Regulation of Thyroid Ornithine Decarboxylase (ODC) by Thyrotropin. I. The Rat1 R. RICHMAN, S. PARK, M. AKBAR, S. YU, AND G. BURKE 2 The Division of Endocrinology, Department of Medicine, Cook County Hospital, and Department of Pediatrics, Abraham Lincoln School of Medicine, Chicago, Illinois 60612 ABMRACT. We studied the effects of TSH on rat thyroid ornithine decarboxylase (ODC) activity. After 1 day of goitrogen treatment, there was an abrupt fall in serum triiodothyronine (T3), a rise in circulating TSH, and a dramatic increase in thyroid ODC activity. Despite the continued rise in TSH and progressive increase in thyroid gland size with further treatment, thyroid ODC activity declined on the third day and remained at submaximal levels. Thyroid ODC activity was also stimulated in a dose-related manner by administration of exogenous TSH. Little TSH effect was noted before 3 h. Maximal ODC activity occurred between 4 and 5 h. The TSH stimulation of ODC could be inhibited by pretreatment with actinomycin D or cycloheximide, suggesting that the increase in ODC activity requires new RNA and protein synthesis. Although pretreatment with agents that alter microtubule structure (e.g., colchicine and vinblastine) prevent stimulation of ODC activity by TSH, additional data suggest, but do not confirm, that hormone secretion and ODC activation may be dissociable. Further studies were undertaken to determine
T
HE polyamines are widely distributed throughout nature (1,2) and their function appears to be related to RNA metabolism (1-4). The initial step in their synthesis from ornithine is catalyzed by the enzyme ornithine decarboxylase (Lornithine carboxylase, EC 4.1.1.17). Its activity has previously been demonstrated to be hormonally regulated in a variety of tissues, including the liver (5,6) and kidney (7). It also appears that the pituitary trophic hormones may regulate ODC3 activity in Received November 8, 1974. This research was supported by Grant AM 17561 from the NIAMDD, USPHS and an Institutional Grant ACS IN 9N #25 to the Abraham Lincoln School of Medicine from the American Cancer Society. 1 Presented in part to the 50th meeting of the American Thyroid Association, St. Louis, Missouri, September 1974. 2 Requests for reprints should be addressed to G. Burke. 3 Abbreviations used in the text; ACTH = adrenocorticotropin; cAMP = cyclic adenosine 3',5'-
whether cyclic AMP (cAMP) or prostaglandins played any role in the regulation of thyroidal ODC activity. Dibutyryl cAMP, alone, or together with aminophylline, did not stimulate thyroidal ODC activity in dosages which concomitantly stimulated adrenal enzyme activity. Likewise, prostaglandin E2 (PGE2) did not stimulate thyroidal ODC activity, but did stimulate adrenal enzyme activity in a dose-related manner. However, pre-treatment of rats with inhibitors of prostaglandin synthesis prevented the activation of thyroidal ODC by TSH. One inhibitor, indomethacin, attenuated the TSH stimulation of enzyme activity in a dose-related manner. Indomethacin pretreatment also resulted in approximately a 10-fold decrease in thyroidal prostaglandin levels. Exogenous PGE2, in dosages as high as 500 pig, did not overcome the inhibitory effect of indomethacin on ODC activation. Although the precise role for endogenous prostaglandins remains to be defined, it does appear that a reduction in thyroidal prostaglandins prevents activation of the enzyme by TSH. (Endocrinology 96: 1403, 1975)
their respective target endocrine glands. This has been confirmed for both the adrenal gland (7,8) and ovary (9). We, therefore, initiated studies to determine if ODC was present in the thyroid gland. If present and regulated by TSH, then ODC activity might serve as a biological marker for the growth-promoting effects of TSH. Since many of these trophic effects are either mimicked or mediated by cyclic AMP (cAMP, 10) and/or prostaglandins (11), it was of interest to determine whether these compounds played any role in the regulation of thyroidal ODC activity. monophosphate; dibutyryl cAMP = 6N-2'-0-dibutyryl cyclic adenosine 3',5'-monophosphate; EDTA = ethylenediaminetetraacetic acid; NIAMDD = National Institute of Arthritis, Metabolism and Digestive Diseases; ODC = ornithine decarboxylase; PGEj = prostaglandin Ej; PGE2 = prostaglandin E2; T3 = 3,5,3'-triiodothyronine; T4 = L-thyroxine; TSH = thyroid-stimulating hormone; TYA = 5,8,11,14-eicosatetraynoic acid.
1403
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RICHMAN ETAL.
1404
Materials and Methods Animals. Male Holtzman rats, weighing 180200 gin, were maintained on standard laboratory chow and tap water. In some experiments, a goitrogen, 4-hydroxy-6-methyl-2-thiopyrimidine (100 mg/liter) (12), was added to their drinking water. To control for fluctuations in blood levels of goitrogen due to variations in drinking patterns, methimazole (1 mg) was also administered subcutaneously at 9:00 AM and 4:00 PM in one experiment. Five animals were assigned to each treatment group. TSH (Thytropar®, Armour Pharmaceutical, Kankakee, 111.) was usually administered between 8 and 10 AM and the animals sacrificed 4 h later. Other pharmacological agents were injected at the times specified. Control animals were usually untreated since basal enzyme activity was unaffected by injection of the vehicle. At the time of sacrifice, the rats were anesthetized with ether and exsanguinated by cardiac puncture. The serum was removed and frozen at —10 C for hormone measurement. Tissue preparation. For each treatment group, thyroid glands from 5 rats were pooled and weighed. They were homogenized with a polytron (Brinkman Instruments, Inc., Westbury, New York) at a concentration of 48 mg/ml in 0.05M Na-K phosphate buffer, pH 7.2 containing 10 mM tetrasodium EDTA and 5 mM dithiothreitol, at 4 C. The homogenates were centrifuged at 20,000 x g for 20 min at 4 C. The supernatant fractions were immediately used for enzyme assay. Enzyme assay. ODC was assayed by a semimicro modification of the method of Russell and Snyder (13). The reaction mixture contained 0.3 /xCi of DL[1 - 14C]ornithine hydrochloride, 58 or 61 mCi/mmol (Amersham-Searle Corp., Arlington Heights, Illinois), 0.1 fimol pyridoxal phosphate, 0.1-0.4 ml of the 20,000 x g thyroid supernatant fraction and buffer (described above) to make a final volume of 0.5 ml. Activity was usually assayed in triplicate. In early experiments, enzyme activity was assayed at 3 different enzyme concentrations (0.1, 0.2, and 0.4 ml of the 20,000 x g supernatant fraction). In later experiments, activity was assayed at one concentration, usually 0.3 ml. The assay was carried out in 15 ml conical centrifuge tubes, sealed with a rubber stopper,
Endo • 1975 Vol 96 • No 6
from which a polyethylene center well (Kontes Glass Co., Vineland, N.J.) was suspended. After incubation at 37 C for 30 min in a Dubnoff metabolic shaking incubator, 0.5 ml of 2M citric acid was injected into the reaction mixture, through the rubber stopper, to stop enzyme activity. 0.2 ml hydroxide of hyamine 10-X (Packard Instrument Co., Downers Grove, 111.) was injected into the center well to trap liberated I4 CO2. The tubes were agitated at 37 C for an additional 30 min. Then each well was removed and placed into a scintillation vial containing 10 ml of scintillation cocktail, consisting of 0.40 ml Permafluor® 25 x concentrated liquid scintillator (Packard Instrument Co., Downers Grove, 111.) and 9.60 ml toluene. Samples were counted in a Searle Mark® II liquid scintillation system. Counting efficiency (90%) was determined by the external standard ratio method. Results are expressed as picomoles of I4 CO 2 liberated from 14 DL[1 — C]ornithine per g wet weight tissue per 30-min incubation. Some of the calculations were performed using a 370 IBM computer, model #145. The amount of 14CO2 liberated from tubes containing no enzyme was subtracted from the value for all other samples. To decrease nonenzymatic liberation of I4CO2, 250 juCi of DL[1 - 14C]ornithine were diluted in 25 ml of 0.1 N HC1, as outlined by WilliamsAshman (14). The ornithine was dried over nitrogen and resuspended in 25 ml of buffer. 5 ml aliquots were stored at —IOC. With this procedure, the no-enzyme blank was reduced to approximately 200 cpm. Putrescine assay. To insure that the decarboxylation reaction catalyzed by the 20,000 X g supernatant fraction resulted in polyamine synthesis, putrescine formation was measured in one experiment. It was assayed according to the method of Raina (15) with DL[5 - 14C]ornithine hydrochloride, 8.19 mCi/mmol (New England Nuclear Co., Boston, Massachusetts) as the substrate. Protein determination. Total protein was measured according to the method of Lowry (16). Hormone determinations. Thyroxine (T4) was measured by a modification of the method of Murphy-Pattee (17), using reagents from Mallinckrodt Chemical Works, St. Louis, Mo. cAMP was measured by radioimmunoassay (18), using
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1405
THYROIDAL ORNITHINE DECARBOXYLASE a kit purchased from Schwarz/Mann. Rat TSH was measured by radioimmunoassay (19), using reagents supplied by the NIAMDD Rat Pituitary Hormone Distribution Program. Serum triiodothyronine (T3) 20) and thyroidal prostaglandin E (PGEJ levels (21) were determined by radioimmunoassay. Antibody to PGE was a gift from Dr. Gordon D. Niswender, Colorado State University, Fort Collins, Colorado. Materials TSH (Thytropar®) and ACTH (Acthar®) were obtained from Armour Pharmaceutical. In addition, a crude preparation of TSH (1 mg = 4 units) was a gift from Dr. J. Fisher (Armour Pharmaceutical). Potency was estimated both by a 3 point dose-response in the McKenzie mouse bioassay (22) and the rat ornithine decarboxylase assay with commercial Thytropar® as the standard. Dibutyryl cAMP and cAMP were purchased from Schwarz/Mann. Prostaglandin E2 (PGE2) was a gift from Dr. John Pike, Upjohn Co., Kalamazoo, Michigan. Methimazole was a gift from Dr. N. J. Hosley, Eli Lilly and Co., Indianapolis, Indiana. 5,8,11,14-eicosatetraynoic acid (TYA) was a gift from Dr. W. E. Scott, Hoffmann-LaRoche, Inc., Nutley, N.J. Indomethacin was purchased from Sigma Chemical Co., St. Louis, Mo., and a purified preparation was a gift from Dr. C. A. Stone, Merck, Sharp and Dohme, West Point, Pa. Naproxen was a gift from Syntex Laboratories, Palo Alto, California, and butazolidin from CIBA-Geigy Corp., Summit, N.J. T3, T4 and cycloheximide were purchased from Sigma Chemical Co., St. Louis, Mo. Human growth hormone (HS1498) was a gift of the Endocrine Study Section, NIAMDD. Colchicine, Vinblastine sulfate (Velban®), and aminophylline were purchased from Eli Lilly and Co. Human chorionic gonadotropin (A.P.L.®) was purchased from Ayerst Laboratories, New York, N.Y., isoproterenol (Isuprel®), from Winthrop Laboratories, New York, N.Y., actinomycin D (Cosmegen®) from Merck and Co., Inc. and hydrocortisone (SoluCorteP) from Upjohn Co.
Results
Characterization of the enzyme. Preliminary studies demonstrated that ODC activity was proportional to the quantity of 20,000 x g supernatant fraction assayed be-
tween 2.4 and 19.2 mg thyroid (0.05-0.4 ml). Enzyme activity was measured from pH 6.0 to 8.5 and maximal activity appeared to plateau between pH 6.9 and 7.9. For all subsequent studies, pH 7.2 was chosen to conform with our previous studies in the adrenal gland (7). The amount of 14CO2 liberated from DL[1 — 14C]ornithine increased in a linear fashion for at least 30 min of incubation. The concentration of unlabeled substrate, L-ornithine, was varied in the reaction mixture, and the data plotted according to the method of Lineweaver and Burk (23). Vmax was found to be 48.1 pmol/0.3 ml/30 min and the Km = 44.4 nM (Fig. 1). Studies on the effect of temperature revealed maximal enzyme activity occurring at 51 C. All other studies reported, however, were carried out at physiological conditions, i.e., temperature 37 C. The effect of exogenous TSH. Groups of rats were sacrificed at varying time inter-
0.05
O.I
0.15
I
[L- ORNITHINE]
0.2
X 10 - 6
FIG. 1. Relationship between substrate concentration and reaction velocity. Increasing amounts of unlabeled substrate (L-ornithine) were added to the reaction mixture and the reaction velocity, amount of CO2 released730 min., determined. Substrate concentration (M) and reaction velocity were plotted as reciprocals according to the method of Lineweaver and Burk (23).
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1406
Endo • 1975 Vol 96 • No 6
RICHMAN ET AL.
vals after the intraperitoneal administration of 4 U of TSH. Little effect on enzyme activity was apparent before three hours (Fig. 2). Maximal ODC activity occurred between 4 and 5 h after TSH administration. In a subsequent series of experiments, varying doses of TSH were injected and the thyroid glands removed 4 h later. No increase in enzyme activity was seen with 0.25 U TSH. A linear log-dose-response curve was obtained between 1 and 8 U of TSH (Fig. 3). To determine if an inhibitor of enzyme activity was present in thyroid glands from untreated animals, the 20,000 x g supernatant fraction from control and TSHstimulated glands were mixed. The resulting enzyme activity of the mixture was comparable to that found in TSHstimulated glands alone (unpublished data). This excluded the possibility that the apparent increase in ODC activity was solely due to TSH decreasing the activity of an enzyme inhibitor. A variety of other hormones including ACTH, human growth hormone, T3, hydrocortisone, human chorionic gonadotropin and isoproterenol were administered. None of the other hormones tested increased thyroid enzyme activity 4 h after injection.
2 3 4 5 6 7 8 HOURS AFTER TSH ADMINISTRATION FIG. 2. The time course of thyroid ODC activity in rats given 4 U of TSH intraperitoneally. Groups of 5 rats each were sacrificed at various times after the administration of TSH. Enzyme activity is expressed as pmoles of 14CO2 liberated per g of thyroid tissue. A representative experiment is shown with each point the mean of 3 determinations.
2000-
0.25
1.0
2.0
8.0
UNITS OF TSH
FIG. 3. Effect of varying doses of exogenous TSH on thyroidal ODC activity. Groups of 5 rats each were given varying doses of TSH intraperitoneally. The animals were sacrificed 4 h later and the thyroid glands removed for determination of ODC activity. Each point represents the mean ± SEM for 2-5 experiments with the number shown in parenthesis.
Also, TSH did not stimulate ODC activity in liver, kidney or adrenal glands. In order to demonstrate the subcellular location of ODC activity in thyroid, crude subcellular fractions were prepared by differential centrifugation. 0.25M sucrose was used to homogenize the tissue instead of buffer. Each pellet was washed a single time with sucrose and then gently resuspended. Enzyme activity was determined for each fraction as described for the 20,000 x g supernatant fraction but was expressed as cpm/mg protein. In addition, each subcellular fraction was assayed in the presence of putrescine (1 mg/reaction tube), a known inhibitor of ODC activity in vitro (14). As seen in Fig. 4, 14CO2 was liberated by several subcellular fractions. However, the 100,000 g supernatant fraction was the only fraction in which the rate of 14CO2 evolution was increased by administration of TSH in vivo and inhibited by the presence of putrescine in vitro. We, therefore, concluded that most, if not all, ODC activity was present in the cytosol. The 14CO2 liberated by the other subcellular fractions probably resulted from biochemical reactions in metabolic path-
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THYROIDAL ORNITHINE DECARBOXYLASE
8000-
R
1407 NO TREATMENT NO PUTRESCINE
7000-
NO TREATMENT PUTRESCINE PRESENT
6000-
TSH TREATED NO PUTRESCINE
5000-
TSH TREATED PUTRESCINE PRESENT
z >o
4000300020001000WHOLE H0M06ENATE
600 g PELLET
5,500 g PELLET
20,000 g PELLET
100,000 g SUPERNATE
SUBCELLULAR FRACTIONS FIG. 4. Subcellular localization of ODC activity. Enzyme activity in various subcellular fractions was compared in control and TSH-stimulated glands. Glands were homogenized in 0.25M sucrose at a concentration of 48 mg/cc. Subcellular fractions were prepared by differential centrifugation. Each pellet was washed a single time and then gently resuspended. 14CO2 release was determined for each fraction, as described in Materials and Methods for the 20,000 x g supernatant fraction. In addition, each fraction was assayed in the presence of putrescine (1 mg/tube), an in vitro inhibitor of the ODC reaction. Enzyme activity is expressed as cpm/mg protein. Since 14CO2 was not released by either 100,000 x ^•pfelket, the data is not shown.
ways other than polyamine synthesis in which ornithine is converted to another compound prior to decarboxylation. In another experiment, the percent of ornithine hydrochloride decarboxylated, as measured by both 14CO2 release from DL[1 — 14C] ornithine and 14C — putrescine formation from Dl_[5 — 14C]ornithine, by 20,000 x g supernatant fractions from control and TSH-stimulated glands was compared. Less than 0.02% of both ornithine substrates was decarboxylated by the supernatant fractions from control glands. The supernatant fractions from the TSHtreated glands converted 0.58% of the [5 — 14C]ornithine to [14C]putrescine and 0.89% of the [1 - 14C]ornithine to 14CO2.
Since the two substrates differed in their specific activities, no attempt was made to demonstrate stoichiometry. In any event, these data indicate that the release of 14CO2 by the 20,000 x g thyroid supernatant fraction correlates well with putrescine formation. Effect of goitrogen treatment on thyroid ODC activity. In this series of experiments, rats were given a goitrogen, 4-hydroxy-6-methyl-2-thiopyrimidine (100 mg/liter) in their drinking water for 1-6 days. On the first day of treatment, there was an abrupt fall in the serum concentration of T3 (Fig. 5). As expected, there was a secondary rise in circulating TSH. There
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Endo ( 1975 Vol96< No 6
RICHMAN ET AL.
1408
700THYROID WEIGHT -100 -800 E
g -120
Z> L
600 w I
8 0 ^ -40
1
2
3 4 DAYS ON 60ITR0GEN
-400"-200
5
FIG. 5. Effect of goitrogen on serum TSH, T3, thyroidal ODC and thyroid gland weight. Groups of animals were given a goitrogen in their drinking water for 0-6 days. All groups of animals were exsanguinated and the thyroid glands excised between 12:30 PM and 2:30 PM on the same afternoon. The glands were weighed and assayed for ODC activity in the usual manner. The serum was removed and stored at —IOC for hormone determination. Gland weights represent the pooled weight (mg) of 10 glands. ODC activity is expressed as the mean 14CO2 pmol/g tissue for 2 representative experiments. T3 and TSH were determined for each serum specimen and expressed as the mean for the group.
was a small, but significant, increase in ODC activity after only 1 day on goitrogen (Fig. 5). On the second day, a dramatic rise in enzyme activity was detected. Despite the continued rise in serum TSH and a progressive increase in thyroid gland size with further treatment, ODC activity declined on the third day and remained at submaximal levels. T 3 levels decreased until the 3rd day of treatment and then plateaued. The effect of inhibitors ofRNA and protein synthesis. The intraperitoneal administration of 0.9 mg of actinomycin D or 10 mg of cycloheximide, 15 min before TSH, totally inhibited the expected increase in thyroidal ODC activity 4 h later (data not shown). Effect of microtubule-active agents. It has been previously demonstrated that agents which alter microtubule structure inhibit
thyroid hormone secretion (24). Two such agents, colchicine and vinblastine, were administered subcutaneously the evening before the experiment and intraperitoneally, 4 h before sacrifice. TSH was also given at this time in a second injection site. Both agents markedly inhibited the TSH stimulation of ODC activity, lowered basal serum T4 levels, and blunted the increase in serum T4 following TSH (Table 1). Effect of dibutyryl cAMP administration. Fifty mg of dibutyryl cAMP were given by the intraperitoneal route. Animals were sacrificed at varying time intervals after injection. Both thyroid lobes were removed from all animals and one adrenal gland from each animal sacrified 4 h after dibutyryl cAMP injection. No increase in thyroid ODC was detected (data not shown). As previously reported (7), enzyme activity was elevated at 4 h in the adrenal glands. In an attempt to demonstrate an
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THYROIDAL ORNITHINE DECARBOXYLASE
1409
effect of dibutyryl cAMP which might have TABLE 1. Effect of pretreatment with colcbicine or on the TSH stimulation of thyroidal ODC been masked by its rapid destruction, 10 vinblastine activity and hormone secretion mg of the phosphodiesterase inhibitor Serum T4* ODC activity aminophylline and 30 mg of dibutyryl jig/100 ml pmol 14CO2/g cAMP were administered simultaneously, - TSH + TSH - TSH + TSH in separate intraperitoneal injection sites. Inhibitor** The animals was sacrificed at varying time None 3.7 ± 0.04 5.6 ± 0.1 48 1,968 intervals thereafter, and the thyroid and Colchicine 20 2.6 ± 0.2 1.9 ± 0.1 0 (0.2 mg) adrenal glands excised. The minimal in14 crease (to 125 pmol CO2/g/30 min) in Vinblastine 1.4 ± 0.2 32 0.4 ± 0.1 0 (lmg) ODC activity in the thyroid gland after 3 h appeared insignificant when compared to * Mean of 5 sera ± SEM were administered in the doses specified, subcutaneously the dramatic increase following TSH ad- 16**hInhibitors before and again intraperitoneally at the same time as 4 U of ministration. In contrast, there was a TSH. The animals were sacrificed 4 h later. marked increase in adrenal enzyme activity Effect of inhibitors of prostaglandin (1106 pmol 14CO2/g/30 min) at 3 h. synthesis. To further investigate any role Effect of PGE2 administration. PGE 2 was that endogenous prostaglandins might play administered intraperitoneally in several in mediating the effect of TSH on thyroid doses up to 500 fig/rat and at varying time ODC, several inhibitors of prostaglandin intervals (through 5 h) prior to the meas- synthesis (25) were administered prior to urement of thyroid and adrenal ODC TSH. Changes in basal levels of ODC were activity. Adrenal ODC activity peaked 3 h not detected when the inhibitors were after 100 /*g PGE 2 (397 pmol 14CO2/g/30 injected alone. As shown in Tables 2 and 3, min) and was stimulated in a dose-related indomethacin and naproxen clearly inhibmanner (data not shown). No effect on ited the TSH stimulation of ODC. The thyroid enzyme activity was apparent. Al- effects of the other inhibitors, at the dosthough prostaglandins modify some effects ages employed, are not as definitive and of TSH when they are administered to- the changes might be due to biological gether (11), no consistent effect on TSH variation. Indomethacin attenuated the stimulated enzyme activity was seen when TSH effect on enzyme activity and serum the PGE 2 was administered simultaneous- T4 levels in a dose-related fashion. Inly, 15 min, 1 h, or 2 h prior to TSH domethacin, at a dose of 1 mg, appeared to inhibit the TSH stimulation of ODC activadministration. TABLE 2. Effects of inhibitors of prostaglandin synthesis on the TSH stimulation of ODC activity and hormone secretion Experiment 2
Experiment 1
Inhibitor**
-TSH
+ TSH
None Aspirin Naproxen Butazolidin TYA***
38 34 24 49 13
3,188 1,970 311 1,914 1,334
ODC activity pmol "COj/g
Serum T4* /zg/100 ml
ODC activity pmol 14CO2/g
-TSH 4.1 4.0 2.9 4.2 3.4
± 0.1 ±0.1 ± 0.2 ± 0.04 ±0.1
+ TSH 7.6 5.5 4.9 4.9 5.6
±0.1 ±0.1 ±0.1 ±0.1 ±0.1
Serum T4* /ig/100 ml
-TSH
+ TSH
-TSH
44 14 0 100
3,691 1,687 65 2,149
4.6: t 0.1 4.0: tO.l 2.6: t 0.1 4.5: t 0.1
+ TSH 7.0 6.3 4.5 5.8
± ± ± ±
0.1 0.2 0.3 0.1
* Mean of 5 sera ± SEM. ** 10 mg of each inhibitor of prostaglandin synthesis were administered subcutaneously 18 h before an intraperitoneal injection of 4 U of TSH. *** TYA = 5, 8, 11, 14-eicosatetraynoic acid.
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Endo i 1975 Vol 96 < No 6
RICHMAN ET AL.
1410
TSH unequivocally stimulated an increase in thyroid ODC activity. Although we have not attempted to define the precise role of *Dose (mg) of ODC activity T4** the enzyme in regulation of thyroid funcTSH indomethacin pmol 14CO2/g Atg/lOO ml tion, the results of our goitrogen treatment suggest that enzyme activity is regulated — — 20 3.6 ± 0.4 + — 1,495 7.7 ± 0.4 by endogenous TSH. Enzyme activity also 1,340 + 0.25 8.8 ± 0.5 increases proportionally to pharmacological 401 + 1.00 8.9 ± 0.5 doses of exogenous TSH. The lag phase of 68 + 2.50 4.9 ± 0.4 11 + 5.00 5.2 ± 0.4 several hours after the administration of TSH appears to have biological impor* Indomethacin was injected at the stated dose subtance. During the first 30 min after horcutaneously 22 h before sacrifice and repeated intraperitoneally 4 h before sacrifice. mone injection, biological effects, such as an ** Mean of 4 - 5 sera ± SEM. increase in endogenous cAMP in the thyroid (26), can be measured. Therefore ity, but not its effect on serum T4 (Table 3). this lag in the appearance of an increase in The inhibition of enzyme activity by 1.5 ODC activity is not simply due to delayed mg of indomethacin, could not be reversed absorption of the hormone from the injecby increasing the dose of TSH adminis- tion site. Since there is no increase in ODC tered (data not shown). Indomethacin pre- activity when actinomycin D or cyclotreatment significantly reduced endogen- heximide is injected 15 min before TSH, ous prostaglandin levels in control and the lag period may be the time required for TSH-stimulated thyroid glands (Table 4). synthesis of new enzyme. The timing of the indomethacin injection Studies with agents promoting dewas very critical. The inhibitory effect was polymerization (colchicine) or aggregation demonstrable if indomethacin was given 1 (vinblastine) of microtubules demonstrated h or more prior to TSH administration. If, that they can inhibit not only TSH-induced however, indomethacin was given 1 h after hormone secretion (24) but also ODC TSH, no inhibition of enzyme activity was stimulation. This suggests that activation of detected. Indomethacin given at the same ODC in thyroid is dependent upon mitime as TSH, did not consistently inhibit hormonal activation of ODC. The increase crotubule integrity and is in some way in serum T4 levels was not clearly atten- related to the intracellular process involved uated when indomethacin was adminis- in hormone secretion. Yet, the observation tered an hour or less before TSH (data not that at least one dose (1.0 mg) of inshown). Exogenous PGE 2 , in high doses as domethacin inhibited the expected inhigh as 500 fig, given either 1 h before or crease in ODC activity, but not the serum 17 h after indomethacin, did not appear to TABLE 4. Effect of indomethacin treatment on influence the inhibitory effect of inthyroidal PGE, levels domethacin on the TSH stimulation of PGE t * enzyme activity. Indomethacin (10 fig/ml) (ng/mg thyroid) added to the reaction mixture, did not alter Time after TSH (4 U) + Indomethacin enzyme activity in vitro.
TABLE 3. Effect of increasing the dose of indomethacin on the TSH stimulation of thyroidal ODC activity and hormone secretion
(min)
- Indomethacin
(2.5 mg)
45 180
0.265 0.280 0.380
0.026 0.033 0.056
Discussion The results of these studies in the thyroid yield additional evidence that the pituitary trophic hormones regulate ODC activity in their target endocrine glands.
* Each value represents the mean for 2 thyroid glands except for the TSH 45 min + indomethacin which represents only a single gland.
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THYROIDAL ORNITHINE DECARBOXYLASE T4 elevation, suggests that hormone secretion and ODC activation may be dissociable. Additional studies are necessary to explore their relationship. It was previously suggested that cAMP may mediate the effect of ACTH on ODC activity in the adrenal gland (7). The data presented here suggest that this is unlikely to obtain for TSH and the thyroid. Whereas dibutyryl cAMP stimulated adrenal ODC activity, this cyclic nucleotide, even in the presence of aminophylline, did not substantially increase thyroid enzyme activity. The data also suggest that endogenous prostaglandins may be important in regulation of thyroidal ODC activity. Since exogenous PGE2 mimics most of the effects of TSH on the thyroid gland (11) and stimulates uterine ODC activity (27), we were concerned about the lack of a direct effect of exogenous PGE2 on thyroid ODC activity. This may have been secondary to either rapid inactivation in the lung, or other organs (28) of the administered PGE2 or to changes in the cardiovascular status of the animals (29). Nevertheless, concomitant studies in the adrenal gland revealed that PGE 2 administration unequivocally elevated adrenal ODC activity. Although this increase in adrenal enzyme activity may be secondary to the previously reported rise in ACTH following prostaglandin administration (30), Dazord et al. (31) recently reported the presence of plasma membrane receptors for PGEX and PGE2 in the adrenal gland. In any event, there is no doubt that PGE2 is biologically effective when injected into the peritoneum. Gallant and Brownie (32) were also unable to show a direct in vivo effect of PGE 2 on adrenal steroidogenesis. Prior treatment with indomethacin, a known inhibitor of prostaglandin synthesis (25), blocked the effect of ACTH on steroidogenesis. PGE2, however, reversed this inhibitory action of indomethacin. In our studies, pretreatment with indomethacin prevented the TSH stimulation of thyroidal ODC activity. PGE 2 administration, however, did not re-
1411
verse the indomethacin blockade. Although indomethacin administration markedly decreased the concentration of E prostaglandins in the thyroid gland, we have not excluded the possibility that the indomethacin effect on thyroidal ODC activity might be unrelated to its role as an antagonist of prostaglandin synthesis. Flores and Sharp (33), using the toad bladder, have shown that indomethacin inhibits phosphodiesterase activity. Since neither dibutyryl cAMP nor dibutyryl cAMP + aminophylline influenced thyroidal ODC activity, any effect of indomethacin on thyroid phosphodiesterase activity would not seem to explain its inhibitory action on thyroidal ODC. Further, naproxen, another chemically unrelated antagonist of prostaglandin synthesis, also blocked TSH-induced activation of thyroidal ODC. Thus, while the precise role for endogenous prostaglandins in the regulation of thyroid ODC activity remains to be defined, it would appear that reduction in thyroidal E prostaglandins prevents activation of the enzyme by TSH. In conclusion: Thyroidal ODC activity is regulated by TSH. The results of our goitrogen treatment suggest that enzyme activity is determined by endogenous, circulating TSH. ODC activity is also stimulated in a dose-related manner by pharmacological doses of exogenous TSH. The lag phase of several hours after the administration of TSH is probably the time required for new enzyme synthesis. Even though agents which interfere with microtubule-mediated functions, e.g., hormone secretion, also prevent the TSH stimulation of ODC, other data suggest, but do not confirm, that hormone secretion and ODC can be dissociated. cAMP does not seem to mediate the TSH stimulation of ODC activity. Although inhibitors of prostaglandin synthesis attenuate the stimulation of ODC by TSH, the precise role endogenous prostaglandins play in enzyme activation remains undefined. It would appear that a reduction in thyroidal prosta-
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RICHMAN ET AL.
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glandins prevents activation of the enzyme by TSH. Acknowledgments The authors gratefully acknowledge the excellent technical assistance of E. Grosvenor and W. Ray.
References 1. Tabor, H., and C. W. Tabor, Pharmacol Rev 16: 245, 1964. 2. Kremzner, L. T., Fed Proc 29: 1560, 1970. 3. Raina, A., and J. Janne, Fed Proc 29: 1568, 1970. 4. Snyder, S. H., and D. H. Russell, Fed Proc 29: 1575, 1970. 5. Russell, D. H., S. H. Snyder, and V. J. Medina, Endocrinology 86: 1414, 1970. 6. Richman, R. A., L. E. Underwood, J. J. Van Wyk, and S. J. Voina, Proc Soc Exp Biol Med 138: 880, 1971 7. Richman, R., C. Dobbins, S. Voina.. L. Underwood, D. Mahaffee, H. J. Gitelman, J. Van Wyk, and R. L. N e y J Clin Invest 52: 2007, 1973. 8. Levine, J. H., W. E. Nicholson, G. W. Liddle, and D. N. Orth, Endocrinology 92: 1089, 1973. 9. Lamprecht, S. A., U. Zor, A. Tsafriri, and H. R. Lindner, J Endocrinol 57: 217, 1973. 10. Pastan, I., and V. MacchiaJ Biol Chem 242: 5757, 1967. 11. Sato, S., M. Szabo, K. Kowalski, and G. Burke, Endocrinology 90: 343, 1972. 12. Adams, D. D., and T. H. Kennedy, J Clin Endocrinol Metab 33: 47, 1971. 13. Russell, D. and S. H. Snyder, Proc Natl Acad Sci USA 60: 1420, 1968. 14. Janne, J., and H. G. Williams-Ashman, / Biol Chem 246: 1725, 1971.
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15. Raina, A., Ada Physiol Scand 60: Suppl. 218, 1963. 16. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. RandallJ Biol Chem 193: 265, 1951. 17. Murphy, B. E., and C. J. PatteeJ Clin Endocrinol Metab 24: 187, 1964. 18. Steiner, A. L., D. M. Kipnis, R. Utiger, and C. Parker, Proc Natl Acad Sci USA 64: 367, 1969. 19. Wilber, J. F., and R. D. Utiger, Endocrinology 81: 145, 1967. 20. Mitsuma, T., J. Colucci, L. Shenkman, and C. S. Hollander, Biochem Biophys Res Commun 46: 2107, 1972. 21. Yu, S. C., L. Chang, and G. Burke, J Clin Invest 51: 1038, 1972. 22. McKenzie, J. M., and A. Williamson, J Clin Endocrinol Metab 26: 518, 1966. 23. Lineweaver, H., and D. Burk, J Am Chem Soc 56: 658, 1934. 24. Williams, J. A., and J. Wolff, Proc Natl Acad Sci USA 67: 1901, 1970. 25. Vane, J. R., Advan Biosci 9: 395, 1972. 26. Zakarija, M., and J. M. McKenzie, Metabolism 22: 1185, 1973. 27. Zor, U., Y. Koch, S. A. Lamprecht, J. Ausher, and H. R. Lindner, 7 Endocrinol 58: 525, 1973. 28. Anggard, E., and B. SamuelssonJ Biol Chem 239: 4097, 1964. 29. Free, M. J., and R. A. Jaffee, Prostaglandins 1: 483, 1972. 30. Peng, T. C., K. M. Six, and P. L. Munson, Endocrinology 86: 202, 1970. 31. Dazord, A., A. M. Morera, J. Bertrand, and J. M. Saez, Endocrinology 96: 352, 1974. 32. Gallant, S., and A. C. Brownie, Biochem Biophys Res Commun 55: 831, 1973. 33. Flores, A. G., and G. W. Sharp, Am J Physiol 233: 1392, 1972.
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T
HE polyamines are widely distributed throughout nature (1,2) and their function appears to be related to RNA metabolism (1-4). The initial step in their synthesis from ornithine is catalyzed by the enzyme ornithine decarboxylase (Lornithine carboxylase, EC 4.1.1.17). Its activity has previously been demonstrated to be hormonally regulated in a variety of tissues, including the liver (5,6) and kidney (7). It also appears that the pituitary trophic hormones may regulate ODC3 activity in Received November 8, 1974. This research was supported by Grant AM 17561 from the NIAMDD, USPHS and an Institutional Grant ACS IN 9N #25 to the Abraham Lincoln School of Medicine from the American Cancer Society. 1 Presented in part to the 50th meeting of the American Thyroid Association, St. Louis, Missouri, September 1974. 2 Requests for reprints should be addressed to G. Burke. 3 Abbreviations used in the text; ACTH = adrenocorticotropin; cAMP = cyclic adenosine 3',5'-
whether cyclic AMP (cAMP) or prostaglandins played any role in the regulation of thyroidal ODC activity. Dibutyryl cAMP, alone, or together with aminophylline, did not stimulate thyroidal ODC activity in dosages which concomitantly stimulated adrenal enzyme activity. Likewise, prostaglandin E2 (PGE2) did not stimulate thyroidal ODC activity, but did stimulate adrenal enzyme activity in a dose-related manner. However, pre-treatment of rats with inhibitors of prostaglandin synthesis prevented the activation of thyroidal ODC by TSH. One inhibitor, indomethacin, attenuated the TSH stimulation of enzyme activity in a dose-related manner. Indomethacin pretreatment also resulted in approximately a 10-fold decrease in thyroidal prostaglandin levels. Exogenous PGE2, in dosages as high as 500 pig, did not overcome the inhibitory effect of indomethacin on ODC activation. Although the precise role for endogenous prostaglandins remains to be defined, it does appear that a reduction in thyroidal prostaglandins prevents activation of the enzyme by TSH. (Endocrinology 96: 1403, 1975)
their respective target endocrine glands. This has been confirmed for both the adrenal gland (7,8) and ovary (9). We, therefore, initiated studies to determine if ODC was present in the thyroid gland. If present and regulated by TSH, then ODC activity might serve as a biological marker for the growth-promoting effects of TSH. Since many of these trophic effects are either mimicked or mediated by cyclic AMP (cAMP, 10) and/or prostaglandins (11), it was of interest to determine whether these compounds played any role in the regulation of thyroidal ODC activity. monophosphate; dibutyryl cAMP = 6N-2'-0-dibutyryl cyclic adenosine 3',5'-monophosphate; EDTA = ethylenediaminetetraacetic acid; NIAMDD = National Institute of Arthritis, Metabolism and Digestive Diseases; ODC = ornithine decarboxylase; PGEj = prostaglandin Ej; PGE2 = prostaglandin E2; T3 = 3,5,3'-triiodothyronine; T4 = L-thyroxine; TSH = thyroid-stimulating hormone; TYA = 5,8,11,14-eicosatetraynoic acid.
1403
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RICHMAN ETAL.
1404
Materials and Methods Animals. Male Holtzman rats, weighing 180200 gin, were maintained on standard laboratory chow and tap water. In some experiments, a goitrogen, 4-hydroxy-6-methyl-2-thiopyrimidine (100 mg/liter) (12), was added to their drinking water. To control for fluctuations in blood levels of goitrogen due to variations in drinking patterns, methimazole (1 mg) was also administered subcutaneously at 9:00 AM and 4:00 PM in one experiment. Five animals were assigned to each treatment group. TSH (Thytropar®, Armour Pharmaceutical, Kankakee, 111.) was usually administered between 8 and 10 AM and the animals sacrificed 4 h later. Other pharmacological agents were injected at the times specified. Control animals were usually untreated since basal enzyme activity was unaffected by injection of the vehicle. At the time of sacrifice, the rats were anesthetized with ether and exsanguinated by cardiac puncture. The serum was removed and frozen at —10 C for hormone measurement. Tissue preparation. For each treatment group, thyroid glands from 5 rats were pooled and weighed. They were homogenized with a polytron (Brinkman Instruments, Inc., Westbury, New York) at a concentration of 48 mg/ml in 0.05M Na-K phosphate buffer, pH 7.2 containing 10 mM tetrasodium EDTA and 5 mM dithiothreitol, at 4 C. The homogenates were centrifuged at 20,000 x g for 20 min at 4 C. The supernatant fractions were immediately used for enzyme assay. Enzyme assay. ODC was assayed by a semimicro modification of the method of Russell and Snyder (13). The reaction mixture contained 0.3 /xCi of DL[1 - 14C]ornithine hydrochloride, 58 or 61 mCi/mmol (Amersham-Searle Corp., Arlington Heights, Illinois), 0.1 fimol pyridoxal phosphate, 0.1-0.4 ml of the 20,000 x g thyroid supernatant fraction and buffer (described above) to make a final volume of 0.5 ml. Activity was usually assayed in triplicate. In early experiments, enzyme activity was assayed at 3 different enzyme concentrations (0.1, 0.2, and 0.4 ml of the 20,000 x g supernatant fraction). In later experiments, activity was assayed at one concentration, usually 0.3 ml. The assay was carried out in 15 ml conical centrifuge tubes, sealed with a rubber stopper,
Endo • 1975 Vol 96 • No 6
from which a polyethylene center well (Kontes Glass Co., Vineland, N.J.) was suspended. After incubation at 37 C for 30 min in a Dubnoff metabolic shaking incubator, 0.5 ml of 2M citric acid was injected into the reaction mixture, through the rubber stopper, to stop enzyme activity. 0.2 ml hydroxide of hyamine 10-X (Packard Instrument Co., Downers Grove, 111.) was injected into the center well to trap liberated I4 CO2. The tubes were agitated at 37 C for an additional 30 min. Then each well was removed and placed into a scintillation vial containing 10 ml of scintillation cocktail, consisting of 0.40 ml Permafluor® 25 x concentrated liquid scintillator (Packard Instrument Co., Downers Grove, 111.) and 9.60 ml toluene. Samples were counted in a Searle Mark® II liquid scintillation system. Counting efficiency (90%) was determined by the external standard ratio method. Results are expressed as picomoles of I4 CO 2 liberated from 14 DL[1 — C]ornithine per g wet weight tissue per 30-min incubation. Some of the calculations were performed using a 370 IBM computer, model #145. The amount of 14CO2 liberated from tubes containing no enzyme was subtracted from the value for all other samples. To decrease nonenzymatic liberation of I4CO2, 250 juCi of DL[1 - 14C]ornithine were diluted in 25 ml of 0.1 N HC1, as outlined by WilliamsAshman (14). The ornithine was dried over nitrogen and resuspended in 25 ml of buffer. 5 ml aliquots were stored at —IOC. With this procedure, the no-enzyme blank was reduced to approximately 200 cpm. Putrescine assay. To insure that the decarboxylation reaction catalyzed by the 20,000 X g supernatant fraction resulted in polyamine synthesis, putrescine formation was measured in one experiment. It was assayed according to the method of Raina (15) with DL[5 - 14C]ornithine hydrochloride, 8.19 mCi/mmol (New England Nuclear Co., Boston, Massachusetts) as the substrate. Protein determination. Total protein was measured according to the method of Lowry (16). Hormone determinations. Thyroxine (T4) was measured by a modification of the method of Murphy-Pattee (17), using reagents from Mallinckrodt Chemical Works, St. Louis, Mo. cAMP was measured by radioimmunoassay (18), using
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1405
THYROIDAL ORNITHINE DECARBOXYLASE a kit purchased from Schwarz/Mann. Rat TSH was measured by radioimmunoassay (19), using reagents supplied by the NIAMDD Rat Pituitary Hormone Distribution Program. Serum triiodothyronine (T3) 20) and thyroidal prostaglandin E (PGEJ levels (21) were determined by radioimmunoassay. Antibody to PGE was a gift from Dr. Gordon D. Niswender, Colorado State University, Fort Collins, Colorado. Materials TSH (Thytropar®) and ACTH (Acthar®) were obtained from Armour Pharmaceutical. In addition, a crude preparation of TSH (1 mg = 4 units) was a gift from Dr. J. Fisher (Armour Pharmaceutical). Potency was estimated both by a 3 point dose-response in the McKenzie mouse bioassay (22) and the rat ornithine decarboxylase assay with commercial Thytropar® as the standard. Dibutyryl cAMP and cAMP were purchased from Schwarz/Mann. Prostaglandin E2 (PGE2) was a gift from Dr. John Pike, Upjohn Co., Kalamazoo, Michigan. Methimazole was a gift from Dr. N. J. Hosley, Eli Lilly and Co., Indianapolis, Indiana. 5,8,11,14-eicosatetraynoic acid (TYA) was a gift from Dr. W. E. Scott, Hoffmann-LaRoche, Inc., Nutley, N.J. Indomethacin was purchased from Sigma Chemical Co., St. Louis, Mo., and a purified preparation was a gift from Dr. C. A. Stone, Merck, Sharp and Dohme, West Point, Pa. Naproxen was a gift from Syntex Laboratories, Palo Alto, California, and butazolidin from CIBA-Geigy Corp., Summit, N.J. T3, T4 and cycloheximide were purchased from Sigma Chemical Co., St. Louis, Mo. Human growth hormone (HS1498) was a gift of the Endocrine Study Section, NIAMDD. Colchicine, Vinblastine sulfate (Velban®), and aminophylline were purchased from Eli Lilly and Co. Human chorionic gonadotropin (A.P.L.®) was purchased from Ayerst Laboratories, New York, N.Y., isoproterenol (Isuprel®), from Winthrop Laboratories, New York, N.Y., actinomycin D (Cosmegen®) from Merck and Co., Inc. and hydrocortisone (SoluCorteP) from Upjohn Co.
Results
Characterization of the enzyme. Preliminary studies demonstrated that ODC activity was proportional to the quantity of 20,000 x g supernatant fraction assayed be-
tween 2.4 and 19.2 mg thyroid (0.05-0.4 ml). Enzyme activity was measured from pH 6.0 to 8.5 and maximal activity appeared to plateau between pH 6.9 and 7.9. For all subsequent studies, pH 7.2 was chosen to conform with our previous studies in the adrenal gland (7). The amount of 14CO2 liberated from DL[1 — 14C]ornithine increased in a linear fashion for at least 30 min of incubation. The concentration of unlabeled substrate, L-ornithine, was varied in the reaction mixture, and the data plotted according to the method of Lineweaver and Burk (23). Vmax was found to be 48.1 pmol/0.3 ml/30 min and the Km = 44.4 nM (Fig. 1). Studies on the effect of temperature revealed maximal enzyme activity occurring at 51 C. All other studies reported, however, were carried out at physiological conditions, i.e., temperature 37 C. The effect of exogenous TSH. Groups of rats were sacrificed at varying time inter-
0.05
O.I
0.15
I
[L- ORNITHINE]
0.2
X 10 - 6
FIG. 1. Relationship between substrate concentration and reaction velocity. Increasing amounts of unlabeled substrate (L-ornithine) were added to the reaction mixture and the reaction velocity, amount of CO2 released730 min., determined. Substrate concentration (M) and reaction velocity were plotted as reciprocals according to the method of Lineweaver and Burk (23).
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1406
Endo • 1975 Vol 96 • No 6
RICHMAN ET AL.
vals after the intraperitoneal administration of 4 U of TSH. Little effect on enzyme activity was apparent before three hours (Fig. 2). Maximal ODC activity occurred between 4 and 5 h after TSH administration. In a subsequent series of experiments, varying doses of TSH were injected and the thyroid glands removed 4 h later. No increase in enzyme activity was seen with 0.25 U TSH. A linear log-dose-response curve was obtained between 1 and 8 U of TSH (Fig. 3). To determine if an inhibitor of enzyme activity was present in thyroid glands from untreated animals, the 20,000 x g supernatant fraction from control and TSHstimulated glands were mixed. The resulting enzyme activity of the mixture was comparable to that found in TSHstimulated glands alone (unpublished data). This excluded the possibility that the apparent increase in ODC activity was solely due to TSH decreasing the activity of an enzyme inhibitor. A variety of other hormones including ACTH, human growth hormone, T3, hydrocortisone, human chorionic gonadotropin and isoproterenol were administered. None of the other hormones tested increased thyroid enzyme activity 4 h after injection.
2 3 4 5 6 7 8 HOURS AFTER TSH ADMINISTRATION FIG. 2. The time course of thyroid ODC activity in rats given 4 U of TSH intraperitoneally. Groups of 5 rats each were sacrificed at various times after the administration of TSH. Enzyme activity is expressed as pmoles of 14CO2 liberated per g of thyroid tissue. A representative experiment is shown with each point the mean of 3 determinations.
2000-
0.25
1.0
2.0
8.0
UNITS OF TSH
FIG. 3. Effect of varying doses of exogenous TSH on thyroidal ODC activity. Groups of 5 rats each were given varying doses of TSH intraperitoneally. The animals were sacrificed 4 h later and the thyroid glands removed for determination of ODC activity. Each point represents the mean ± SEM for 2-5 experiments with the number shown in parenthesis.
Also, TSH did not stimulate ODC activity in liver, kidney or adrenal glands. In order to demonstrate the subcellular location of ODC activity in thyroid, crude subcellular fractions were prepared by differential centrifugation. 0.25M sucrose was used to homogenize the tissue instead of buffer. Each pellet was washed a single time with sucrose and then gently resuspended. Enzyme activity was determined for each fraction as described for the 20,000 x g supernatant fraction but was expressed as cpm/mg protein. In addition, each subcellular fraction was assayed in the presence of putrescine (1 mg/reaction tube), a known inhibitor of ODC activity in vitro (14). As seen in Fig. 4, 14CO2 was liberated by several subcellular fractions. However, the 100,000 g supernatant fraction was the only fraction in which the rate of 14CO2 evolution was increased by administration of TSH in vivo and inhibited by the presence of putrescine in vitro. We, therefore, concluded that most, if not all, ODC activity was present in the cytosol. The 14CO2 liberated by the other subcellular fractions probably resulted from biochemical reactions in metabolic path-
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THYROIDAL ORNITHINE DECARBOXYLASE
8000-
R
1407 NO TREATMENT NO PUTRESCINE
7000-
NO TREATMENT PUTRESCINE PRESENT
6000-
TSH TREATED NO PUTRESCINE
5000-
TSH TREATED PUTRESCINE PRESENT
z >o
4000300020001000WHOLE H0M06ENATE
600 g PELLET
5,500 g PELLET
20,000 g PELLET
100,000 g SUPERNATE
SUBCELLULAR FRACTIONS FIG. 4. Subcellular localization of ODC activity. Enzyme activity in various subcellular fractions was compared in control and TSH-stimulated glands. Glands were homogenized in 0.25M sucrose at a concentration of 48 mg/cc. Subcellular fractions were prepared by differential centrifugation. Each pellet was washed a single time and then gently resuspended. 14CO2 release was determined for each fraction, as described in Materials and Methods for the 20,000 x g supernatant fraction. In addition, each fraction was assayed in the presence of putrescine (1 mg/tube), an in vitro inhibitor of the ODC reaction. Enzyme activity is expressed as cpm/mg protein. Since 14CO2 was not released by either 100,000 x ^•pfelket, the data is not shown.
ways other than polyamine synthesis in which ornithine is converted to another compound prior to decarboxylation. In another experiment, the percent of ornithine hydrochloride decarboxylated, as measured by both 14CO2 release from DL[1 — 14C] ornithine and 14C — putrescine formation from Dl_[5 — 14C]ornithine, by 20,000 x g supernatant fractions from control and TSH-stimulated glands was compared. Less than 0.02% of both ornithine substrates was decarboxylated by the supernatant fractions from control glands. The supernatant fractions from the TSHtreated glands converted 0.58% of the [5 — 14C]ornithine to [14C]putrescine and 0.89% of the [1 - 14C]ornithine to 14CO2.
Since the two substrates differed in their specific activities, no attempt was made to demonstrate stoichiometry. In any event, these data indicate that the release of 14CO2 by the 20,000 x g thyroid supernatant fraction correlates well with putrescine formation. Effect of goitrogen treatment on thyroid ODC activity. In this series of experiments, rats were given a goitrogen, 4-hydroxy-6-methyl-2-thiopyrimidine (100 mg/liter) in their drinking water for 1-6 days. On the first day of treatment, there was an abrupt fall in the serum concentration of T3 (Fig. 5). As expected, there was a secondary rise in circulating TSH. There
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Endo ( 1975 Vol96< No 6
RICHMAN ET AL.
1408
700THYROID WEIGHT -100 -800 E
g -120
Z> L
600 w I
8 0 ^ -40
1
2
3 4 DAYS ON 60ITR0GEN
-400"-200
5
FIG. 5. Effect of goitrogen on serum TSH, T3, thyroidal ODC and thyroid gland weight. Groups of animals were given a goitrogen in their drinking water for 0-6 days. All groups of animals were exsanguinated and the thyroid glands excised between 12:30 PM and 2:30 PM on the same afternoon. The glands were weighed and assayed for ODC activity in the usual manner. The serum was removed and stored at —IOC for hormone determination. Gland weights represent the pooled weight (mg) of 10 glands. ODC activity is expressed as the mean 14CO2 pmol/g tissue for 2 representative experiments. T3 and TSH were determined for each serum specimen and expressed as the mean for the group.
was a small, but significant, increase in ODC activity after only 1 day on goitrogen (Fig. 5). On the second day, a dramatic rise in enzyme activity was detected. Despite the continued rise in serum TSH and a progressive increase in thyroid gland size with further treatment, ODC activity declined on the third day and remained at submaximal levels. T 3 levels decreased until the 3rd day of treatment and then plateaued. The effect of inhibitors ofRNA and protein synthesis. The intraperitoneal administration of 0.9 mg of actinomycin D or 10 mg of cycloheximide, 15 min before TSH, totally inhibited the expected increase in thyroidal ODC activity 4 h later (data not shown). Effect of microtubule-active agents. It has been previously demonstrated that agents which alter microtubule structure inhibit
thyroid hormone secretion (24). Two such agents, colchicine and vinblastine, were administered subcutaneously the evening before the experiment and intraperitoneally, 4 h before sacrifice. TSH was also given at this time in a second injection site. Both agents markedly inhibited the TSH stimulation of ODC activity, lowered basal serum T4 levels, and blunted the increase in serum T4 following TSH (Table 1). Effect of dibutyryl cAMP administration. Fifty mg of dibutyryl cAMP were given by the intraperitoneal route. Animals were sacrificed at varying time intervals after injection. Both thyroid lobes were removed from all animals and one adrenal gland from each animal sacrified 4 h after dibutyryl cAMP injection. No increase in thyroid ODC was detected (data not shown). As previously reported (7), enzyme activity was elevated at 4 h in the adrenal glands. In an attempt to demonstrate an
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THYROIDAL ORNITHINE DECARBOXYLASE
1409
effect of dibutyryl cAMP which might have TABLE 1. Effect of pretreatment with colcbicine or on the TSH stimulation of thyroidal ODC been masked by its rapid destruction, 10 vinblastine activity and hormone secretion mg of the phosphodiesterase inhibitor Serum T4* ODC activity aminophylline and 30 mg of dibutyryl jig/100 ml pmol 14CO2/g cAMP were administered simultaneously, - TSH + TSH - TSH + TSH in separate intraperitoneal injection sites. Inhibitor** The animals was sacrificed at varying time None 3.7 ± 0.04 5.6 ± 0.1 48 1,968 intervals thereafter, and the thyroid and Colchicine 20 2.6 ± 0.2 1.9 ± 0.1 0 (0.2 mg) adrenal glands excised. The minimal in14 crease (to 125 pmol CO2/g/30 min) in Vinblastine 1.4 ± 0.2 32 0.4 ± 0.1 0 (lmg) ODC activity in the thyroid gland after 3 h appeared insignificant when compared to * Mean of 5 sera ± SEM were administered in the doses specified, subcutaneously the dramatic increase following TSH ad- 16**hInhibitors before and again intraperitoneally at the same time as 4 U of ministration. In contrast, there was a TSH. The animals were sacrificed 4 h later. marked increase in adrenal enzyme activity Effect of inhibitors of prostaglandin (1106 pmol 14CO2/g/30 min) at 3 h. synthesis. To further investigate any role Effect of PGE2 administration. PGE 2 was that endogenous prostaglandins might play administered intraperitoneally in several in mediating the effect of TSH on thyroid doses up to 500 fig/rat and at varying time ODC, several inhibitors of prostaglandin intervals (through 5 h) prior to the meas- synthesis (25) were administered prior to urement of thyroid and adrenal ODC TSH. Changes in basal levels of ODC were activity. Adrenal ODC activity peaked 3 h not detected when the inhibitors were after 100 /*g PGE 2 (397 pmol 14CO2/g/30 injected alone. As shown in Tables 2 and 3, min) and was stimulated in a dose-related indomethacin and naproxen clearly inhibmanner (data not shown). No effect on ited the TSH stimulation of ODC. The thyroid enzyme activity was apparent. Al- effects of the other inhibitors, at the dosthough prostaglandins modify some effects ages employed, are not as definitive and of TSH when they are administered to- the changes might be due to biological gether (11), no consistent effect on TSH variation. Indomethacin attenuated the stimulated enzyme activity was seen when TSH effect on enzyme activity and serum the PGE 2 was administered simultaneous- T4 levels in a dose-related fashion. Inly, 15 min, 1 h, or 2 h prior to TSH domethacin, at a dose of 1 mg, appeared to inhibit the TSH stimulation of ODC activadministration. TABLE 2. Effects of inhibitors of prostaglandin synthesis on the TSH stimulation of ODC activity and hormone secretion Experiment 2
Experiment 1
Inhibitor**
-TSH
+ TSH
None Aspirin Naproxen Butazolidin TYA***
38 34 24 49 13
3,188 1,970 311 1,914 1,334
ODC activity pmol "COj/g
Serum T4* /zg/100 ml
ODC activity pmol 14CO2/g
-TSH 4.1 4.0 2.9 4.2 3.4
± 0.1 ±0.1 ± 0.2 ± 0.04 ±0.1
+ TSH 7.6 5.5 4.9 4.9 5.6
±0.1 ±0.1 ±0.1 ±0.1 ±0.1
Serum T4* /ig/100 ml
-TSH
+ TSH
-TSH
44 14 0 100
3,691 1,687 65 2,149
4.6: t 0.1 4.0: tO.l 2.6: t 0.1 4.5: t 0.1
+ TSH 7.0 6.3 4.5 5.8
± ± ± ±
0.1 0.2 0.3 0.1
* Mean of 5 sera ± SEM. ** 10 mg of each inhibitor of prostaglandin synthesis were administered subcutaneously 18 h before an intraperitoneal injection of 4 U of TSH. *** TYA = 5, 8, 11, 14-eicosatetraynoic acid.
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Endo i 1975 Vol 96 < No 6
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TSH unequivocally stimulated an increase in thyroid ODC activity. Although we have not attempted to define the precise role of *Dose (mg) of ODC activity T4** the enzyme in regulation of thyroid funcTSH indomethacin pmol 14CO2/g Atg/lOO ml tion, the results of our goitrogen treatment suggest that enzyme activity is regulated — — 20 3.6 ± 0.4 + — 1,495 7.7 ± 0.4 by endogenous TSH. Enzyme activity also 1,340 + 0.25 8.8 ± 0.5 increases proportionally to pharmacological 401 + 1.00 8.9 ± 0.5 doses of exogenous TSH. The lag phase of 68 + 2.50 4.9 ± 0.4 11 + 5.00 5.2 ± 0.4 several hours after the administration of TSH appears to have biological impor* Indomethacin was injected at the stated dose subtance. During the first 30 min after horcutaneously 22 h before sacrifice and repeated intraperitoneally 4 h before sacrifice. mone injection, biological effects, such as an ** Mean of 4 - 5 sera ± SEM. increase in endogenous cAMP in the thyroid (26), can be measured. Therefore ity, but not its effect on serum T4 (Table 3). this lag in the appearance of an increase in The inhibition of enzyme activity by 1.5 ODC activity is not simply due to delayed mg of indomethacin, could not be reversed absorption of the hormone from the injecby increasing the dose of TSH adminis- tion site. Since there is no increase in ODC tered (data not shown). Indomethacin pre- activity when actinomycin D or cyclotreatment significantly reduced endogen- heximide is injected 15 min before TSH, ous prostaglandin levels in control and the lag period may be the time required for TSH-stimulated thyroid glands (Table 4). synthesis of new enzyme. The timing of the indomethacin injection Studies with agents promoting dewas very critical. The inhibitory effect was polymerization (colchicine) or aggregation demonstrable if indomethacin was given 1 (vinblastine) of microtubules demonstrated h or more prior to TSH administration. If, that they can inhibit not only TSH-induced however, indomethacin was given 1 h after hormone secretion (24) but also ODC TSH, no inhibition of enzyme activity was stimulation. This suggests that activation of detected. Indomethacin given at the same ODC in thyroid is dependent upon mitime as TSH, did not consistently inhibit hormonal activation of ODC. The increase crotubule integrity and is in some way in serum T4 levels was not clearly atten- related to the intracellular process involved uated when indomethacin was adminis- in hormone secretion. Yet, the observation tered an hour or less before TSH (data not that at least one dose (1.0 mg) of inshown). Exogenous PGE 2 , in high doses as domethacin inhibited the expected inhigh as 500 fig, given either 1 h before or crease in ODC activity, but not the serum 17 h after indomethacin, did not appear to TABLE 4. Effect of indomethacin treatment on influence the inhibitory effect of inthyroidal PGE, levels domethacin on the TSH stimulation of PGE t * enzyme activity. Indomethacin (10 fig/ml) (ng/mg thyroid) added to the reaction mixture, did not alter Time after TSH (4 U) + Indomethacin enzyme activity in vitro.
TABLE 3. Effect of increasing the dose of indomethacin on the TSH stimulation of thyroidal ODC activity and hormone secretion
(min)
- Indomethacin
(2.5 mg)
45 180
0.265 0.280 0.380
0.026 0.033 0.056
Discussion The results of these studies in the thyroid yield additional evidence that the pituitary trophic hormones regulate ODC activity in their target endocrine glands.
* Each value represents the mean for 2 thyroid glands except for the TSH 45 min + indomethacin which represents only a single gland.
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THYROIDAL ORNITHINE DECARBOXYLASE T4 elevation, suggests that hormone secretion and ODC activation may be dissociable. Additional studies are necessary to explore their relationship. It was previously suggested that cAMP may mediate the effect of ACTH on ODC activity in the adrenal gland (7). The data presented here suggest that this is unlikely to obtain for TSH and the thyroid. Whereas dibutyryl cAMP stimulated adrenal ODC activity, this cyclic nucleotide, even in the presence of aminophylline, did not substantially increase thyroid enzyme activity. The data also suggest that endogenous prostaglandins may be important in regulation of thyroidal ODC activity. Since exogenous PGE2 mimics most of the effects of TSH on the thyroid gland (11) and stimulates uterine ODC activity (27), we were concerned about the lack of a direct effect of exogenous PGE2 on thyroid ODC activity. This may have been secondary to either rapid inactivation in the lung, or other organs (28) of the administered PGE2 or to changes in the cardiovascular status of the animals (29). Nevertheless, concomitant studies in the adrenal gland revealed that PGE 2 administration unequivocally elevated adrenal ODC activity. Although this increase in adrenal enzyme activity may be secondary to the previously reported rise in ACTH following prostaglandin administration (30), Dazord et al. (31) recently reported the presence of plasma membrane receptors for PGEX and PGE2 in the adrenal gland. In any event, there is no doubt that PGE2 is biologically effective when injected into the peritoneum. Gallant and Brownie (32) were also unable to show a direct in vivo effect of PGE 2 on adrenal steroidogenesis. Prior treatment with indomethacin, a known inhibitor of prostaglandin synthesis (25), blocked the effect of ACTH on steroidogenesis. PGE2, however, reversed this inhibitory action of indomethacin. In our studies, pretreatment with indomethacin prevented the TSH stimulation of thyroidal ODC activity. PGE 2 administration, however, did not re-
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verse the indomethacin blockade. Although indomethacin administration markedly decreased the concentration of E prostaglandins in the thyroid gland, we have not excluded the possibility that the indomethacin effect on thyroidal ODC activity might be unrelated to its role as an antagonist of prostaglandin synthesis. Flores and Sharp (33), using the toad bladder, have shown that indomethacin inhibits phosphodiesterase activity. Since neither dibutyryl cAMP nor dibutyryl cAMP + aminophylline influenced thyroidal ODC activity, any effect of indomethacin on thyroid phosphodiesterase activity would not seem to explain its inhibitory action on thyroidal ODC. Further, naproxen, another chemically unrelated antagonist of prostaglandin synthesis, also blocked TSH-induced activation of thyroidal ODC. Thus, while the precise role for endogenous prostaglandins in the regulation of thyroid ODC activity remains to be defined, it would appear that reduction in thyroidal E prostaglandins prevents activation of the enzyme by TSH. In conclusion: Thyroidal ODC activity is regulated by TSH. The results of our goitrogen treatment suggest that enzyme activity is determined by endogenous, circulating TSH. ODC activity is also stimulated in a dose-related manner by pharmacological doses of exogenous TSH. The lag phase of several hours after the administration of TSH is probably the time required for new enzyme synthesis. Even though agents which interfere with microtubule-mediated functions, e.g., hormone secretion, also prevent the TSH stimulation of ODC, other data suggest, but do not confirm, that hormone secretion and ODC can be dissociated. cAMP does not seem to mediate the TSH stimulation of ODC activity. Although inhibitors of prostaglandin synthesis attenuate the stimulation of ODC by TSH, the precise role endogenous prostaglandins play in enzyme activation remains undefined. It would appear that a reduction in thyroidal prosta-
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glandins prevents activation of the enzyme by TSH. Acknowledgments The authors gratefully acknowledge the excellent technical assistance of E. Grosvenor and W. Ray.
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