, D. J., and H. N. Munro, Nature 207: 483, M. E. Geroch, and A. Schenone, Adv Enzyme 1965. Regul 10: 225, 1971. 31. Lecocq, R. E., and J. E. Dumont, Biochem J 104: 13A, 1967. 5. Holtta, E., and A. Raina, Ada Endocrinol (Kbh) 73: 794, 1973. 32. El-Khatib, S. M., J. Haldar, and J. L. Starr, Proc 6. Levine, J. H., W. E. Nicholson, A. Peytremann, and Soc Exp Biol Med 143: 869, 1973. D. N. Orth, Endocrinology 97: 136, 1975. 33. Zor, U., T. Kaneko, I. P. Lowe, G. Bloom, and 7. Richman, R., C. Dobbins, S. Voina, L. UnderJ. B. FieldJ Biol Chem 244: 5189, 1969. wood, D. Mahafee, H. J. Gitelman, J. Van Wyk, 34. Theoharides, T. C , and Z. N. Canellakis,; Biol and R. L. N e y J Clin Invest 52: 2007, 1973. Chem 251: 1781, 1976. 8. Oka, T., and J. W. Perry, J Biol Chem 251: 1738, 35. Knopp, J., V. Stole, and W. Tong, ; Biol Chem 1976. 245: 4403, 1970. 9. Zusman, D. R., and G. N. Burrow, Endocrinology 36. Pavlovic-Hournac, M., L. Rappaport, and J. Nunes, 97: 1089, 1975. Endocrinology 89: 1477, 1971. 10. Richman, R., S. Park, M. Akbar, S. Yu, and G. Burke, 37. Sherwin, J. R., and W. Tong, Biochim Biophys Endocrinology 96: 1403, 1975. Ada 425: 502, 1976.
appeared to precipitate protein as it inactivated ODC, suggesting that TSH stimulation of ODC activity did in fact represent induced synthesis of enzyme protein. TSH has several immediate effects on the thyroid such as the stimulation of adenylate cyclase and the release of thyroid hormones (25,34). Continued TSH stimulation results in additional effects including enhanced uptake of iodide and thyroglobulin synthesis (35,36). These delayed effects appear to differ from the immediate ones in that they require synthesis of protein and RNA (37). Polyamines might play a role in modulating this latter phase of TSH stimulation in the thyroid.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:36 For personal use only. No other uses without permission. . All rights reserved.
